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Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: Modulation by glucocorticoids and inflammation
British Journal of Pharmacology
Abstract
Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N‐terminus peptide, cell‐associated LC1‐like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane.
Treatment of mice with dexamethasone (Dex; 0.5‐5 μg per mouse corresponding to ∼ 0.015‐1.5 mg kg ⁻¹ ) increased LC1 levels in neutrophils and monocytes. The 2–3 fold increase in LC1 levels was time‐dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg ⁻¹ orally) decreased LC1‐like immunoreactivity in all three types of circulating leukocytes by ≥50%.
Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (≥50%) in LC1 levels compared with circulating neutrophils. A high LC1‐like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane‐associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane‐associated LC1 to a significant extent, i.e. up to 70%.
In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.
... The cellular in¯ux into the air-pouch 24 h after Ova challenge consisted of a mixed population of leucocytes. We wished to dierentiate between the mononuclear and PMN populations and, to characterize the PMN population by use of¯ow cytometry as previously described (Perretti & Flower, 1996). For this purpose we utilized mAbs directed against speci®c surface antigens present on murine leucocytes. ...
... The eect of Dex on intracellular LC1 levels of circulating eosinophils was measured by use of a protocol previously described (Perretti & Flower, 1996). As the percentage of circulating eosinophils in BALB/c mice is very low, CBA/Ca mice overexpressing the human IL-5 gene were used for these experiments (Dent et al., 1990). ...
... Different cell populations were discriminated by their forward and side scatter characteristics and the MFI values were calculated in the FL-1 channel. The number of LC1 molecules was then calculated from the MFI values by use of reference microbeads labelled with standard numbers of FITC molecules (Flow Cytometry Standards Corp) as described previously (Perretti & Flower, 1996). The results are presented as the number of LC1like immunoreactive molecules per cell. ...
We have developed a novel model of allergen-induced eosinophil extravasation into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism(s) underlying the anti-inflammatory action of the glucocorticoid hormone dexamethasone (Dex).
Injection of 10 μg Ova into 6-day-old dorsal air-pouches of mice sensitized to the same antigen provoked an intense cell accumulation as early as 6 h post-challenge (0.08±0.03 and 4.0±1.0×105 leucocytes in saline and Ova-treated air-pouches, respectively), maximal at 24 h (0.02±0.01 and 6.0±0.8×105 leucocytes in saline and Ova-treated air-pouches, respectively) and persisted up to 48 h. At the 24 h time-point, the cellular infiltrate consisted of 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as assessed by histological examination. The same ratio of eosinophil/neutrophil was obtained by fluorescence-activated cell sorting (FACS) analysis, since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4 (VLA-4) expression.
Subcutaneous (s.c.) administration of Dex (50 or 100 μg per mouse, −1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P<0.05) and 75% (P<0.05), respectively, when compared to controls. Cell accumulation measured at 48 h after Ova injection was also significantly reduced (−75%) by Dex administration at the 24 h time-point (n=12, P<0.05).
Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eosinophil numbers in the bone marrow (naive mice: 2.7±0.3×105; sensitized mice: 8.7±1.7×105, P<0.05) and blood (naive mice: 0.5±0.2×105; sensitized mice: 1.5±0.3×105, P<0.01). However, 24 h following Ova challenge, the eosinophil numbers in the bone marrow had dropped (3.7±0.8×105) with no change in the circulating pool, suggesting an equilibrium within the eosinophil pools had been reached.
Dex administration provoked a profound eosinopaenia in the blood of naive (5.2±1.5 to 0.9±0.6×104) and sensitized mice (1.5±0.3 to 0.08±0.02×105) at 4 h. This effect was reversed within 24 h. Dex also inhibited the release of eosinophils from the bone marrow in response to Ova challenge.
We show for the first time that eosinophils express the steroid-inducible protein lipocortin 1 (LC1). FACS analysis of eosinophils emigrated into the Ova challenged air-pouches revealed detectable LC1-like immunoreactivity (373×103). These data were also substantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 μg) did not alter LC1 levels in blood eosinophils, such that 235±21×103 LC1-like molecules per cell were measured after vehicle treatment (n=5), and 224±8×103 LC1-like molecules per cell were associated with this cell type 1 h after steroid treatment (n=5, not significant). Finally, resident eosinophils (in the pleural cavity) were found to have much higher LC1 levels than that found in the blood circulation (2 fold increase, P<0.05).
Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modify the anti-migratory activity exerted by Dex towards eosinophil extravasation into Ova-challenged air-pouches. The steroid (50 μg s.c., −1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) and LCS3-treated mice (−56% and 59%, respectively, n=15–21, not significant).
In conclusion, the air-pouch provides a novel and convenient cavity to study allergen-induced cell recruitment which is sensitive to glucocorticoid hormone treatment. The effect of Dex on eosinophil distribution in these experimental conditions has been studied in detail and we failed to find an important role for endogenous LC1 in these actions of the steroid.
British Journal of Pharmacology (1997) 121, 97–104; doi:10.1038/sj.bjp.0701122
... AnxA1 is found in many tissues, including the lungs, bone marrow, and intestine, at concentrations of <50 ng/mL, with the highest levels reported to be in the seminal fluid (150 µg/mL). Although found in many differentiated cells and tissues, AnxA1 makes up 2-4% of the total cytosolic protein in some cells, such as PMN [84]. ...
... An intricate association between AnxA1 and GCs was uncovered when it was shown that AnxA1 modulates the GC-induced secretion of the adrenocorticotrophic hormone (ACTH) from the anterior pituitary gland [86,87]. Congruently, it was also demonstrated that AnxA1 levels in murine peripheral blood leukocytes were raised two-to threefold within 2 h of steroid treatment, and this effect was blocked by the GC receptor antagonist RU486 [84]. ...
Our interest in inflammation and its treatment stems from ancient times. Hippocrates used willow bark to treat inflammation, and many centuries later, salicylic acid and its derivative aspirin’s ability to inhibit cyclooxygenase enzymes was discovered. Glucocorticoids (GC) ushered in a new era of treatment for both chronic and acute inflammatory disease, but their potentially dangerous side effects led the pharmaceutical industry to seek other, safer, synthetic GC drugs. The discovery of the GC-inducible endogenous anti-inflammatory protein annexin A1 (AnxA1) and other endogenous proresolving mediators has opened a new era of anti-inflammatory therapy. This review aims to recapitulate the last four decades of research on NSAIDs, GCs, and AnxA1 and their anti-inflammatory effects.
... The protein is present in many differentiated cell types, predominantly abundant in cells of the myeloid lineage including macrophages, mast cells, eosinophils, and neutrophils. Many studies have shown that the synthesis and secretion of Anx-A1 is triggered by glucocorticoids (Goulding et al., 1990;Peers et al., 1993;Ahluwalia et al., 1994;Perretti and Flower, 1996), and this evidence is supported by the strong correlation between peripheral blood Anx-A1 concentrations and plasma cortisol/corticosterone in rodents and man (Perretti and Flower, 1996;Mulla et al., 2005). Glucocorticoids not only stimulate the transcription of Anx-A1 but also induce the release of preexisting pools of Anx-A1 in the cytoplasm via a receptor-dependent, non-genomic pathway (Oliani et al., 2000), which is preceded by phosphorylation at key sites in the N-terminus and other sites, catalyzed by protein kinase C (PKC) (Croxtall et al., 2000;John et al., 2003;Solito et al., 2003). ...
... The protein is present in many differentiated cell types, predominantly abundant in cells of the myeloid lineage including macrophages, mast cells, eosinophils, and neutrophils. Many studies have shown that the synthesis and secretion of Anx-A1 is triggered by glucocorticoids (Goulding et al., 1990;Peers et al., 1993;Ahluwalia et al., 1994;Perretti and Flower, 1996), and this evidence is supported by the strong correlation between peripheral blood Anx-A1 concentrations and plasma cortisol/corticosterone in rodents and man (Perretti and Flower, 1996;Mulla et al., 2005). Glucocorticoids not only stimulate the transcription of Anx-A1 but also induce the release of preexisting pools of Anx-A1 in the cytoplasm via a receptor-dependent, non-genomic pathway (Oliani et al., 2000), which is preceded by phosphorylation at key sites in the N-terminus and other sites, catalyzed by protein kinase C (PKC) (Croxtall et al., 2000;John et al., 2003;Solito et al., 2003). ...
Mast cell stabilizers like cromoglycate and nedocromil are mainstream treatments for ocular allergy. Biochemical studies in vitro suggest that these drugs prevent mast cell degranulation through the release of Annexin-A1 (Anx-A1) protein. However, the direct effect of Anx-A1 gene deletion on mast cell function in vitro and in vivo is yet to be fully investigated. Hence, we aim to elucidate the role of Anx-A1 in mast cell function, both in vivo and in vitro, using a transgenic mouse model where the Anx-A1 gene has been deleted. Bone marrow-derived mast cells (BMDMCs) were cultured from wild-type animals and compared throughout their development to BMDMCs obtained from mice lacking the Anx-A1 gene. The mast cell differentiation, maturity, mediator, and cytokine release were explored using multiple biochemical techniques, such as Western blots, ELISA, and flow cytometry analysis. Electron microscopy was used to identify metachromatic granules content of cells. For in vivo studies, Balb/C wild-type and Anx-A1-deficient mice were divided into the following groups: group 1, a control receiving only saline, and group 2, which had been sensitized by prior exposure to short ragweed (SRW) pollen by topical contact with the conjunctival mucosae. Allergic conjunctivitis was evaluated blind after 24 h by trained observers scoring clinical signs. Electron micrographs of BMDMCs from Anx-A1-null mice revealed more vacuoles overall and more fused vacuoles than wild-type cells, suggesting enhanced secretory activity. Congruent with these observations, BMDMCs lacking the Anx-A1 gene released significantly increased amounts of histamine both spontaneously as well as in response to Ig-E-FcεRI cross-linking compared to those from wild-type mice. Interestingly, the spontaneous release of IL-5, IL-6, IL-9, and monocyte chemoattractant protein-1 (MCP-1) were also markedly increased with a greater production observed upon IgE cross-linking. This latter finding is congruent with augmented calcium mobilization in BMDMCs lacking the Anx-A1 gene. In vivo, when compared to wild-type animals, Anx-A1-deficient mice exposed to SRW pollen displayed exacerbated signs and symptoms of allergic conjunctivitis. Taken together, these results suggest Anx-A1 is an important non-redundant regulator of mast cell reactivity and particularly in allergen mediated allergic reactions.
... LC1 is the first member of the annexin or lipocortin superfamily of phospholipid-and calcium-binding proteins to have been cloned (36), and it is widely accepted that its synthesis and disposition can be modulated by glucocorticoids as well as by the cytokine interleukin-6 (36)(37)(38)(39). Recently, we have demonstrated that administration of LC1 to experimental animals reduced the extravasation of blood-borne neutrophils in simple models of acute inflammation (16,40), apparently by interfering with the process of neutrophil interaction with the activated endothelium (19). ...
... The profile of effect was similar to that attained with LC1 (i.e., inhibition of IS and IS/LV, reduction in MPO values, TNF-α and MIP-1α levels, and no effect on the parameters of systemic cell activation). Treatment with DEX is likely to increase LC1 levels in circulating leukocytes (18,38,54), thereby augmenting the inhibitory action of endogenous LC1 on neutrophils adherent to the endothelium (15,55,56). Further investigation is required to study the potential role of endogenous LC1 in a systematic manner. ...
... The function of Anx-A1 in mammals includes the ability to reduce inflammation [1,2] and to activate many pro-resolution pathways [3]. Both the release of preformed Anx-A1 and the transcription of Anx-A1 mRNA are further enhanced in cells of the innate immune system by endogenous cortisol or exogenous glucocorticoids, which in this regard act to amplify the 'alarm signals' [4]. Extensive work using anti-Anx-A1 neutralizing antibodies [5], anti-sense RNA [6] or transgenic Anx-A1 null mice [7], has demonstrated conclusively that this protein is a major player in the anti-inflammatory/pro-resolution arm of the innate immune response. ...
... Anx-A1 is expressed in human and mouse T cells but to a lesser extent than in circulating innate immune cells such as neutrophils and monocytes [4,8]. For this reason, the biological function of Anx-A1 in T cells was long neglected and has only been interrogated during the last decade. ...
Annexin-A1 (Anx-A1) is an endogenous anti-inflammatory molecule and while described as a repressor of innate immune responses, the role of Anx-A1 in adaptive immunity, and in particular in T helper (Th) cell responses, remains controversial. We have used a T-cell mediated mouse model of retinal autoimmune disease to unravel the role of Anx-A1 in the development of autoreactive Th cell responses and pathology. RBP1–20-immunized C57BL/6 Anx-A1−/− mice exhibit significantly enhanced retinal inflammation and pathology as a result of an uncontrolled proliferation and activation of Th17 cells. This is associated with a limited capacity to induce SOCS3, resulting in un-restricted phosphorylation of STAT3. RBP1–20-specific CD4+ cells from immunized Anx-A1−/− animals generated high levels of Th17 cells-associated cytokines. Following disease induction, daily systemic administration of human recombinant Anx-A1 (hrAnx-A1), during the afferent phase of disease, restrained autoreactive CD4+ cell proliferation, reduced expression of pro-inflammatory cytokines IL-17, IFN-γ and IL-6 and attenuated autoimmune retinal inflammatory disease. Furthermore, in man, Anx-A1 serum levels when measured in active uveitis patient sera were low and associated with the detection of IgM and IgG anti-Anx-A1 antibodies when compared to healthy individuals. This data supports Anx-A1 as an early and critical regulator of Th17 cell driven autoimmune diseases such as uveitis.
... Anx-A1 is present in many differentiated cell types in man and animals but is particularly abundant in cells of the myeloid lineage including neutrophils, eosinophils, macrophages and mast cells. Glucocorticoids (GCs) potently stimulate synthesis and secretion35363738 of Anx-A1 and there is a correlation between plasma corticosterone/cortisol and peripheral blood Anx-A1 concentra- tions [38,39]. Not only do GCs increase the transcription of the Anx-A1 gene in target cells including mast cells [40] but also stimulate the release of pre-existing cytosolic pools of Anx-A1 through a receptor-dependent, non-genomic mechanism. ...
... Anx-A1 is present in many differentiated cell types in man and animals but is particularly abundant in cells of the myeloid lineage including neutrophils, eosinophils, macrophages and mast cells. Glucocorticoids (GCs) potently stimulate synthesis and secretion35363738 of Anx-A1 and there is a correlation between plasma corticosterone/cortisol and peripheral blood Anx-A1 concentra- tions [38,39]. Not only do GCs increase the transcription of the Anx-A1 gene in target cells including mast cells [40] but also stimulate the release of pre-existing cytosolic pools of Anx-A1 through a receptor-dependent, non-genomic mechanism. ...
Although the 'cromones' (di-sodium cromoglycate and sodium nedocromil) are used to treat allergy and asthma, their 'mast cell stabilising' mechanism of pharmacological action has never been convincingly explained. Here, we investigate the hypothesis that these drugs act by stimulating the release of the anti-inflammatory protein Annexin-A1 (Anx-A1) from mast cells.
We used biochemical and immuno-neutralisation techniques to investigate the mechanism by which cromones suppress histamine and eicosanoid release from cord-derived human mast cells (CDMCs) or murine bone marrow-derived mast cells (BMDMCs) from wild type and Anx-A1 null mice.
CDMCs activated by IgE-FcRε1 crosslinking, released histamine and prostaglandin (PG) D2, which were inhibited (30-65%) by 5 min pre-treatment with cromoglycate (10 nM) or nedocromil (10 nM), as well as dexamethasone (2 nM) and human recombinant Anx-A1 (1-10 nM). In CDMCs cromones potentiated (2-5 fold) protein kinase C (PKC) phosphorylation and Anx-A1 phosphorylation and secretion (3-5 fold). Incubation of CDMCs with a neutralising anti-Anx-A1 monoclonal antibody reversed the cromone inhibitory effect. Nedocromil (10 nM) also inhibited (40-60%) the release of mediators from murine bone marrow derived-mast cells from wild type mice activated by compound 48/80 and IgE-FcRε1 cross-linking, but were inactive in such cells when these were prepared from Anx-A1 null mice or when the neutralising anti-Anx-A1 antibody was present.
We conclude that stimulation of phosphorylation and secretion of Anx-A1 is an important component of inhibitory cromone actions on mast cells, which could explain their acute pharmacological actions in allergy. These findings also highlight a new pathway for reducing mediator release from these cells.
... It is reasonable to assume that for AnxA1 to participate in development of the adaptive response, that the key cellular players would express AnxA1 and/or FPR2/ALXR. As such, it was recognized many years ago that AnxA1 is expressed constitutively by T cells, although at ~25% of the levels expressed in neutrophils (Goulding et al., 1990; Morand et al., 1995; Perretti and Flower, 1996; Paschalidis et al., 2009; Spurr et al., 2011). In addition, while unstimulated T cells have been shown to express FPR2/ALXR at low or negligible levels, following stimulation they increase surface expression of FPR2/ALXR within 30 min, maintaining elevated expression for several hours (D’Acquisto et al., 2007a). ...
... T cells and mast cells express the protein, although B cells express it at low levels, and platelets do not (Cirino et al., 1987; Morand et al., 1995; Oliani et al., 2000; Rescher and Gerke, 2004). Cell differentiation (such as monocytes maturing into macrophages) tends to be associated with higher levels of expression of AnxA1, as demonstrated in studies showing that levels of AnxA1 expression are lower in monocytes relative to those in macrophages from the same donor (Perretti and Flower, 1996). ...
Inflammation is the body’s way of defending itself against noxious stimuli and pathogens. Under normal circumstances, the body is able to eliminate the insult and subsequently promote the resolution of inflammation and the repair of damaged tissues. The concept of homeostasis is one that not only requires a fine balance between both pro-inflammatory mediators and pro-resolving/anti-inflammatory mediators, but also that this balance occurs in a time and space-specific manner. This review examines annexin A1, an anti-inflammatory protein that, when used as an exogenous therapeutic, has been shown to be very effective in limiting inflammation in a diverse range of experimental models, including myocardial ischemia/reperfusion injury, arthritis, stroke, multiple sclerosis, and sepsis. Notably, this glucocorticoid-inducible protein, along with another anti-inflammatory mediator, lipoxin A4, is starting to help explain and shape our understanding of the resolution phase of inflammation. In so doing, these molecules are carving the way for innovative drug discovery, based on the stimulation of endogenous pro-resolving pathways.
... Control animals received an equivalent volume of saline. Where indicated, animals were orally given RU 486 (20 mg kg 71 , Perretti & Flower, 1996) in 0.5% methylcellulose, with the help of a spherical end stainless steel catheter, 2 h before dexamethasone administration. Control animals received an equivalent volume of 0.5% methylcellulose. ...
... Both eects were synergistic, because dexamethasone did not induce colony formation nor eosinophil maturation by itself. The eects of dexamethasone were blocked by RU 486, suggesting the involvement of the glucocorticoid receptor (Perretti & Flower, 1996;Hardy et al., 1996). ...
Since the production of eosinopoietic cytokines (GM-CSF, IL-3, IL-5) is inhibited by glucocorticoids, while responsiveness to these cytokines is enhanced in bone-marrow of allergic mice, we studied the ability of glucocorticoids to modulate murine bone-marrow eosinopoiesis.
Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized and challenged mice or (c) dexamethasone (1–5 mg kg−1) injected mice. Cultures were established with GM-CSF (2 ng ml−1) or IL-5 (1 ng ml−1), respectively, alone or associated with dexamethasone, hydrocortisone or corticosterone. Total myeloid colony numbers, frequency and size of eosinophil colonies, and numbers of eosinophil-peroxidase-positive cells were determined at day 7.
In BALB/c mice, dexamethasone (10−7 M) increased GM-CSF-stimulated myeloid colony formation (P=0.01), as well as the frequency (P=0.01) and size (P<0.01) of eosinophil colonies. Dexamethasone (10−7 M) alone had no effect. Dexamethasone (10−7–10−10 M) increased (P<0.002) eosinophil precursor responses to IL-5. Potentiation by dexamethasone was still detectable: (a) on low density, immature, nonadherent BALB/c bone-marrow cells, (b) on bone-marrow from other strains, and (c) on cells from allergic mice. Hydrocortisone and corticosterone had similar effects. Dexamethasone administered in vivo, 24 h before bone-marrow harvest, increased subsequent progenitor responses to GM-CSF (P=0.001) and precursor responses to IL-5 (P<0.001). These effects were blocked by RU 486 (20 mg kg−1, orally, 2 h before dexamethasone, or added in vitro at 10 μM, P<0.001).
Glucocorticoids, acting in vivo or in vitro, through glucocorticoid receptors, enhance bone-marrow eosinopoiesis in naïve and allergic mice.
British Journal of Pharmacology (2000) 129, 1543–1552; doi:10.1038/sj.bjp.0703145
... Most annexins are abundant intracellular proteins (Raynal and Pollard, 1994), and in polymorphonuclear leukocytes (PMNs) annexin A1 comprises ~2-4% of total cellular protein, which is almost half of the amount of actin (Lim and Pervaiz, 2007;Perretti and Flower, 1996;Raynal and Pollard, 1994). In PMNs and monocytes, ~17% and ~33%, respectively, of total annexin A1 is associated with the plasma membrane (Perretti and Flower, 1996). ...
... Most annexins are abundant intracellular proteins (Raynal and Pollard, 1994), and in polymorphonuclear leukocytes (PMNs) annexin A1 comprises ~2-4% of total cellular protein, which is almost half of the amount of actin (Lim and Pervaiz, 2007;Perretti and Flower, 1996;Raynal and Pollard, 1994). In PMNs and monocytes, ~17% and ~33%, respectively, of total annexin A1 is associated with the plasma membrane (Perretti and Flower, 1996). From our immunofluorescence analysis, we could also clearly see that there was a high level of annexin A1 both in the cytoplasm and in ruffles of the plasma membranes (Fig. 8). ...
Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin-binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca(2+) and facilitates the F-actin-LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation ('actin flashing'). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation.
... However, what causes the dysregulated activity of these T cells is currently unknown. Several groups have reported an increased level of ANXA1 in an inflammatory setting, in both animal models of disease [68,69] and in human samples [70]. Experimental autoimmune encephalomyelitis (EAE) mouse models of inflammation have shown that ANXA1 levels correlated with disease severity. ...
Annexin‐A1 has a well‐defined anti‐inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid‐induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin‐A1 primarily signals, have also been implicated in these diseases. In contrast, treatment with recombinant annexin‐A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin‐A1 in heath and disease and discuss the potential of this protein as a biomarker and therapeutic target.
... It is synthetized in some immune cells, mainly myeloid cells including macrophages, MCs, eosinophils, and neutrophils, beyond the neuroendocrine system (15)(16)(17). Glucocorticoids not only stimulate AnxA1 transcription, but also induce the release into the cytoplasm of its pre-existing forms via a receptor-dependent, non-genomic pathway, preceded by phosphorylation at key sites in the N-terminus and other sites (18). ...
Mast cells (MCs) are main effector cells in allergic inflammation and after activation, they release stored (histamine, heparin, proteases) and newly synthesized (lipid mediators and cytokines) substances. In the gastrointestinal tract the largest MC population is located in the lamina propria and submucosa whereas several signals such as the cytokine IL-4, seem to increase the granule content and to stimulate a remarkable expansion of intestinal MCs. The broad range of MC-derived bioactive molecules may explain their involvement in many different allergic disorders of the gastrointestinal tract. Annexin A1 (AnxA1) is a 37 KDa glucocorticoid induced monomeric protein selectively distributed in certain tissues. Its activity can be reproduced by mimetic peptides of the N-terminal portion, such as Ac2-26, that share the same receptor FPR-L1. Although previous reports demonstrated that AnxA1 inhibits MC degranulation in murine models, the effects of exogenous peptide Ac2-26 on intestinal MCs or the biological functions of the Ac2-26/FPR2 system in human MCs have been poorly studied. To determine the effects of Ac2-26 on the function of MCs toward the possibility of AnxA1-based therapeutics, we treated WT and IL-4 knockout mice with peptide Ac2-26, and we examined the spontaneous and compound 48/80 stimulated colonic MC degranulation and cytokine production. Moreover, in vitro, using human mast cell line HMC-1 we demonstrated that exogenous AnxA1 peptide is capable of interfering with the HMC-1 degranulation in a direct pathway through formyl peptide receptors (FPRs). We envisage that our results can provide therapeutic strategies to reduce the release of MC mediators in inflammatory allergic processes.
... The secretion of AnxA1 is key for anti-inflammatory activity, as this protein binds in an autocrine or paracrine manner to specific receptors on the outer leaflet of the plasma membrane of target cells, reducing proinflammatory activity [17][18][19]. AnxA1 receptor (formyl peptide receptor: FPR1, FPR2 and FPR3) expression is particularly high on the plasma membranes of macrophages, monocytes and neutrophils [19][20][21][22]. ...
Background
Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients.
Methods
This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 and formyl peptide receptor ( FPR1 , FPR2 and FPR3 ) expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA).
Results
ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls (AS, p = 0.0032; HAM/TSP, p < 0.0001). Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group ( p = 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals ( p = 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP (AUC = 0.851 and AUC = 0.937, respectively, to AUC = 1000).
Conclusions
Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.
... 16 Interestingly, AnxA1 plasma levels in STM mice were similar to those in control mice. 11 Although AnxA1 distribution is similar between human and murine neutrophils, 37 differences in AnxA1 levels between species could be related to variances in neutrophil populations (eg, human neutrophils constitute 65%-75% of all peripheral blood leukocytes, unlike in the mouse, where ;10%-25% of all leukocytes are neutrophils). In addition, it was recently shown that resolvin (Rv)D1 (another endogenous proresolving mediator) levels were similar in SCD mice and controls. ...
Neutrophils plays a crucial role in the intertwined processes of thrombosis and inflammation. Altered neutrophil phenotype may contribute to inadequate resolution which is known to be a major pathophysiological contributor of thrombo-inflammatory conditions such as Sickle Cell Disease (SCD). The endogenous protein Annexin A1 (AnxA1) facilitates inflammation resolution via Formyl Peptide Receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting neutrophil dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of FPR2/ALX (a fundamental receptor involved in resolution) pathway. We demonstrated direct evidence that neutrophils with SCD phenotype play a key role in contributing to thrombo-inflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. Herein, we present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thrombo-inflammatory conditions such as SCD.
... The secretion of AnxA1 is key for anti-in ammatory activity, as this protein binds in an autocrine or paracrine manner to speci c receptors on the outer lea et of the plasma membrane of target cells, reducing proin ammatory activity [17][18][19]. AnxA1 receptor (formyl peptide receptor: FPR1, FPR2 and FPR3) expression is particularly high on the plasma membranes of macrophages, monocytes and neutrophils [19][20][21][22]. ...
Background: Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients.
Methods: This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 and formyl peptide receptor (FPR1, FPR2 and FPR3) expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA).
Results: ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls (AS, p= 0.0032; HAM/TSP, p< 0.0001). Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group (p= 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals (p= 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP (AUC = 0.851 and AUC = 0.937, respectively, to AUC=1,000).
Conclusions: Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.
... It forms 2-4% of total cytosolic protein and is found in gelatinase granules in neutrophils. Although neutrophils released out have low levels of AnxA1, when the gelatinase granules are released, AnxA1 is also released by endothelial vascular adhesion (34). It is thought that when AnxA1 is released, it binds to its receptor and mediates the cellular detachment of neutrophils and blocks the inflammatory response by inhibiting transmigration of leukocytes (35). ...
... Annexin A1 effects on T cells are still debated and many conclusions on the interconnection between Annexin A1 and adaptive immunity are still under way. One reason for the lack of agreement on this point is that T cells express low levels of Annexin A1 [22], albeit this expression is up-regulated during inflammation [23]. Moreover, in standing conditions, the expression of the Annexin A1 receptor ALXR by T cells is also negligible [9] and increases after stimulation. ...
Annexin A1 is a protein with multifunctional roles in innate and adaptive immunity mainly devoted to the regulation of inflammatory cells and the resolution of inflammation. Most of the data regarding Annexin A1 roles in immunity derive from cell studies and from mice models lacking Annexin A1 for genetic manipulation (Annexin A1−/−); only a few studies sought to define how Annexin A1 is involved in human diseases. High levels of anti-Annexin A1 autoantibodies have been reported in systemic lupus erythematosus (SLE), suggesting this protein is implicated in auto-immunity. Here, we reviewed the evidence available for an association of anti-Annexin A1 autoantibodies and SLE manifestations, in particular in those cases complicated by lupus nephritis. New studies show that serum levels of Annexin A1 are increased in patients presenting renal complications of SLE, but this increment does not correlate with circulating anti-Annexin A1 autoantibodies. On the other hand, high circulating Annexin A1 levels cannot explain per se the development of autoantibodies since post-translational modifications are necessary to make a protein immunogenic. A hypothesis is presented here and discussed regarding the possibility that Annexin A1 undergoes post-translational modifications as a part of neutrophil extracellular traps (NETs) that are produced in response to viral, bacterial, and/or inflammatory triggers. In particular, focus is on the process of citrullination of Annexin A1, which takes place within NETs and that mimics, to some extent, other autoimmune conditions, such as rheumatoid arthritis, that are characterized by the presence of anti-citrullinated peptides in circulation. The description of pathologic pathways leading to modification of Annexin A1 as a trigger of autoimmunity is a cognitive evolution, but requires more experimental data before becoming a solid concept for explaining autoimmunity in human beings.
... Moreover, ANXA1 has been recently identified as one of the "eat me" signals on apoptotic cells to be recognized and ingested by phagocytes [7]. Studies on the expression of ANXA1 in human and mouse leukocytes have shown that this protein is also expressed at higher levels in cells of the adaptive immune response, such as T and B lymphocytes [8][9][10]. Further research indicates that ANXA1 increases T cells activation and favors their differentiation to Th1 cells by modulating T cell receptor (TCR) signaling [4]. ...
Decreased Th1/Th2 ratio is one of the major characteristics of immunosuppression in sepsis. Both membrane adhesive protein Annexin-A1 (ANXA1) and transcription factor GATA-3 have been reported to play important roles in T cell differentiation. However, the relationship between ANXA1 and GATA-3 in Th1/Th2 shift is unknown. Our study investigated the interaction effects of ANXA1 and GATA-3 to influence T cell differentiation in CD4 ⁺ T cells. We found that GATA-3 and ANXA1 were coexpressed on Th0/Th1/Th2 cytoplasm and nuclear. Overexpressed ANXA1 significantly increased the expression of IFN γ and reduced IL-4 expression in T cells, while ANXA1-silenced T cells exhibited decreased production of IFN γ and increased production of IL-4. Knockdown of ANXA1 promoted higher expression level of GATA-3 and low level of T-box transcription factor (T-bet/Tbx21). Further study demonstrated that ANXA1 regulated GATA-3 expression through the formyl peptide receptor like-1 (FPRL-1) downstream signaling pathways ERK and PKB/Akt. These results suggested that ANXA1 modulates GATA-3/T-bet expression induced Th0/Th1 differentiation. Moreover, we found that GATA-3 inhibited ANXA1 expression by binding to its promoter for the first time. It is proposed that the interactions between ANXA1 and GATA-3 may provide clues to understand the immunosuppression and have potential as new therapeutic targets in immunotherapy after sepsis.
... However, the role of ANXA1 in adaptive immunity is poorly defi ned. Published data indicate that ANXA1 expression is lower in lymphocytes than in neutrophils and monocytes (24) (25) , and imply that ANXA1 modulates T cell-mediated immune response (26) by activating specifi c signaling pathways and suppressing transcription factors (26) (27) . ...
INTRODUCTION:
The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.
METHODS:
Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.
RESULTS:
All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 ⁺ macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 ⁺ cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 ⁺ T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.
CONCLUSIONS:
Annexin A1 is differentially expressed in CD163 ⁺ macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.
... Várias outras proteínas são também positivamente reguladas pelos glicocorticóides. A expressão da proteína endógena anti-inflamatória anexina-1, também conhecida como lipocortina-1, tem sido descrita como induzida por glicocorticóides em neutrófilos murinos (Perretti & Flower 1996). Cunha & Ferreira (1986) (Newton et al, 1998). ...
Leukocytes are accumulated, in the inflammatory process, because to action of the wide array of stimuli. This event envolved several and coodenated steps whose inhibited by glucocoticoids, such as dexamethasone. Dexamethasone affects human neutrophils in different ways. It shows negative effects (on synthesis and secretion of pro-inflammatory mediators) and positive effects (for example, on annexin I). Among the inducers of leukocyte migration, MNCF, a galactose-binding lectin, has been described as an agonist and chemoattractant for neutrophils, both in vivo and in vitro. MNCF shows as peculiar activity the migration of neutrophils resistant to dexamethasone actions which awakes great interest in undertanding the mechanism of action on polymorphonuclears by this lectin. Our first step was study human MNCF-stimulated neutrophils pre-incubated with dexamethasone. In these conditions, MNCF, in the absence of F-actin polymerization: (a) protects neutrophils from spontaneous apoptosis, (b) induces tyrosine and p38 MAP quinases phosphorylation, (c) induces CD62L shedding, (d) degranulates secretory vesicles and secundary granules, but not azurophilic granules, in the dependent manner of tyrosine and MAP quinases and Src family, (e) translocates the transcription factor NF-kB and, (f) induces transcription and secretion of pro-inflammatory cytokines and chemokines. In parallel, human neutrophils pre-incubated with dexamethasone and stimulated with MNCF did not show CD62L shedding and F-actin polarization, but the in vitro migration was maintained. Besides, we already observe translocation of NF-kB from cytoplasm to nucleous which it activates the genic transcription and secretion of inflammatory mediators, such as CXCL8. The results, showed here, strengthens previous results, demonstranting that MNCF as a agonist to neutrophils, beyond increase the half life them. Although, dexamethasone modifies some effects of MNCF on neutrophis, this glucocorticoid does not inhibit the cellular response to the studied lectin. Thus, the maintenance of inflammatory mediators, dependents to NF-kB, during the inflammatory process, could explain, even parcialy, the break in the resitance to glucocorticoids actions by MNCF in the neutrophil migration.
... Protein expression was then investigated as a possible molecular mechanism for glucocorticoid action. Total CD11b protein levels were measured after a validated permeabilization protocol (15). In vitro treatment with DEX for 48 h failed to reduce the intracellular level of this protein. ...
In vitro incubation of mouse blood eosinophils with dexa-methasone (DEX) resulted in concentration-and time-dependent reduction in CD11b and CD49d cell-surface expression as detected by flow cytometry. This inhibitory effect ranged between 20 and 40% for both integrins, and it was not related to alteration of cell survival. DEX was maximally effective at 1 M, and it was prevented by coaddition of the glucocorticoid receptor antagonist RU486 (mifepristone; 10 M). Budes-onide, hydrocortisone, and prednisolone, but not the sex ste-roids testosterone and progesterone, reduced CD11b and CD49d cell-surface expression to a similar extent. Subchronic treatment of mice with 1 mg/kg DEX again reduced both CD11b and CD49d expression on circulating eosinophils, without alterations in CD11b messenger RNA expression as assessed by polymerase chain reaction analysis. In contrast, membrane but not intracellular protein expression of either CD11b or CD49d was inhibited by eosinophil incubation with DEX in vitro ; thus, an interference with exportation of these adhesion molecules to the cell surface is proposed as the mechanism of action of the glucocorticoid. Finally, steroid effects on integrin expression were linked to a reduced eosino-phil function as indicated by a lower degree of cell chemotaxis after incubation with DEX, an effect which was again prevented by 10 M RU486. These observations may explain part of the therapeutic efficacy displayed by glucocorticoid hormones in the clinical control of tissue eosinophilia in allergic disease conditions.
... Upon cellular activation, ANXA1 is released from its storage sites and translocates immediately to the membrane. In neutrophils, ANXA1 is stored in gelatinase granules, and upon neutrophilactivating events, such as adhesion to the endothelium, it is rapidly exteriorized (47), exposing the N-terminal region that mediates its biological response ( Figure 3D). Neutrophil ANXA1 externalization can occur without interaction with endothelial cells, indicating that cellular adhesion to the endothelium is not required for release of ANXA1 (48). ...
Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.
... In parallel studies, it was demonstrated that treatment with glucocorticoids increased Annexin A1 content in circulating leucocytes in man (Goulding, Godolphin et al. 1990) and rodents (Perretti and Flower 1996). As cytosolic Annexin A1 is actively mobilized when the neutrophil adheres to the endothelium, glucocorticoid-dependent increase in cellular Annexin A1 content would increase the amount of the protein externalized on the cell surface once the leukocyte adheres to the inflamed vascular endothelium. ...
... One reason that annexin A1 research has focused on the innate rather than the adaptive immune responses might be that annexin A1 is expressed at much lower levels in T cells than in neutrophils, both in humans and in mice. [38][39][40][41] Moreover, conflicting data from the limited number of studies published to date have resulted in a lack of agreement as to the effects of annexin A1 on T-cell activation and T-cell-dependent disease models. Deficiency of annexin A1 has been reported to result in reduced disease severity in experimental auto immune encephalomyelitis, a T-cell-dependent mouse model of multiple sclerosis. ...
Glucocorticoids have broad-ranging and powerful anti-inflammatory and immunomodulatory effects. Unsurprisingly, therefore, glucocorticoids are widely and persistently used to treat a large number of inflammatory diseases, including rheumatoid arthritis (RA), despite the well-described adverse effects of these drugs. Annexin A1 is a glucocorticoid-induced molecule that is known to replicate many of the described anti-inflammatory effects of glucocorticoids. In addition to the well-documented roles of this protein in neutrophil function, emerging evidence suggests that annexin A1 is involved in the modulation of T-cell function and the adaptive immune responses relevant to RA. Interest in annexin A1 was renewed after the delineation of the receptors for this protein. This breakthrough also led to advances in our understanding of anti-inflammatory annexin A1 mimetic peptides and agonistic compounds targeting these receptors, particularly those specific for the receptor N-formyl peptide receptor 2 (FPR2). Herein, we review the current knowledge of the biological activities of annexin A1 and their relevance to RA pathogenesis. We also discuss the potential of annexin A1 mimics and strategies aimed at potentiating annexin A1 signalling to become viable approaches to minimizing glucocorticoid use in RA and other inflammatory disorders.
... 16 This occurred through inhibition of phosphatase PP2A, which constitutively limits PKC activity, thereby potentiating the phosphorylation and release of Anx-A1. 16 Anx-A1 is found in high amounts in circulating neutrophils and monocytes, 17 from which the protein is externalized and cleaved after leukocyte adhesion to the endothelium in vitro. 18 The protein strongly inhibits neutrophil recruitment in vivo 19 and in vitro. ...
To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism.
Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (P<0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1(-/-) mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (P<0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs.
We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.
... 4 In our previous work, we have also identified up-regulated expression of FPR1 in response to GC treatment. Interestingly, annexin A1, which is one of the most important mediators of GC-mediated effects, [35][36][37] has been suggested to signal through receptors belonging to the FPR family. 38,39 However, downstream signaling after annexin A1 binding to its receptor involves rapid and transient activation of ERK1/2 and p38, which stays in strict contrast to the prolonged ERK-activation responsible for the antiapoptotic effect described in our study. ...
Active resolution of inflammation is a previously unrecognized process essential for tissue homeostasis. Monocytes play a pivotal role in the generation as well as resolution of inflammation. Glucocorticoids (GCs) are widely used anti-inflammatory agents. We demonstrate that GCs exhibit antiapoptotic effects in monocytes resulting in differentiation to an anti-inflammatory phenotype. The molecular basis of this novel antiapoptotic effect is a prolonged activation of the extracellular signal regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway resulting in inhibition of caspase activities and expression of antiapoptotic genes via activation of c-Myc. We identified up-regulation and activation of A3 adenosine receptor (A3AR) as the initial trigger of this antiapoptotic pathway. In summary, we deciphered a novel molecular pathway promoting survival of anti-inflammatory monocytes. Specific activation of A3AR or its downstream signaling pathways may thus be a novel strategy to modulate inflammation in autoimmune disorders with fewer side effects via induction of inflammatory resolution rather than immunosuppression.
... Anx-A1, a 37 kDa member of the annexin super-family (13 proteins in mammals), and its Nterminal peptide N-acetyl 2-26 [1,2], have been shown by us, and by other laboratories to possess powerful anti-inflammatory actions in a wide variety of animal models of acute34567891011121314151617 or chronic181920 inflammation. ...
Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the anti-inflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5min) concentration-dependent activation of PKCalpha/beta (Protein Kinase C; EC 2.7.11.13) and phosphorylation of Anx-A1 on Ser(27). Both phosphoproteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B(2) generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCalpha/beta phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB(2) generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A (phosphoprotein phosphatase; EC 3.1.3.16), which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators.
Chikungunya (CHIKV) is an arthritogenic alphavirus that causes a self-limiting disease usually accompanied by joint pain and/or polyarthralgia with disabling characteristics. Immune responses developed during the acute phase of CHIKV infection determine the rate of disease progression and resolution. Annexin A1 (AnxA1) is involved in both initiating inflammation and preventing over-response, being essential for a balanced end of inflammation. In this study, we investigated the role of the AnxA1-FPR2/ALX pathway during CHIKV infection. Genetic deletion of AnxA1 or its receptor enhanced inflammatory responses driven by CHIKV. These knockout mice showed increased neutrophil accumulation and augmented tissue damage at the site of infection compared with control mice. Conversely, treatment of wild-type animals with the AnxA1 mimetic peptide (Ac2–26) reduced neutrophil accumulation, decreased local concentration of inflammatory mediators and diminished mechanical hypernociception and paw edema induced by CHIKV-infection. Alterations in viral load were mild both in genetic deletion or with treatment. Combined, our data suggest that the AnxA1-FPR2/ALX pathway is a potential therapeutic strategy to control CHIKV-induced acute inflammation and polyarthralgia.
Atherosclerosis, a silent chronic vascular pathology, is the cause of the majority of cardiovascular ischaemic events. Atherosclerosis is characterized by a series of deleterious changes in cellularity, including endothelial dysfunction, transmigration of circulating inflammatory cells into the arterial wall, pro-inflammatory cytokines production, lipid accumulation in the intima, vascular local inflammatory response, atherosclerosis-related cells apoptosis and autophagy. Proteins of Annexin A (AnxA) family, the well-known Ca²⁺ phospholipid-binding protein, have many functions in regulating inflammation-related enzymes and cell signaling transduction, thus influencing cell adhesion, migration, differentiation, proliferation and apoptosis. There is now accumulating evidence that some members of the AnxA family, such as AnxA1, AnxA2, AnxA5 and AnxA7, play major roles in the development of atherosclerosis. This article discusses the major roles of AnxA1, AnxA2, AnxA5 and AnxA7, and the multifaceted mechanisms of the main biological process in which they are involved in atherosclerosis. Considering these evidences, it has been proposed that AnxA are drivers- and not merely participator- on the road to atherosclerosis, thus the progression of atherosclerosis may be prevented by targeting the expression or function of the AnxA family proteins.
Mast cell-activating signals in cold urticaria are not yet well defined and are likely to be heterogeneous. Cold agglutinins and cryoglobulins have been described as factors possibly associated with cold urticaria, but their relevance has not been explained. We performed a single-center prospective cohort study of 35 cold urticaria patients. Cold agglutinin and cryoglobulin test results, demographics, detailed history data, cold stimulation test results, complete blood count values, C-reactive protein, total immunoglobulin E levels, and basal serum tryptase levels were analyzed. Forty six percent (n = 16) of 35 tested patients had a positive cold agglutinin test and 27% (n = 9) of 33 tested patients had a positive cryoglobulin test. Cold agglutinin positive patients, when compared to cold agglutinin negative ones, were mainly female (P = 0.030). No gender-association was found for cryoglobulins. A positive cold agglutinin test, but not a positive cryoglobulin test, was associated with a higher rate of reactions triggered by cold ambient air (P = 0.009) or immersion in cold water (P = 0.041), and aggravated by increased summer humidity (P = 0.007). Additionally, patients with a positive cold agglutinin test had a higher frequency of angioedema triggered by ingestion of cold foods or drinks (P = 0.043), and lower disease control based on Urticaria Control Test (P = 0.023). Cold agglutinin levels correlated with erythrocyte counts (r = −0.372, P = 0.028) and monocyte counts (r = −0.425, P = 0.011). Cryoglobulin concentrations correlated with basal serum tryptase levels (r = 0.733, P = 0.025) and cold urticaria duration (r = 0.683, P = 0.042). Results of our study suggest that cold agglutinins and cryoglobulins, in a subpopulation of cold urticaria patients, are linked to the course and possibly the pathogenesis of their disease.
Background: Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients.
Methods: This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 and formyl peptide receptor (FPR1, FPR2 and FPR3) expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA).
Results: ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls (AS, p= 0.0032; HAM/TSP, p< 0.0001). Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group (p= 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals (p= 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP (AUC = 0.851 and AUC = 0.937, respectively, to AUC=1,000).
Conclusions: Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.
Members of the Annexin family of calcium-binding proteins are generally expressed in the cytosol of most eukaryotic cells from plants to humans. In vertebrates there are at least ten members of the family of which any one cell type expresses a selection. Annexins are characterised by the ability to bind calcium-dependently to negatively charged phospholipids, and by the presence of a 70 aa motif usually repeated four times. Numerous functions have been attributed to the annexins including calcium channel activity, regulation of exocytosis, phospholipase A2 inhibition and involvement in endocytosis. None of these postulated roles has been proved conclusively. This thesis describes work designed to define more rigorously the roles of members of this gene family in vivo by targeted disruption of the loci encoding the genes in the chick pre-B-cell DT40 lineage and mice. As a first step, targeting constructs were generated by a novel PCR-based method. Mice were then generated that have lost expression of Annexin VI, a unique member of the family in that it contains two instead of one set of four 70 aa repeats. One allele was disrupted in embryonic stem (ES) cells using the now well-documented method of homologous recombination, and genetically modified ES cells, identified by PCR, were then injected into blastocysts and reimplanted into recipient females. Chimaeric offspring that gave germline transmission were then bred to produce hetero- and finally homozygous null-mutant mice. Mice lacking Annexin VI are viable and fertile, and show no gross morphological defects. While no defects in B and T-cell development, or heart function were identified, adult male annexin VI knock-out mice gain weight at about half the rate of their wild-type counterparts, suggesting a novel role for annexin VI in animal physiology. In addition, clones of DT40 cells were generated with disruptions at both alleles of annexins II and V. The chick pre-B-cell DT40 lineage expresses several annexins including annexins I, II, V and VI. Targeted disruption is facilitated in this cell line by the expression of high levels of recombinase during IgG chain rearrangement. Loss of annexin II or V is also not lethal, but does lead to complex but distinct phenotypes involving calcium signalling, apoptosis and cell clumping. Detailed analysis of knock-out DT40 cells shows that these genes have non-redundant functions and play important roles in the cell. This work provides the experimental systems and information necessary to make significant progress in the understanding of the true roles of Annexins in cellular physiology.
A complex intracellular signaling governs different cellular responses in inflammation. Extracellular stimuli are sensed, amplified, and transduced through a dynamic cellular network of messengers converting the first signal into a proper response: production of specific mediators, cell activation, survival, or death. Several overlapping pathways are coordinated to ensure specific and timely induction of inflammation to neutralize potential harms to the tissue. Ideally, the inflammatory response must be controlled and self-limited. Resolution of inflammation is an active process that culminates with termination of inflammation and restoration of tissue homeostasis. Comparably to the onset of inflammation, resolution responses are triggered by coordinated intracellular signaling pathways that transduce the message to the nucleus. However, the key messengers and pathways involved in signaling transduction for resolution are still poorly understood in comparison to the inflammatory network. cAMP has long been recognized as an inducer of anti-inflammatory responses and cAMP-dependent pathways have been extensively exploited pharmacologically to treat inflammatory diseases. Recently, cAMP has been pointed out as coordinator of key steps of resolution of inflammation. Here, we summarize the evidence for the role of cAMP at inducing important features of resolution of inflammation.
Glucocorticoid hormones regulate essential body functions in mammals, control cell metabolism, growth, differentiation, and apoptosis. Importantly, they are potent suppressors of inflammation, and multiple immune-modulatory mechanisms involving leukocyte apoptosis, differentiation, and cytokine production have been described. Due to their potent anti-inflammatory and immune-suppressive activity, synthetic glucocorticoids (GCs) are the most prescribed drugs used for treatment of autoimmune and inflammatory diseases. It is long been noted that males and females exhibit differences in the prevalence in several autoimmune diseases (AD). This can be due to the role of sexual hormones in regulation of the immune responses, acting through their endogenous nuclear receptors to mediate gene expression and generate unique gender-specific cellular environments. Given the fact that GCs are the primary physiological anti-inflammatory hormones, and that sex hormones may also exert immune-modulatory functions, the link between GCs and sex hormones may exist. Understanding the nature of this possible crosstalk is important to unravel the reason of sexual disparity in AD and to carefully prescribe these drugs for the treatment of inflammatory diseases. In this review, we discuss similarities and differences between the effects of sex hormones and GCs on the immune system, to highlight possible axes of functional interaction.
Purpose
The purpose of this study was to examine the expression of the anti-inflammatory protein Annexin A1 (AnxA1) in mice and human retinae during uveitis and to determine whether local administration of human recombinant AnxA1 (hrAnxA1) can suppress uveitis in mice.
Methods
Retinal sections from mice (healthy normal and uveitis) and postmortem human (no history of eye disease (n = 5) and uveitis (n = 7)) were stained for AnxA1 expression and imaged by immunofluorescence microscopy. AnxA1 cellular expression was determined by colabeling with CD45, glial fibrillary acidic protein (GFAP), and Iba-1 cells, with additional staining of AnxA1 receptors formyl peptide receptor 1 (FPR1) and FPRL1/FPR2. Mice with acute endotoxin-induced uveitis and chronic experimental autoimmune uveitis were treated locally by intravitreal injection with hrAnxA1, and disease was assessed by clinical scoring and quantification of leukocyte infiltrate via flow cytometry.
Results
Constitutive expression of AnxA1 was observed in both healthy mouse and human retinae, and its expression increased during uveitis compared to healthy controls. AnxA1 colocalizes predominantly with CD45⁺ cells, GFAP⁺ macroglia, and to a lesser extent, Iba-1⁺ myeloid cells. We also demonstrate that local treatment with hrAnxA1 attenuates the severity of uveitis in mice.
Conclusions
These data indicate that locally expressed AnxA1 is elevated in the retina during intraocular inflammation. We demonstrate that local administration of hrAnxA1 to augment levels results in suppression of uveitis in mice.
Translational Relevance
Our data suggest that elevated expression of retinal AnxA1 in human uveitis may be immunoregulatory and that local supplementation with hrAnxA1 may provide a potential novel treatment for inflammatory eye diseases such as noninfectious uveitis.
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.
Over fifty years have now elapsed since glucocorticoids were first employed as anti-inflammatory drugs in the clinic. At that time, Dr Philip Hench and his colleagues at the Mayo Clinic made the seminal observation that administering an adrenal cortical steroid extract to a patient with progressive, active rheumatoid arthritis stopped the disease dead in its tracks [1], This soon led to synthetic adrenal cortical steroids gaining a remarkable reputation in the treatment of a wide range of inflammatory and autoimmune disorders. However, it soon became apparent that this efficacy did not come without a cost in terms of potentially serious adverse effects. The fascinating history of glucocorticoid biology from Thomas Addison to the glucocorticoid receptor transgenic mouse is covered in the next chapter (A. Munck, this volume).
In previous studies, we found that Annexin I (Anx I) was co‐secreted with insulin in response to glucose, and that extracellular Anx I stimulated the release of insulin via the Anx I binding site in rat pancreatic islets and the β‐cell line. However, the role that Anx I plays in the insulin secretion was not established. Therefore, in this study, we evaluated the insulin secretion pattern in response to Anx I and the involvement of the cytoskeleton or PKC in Anx I‐stimulated insulin secretion in MIN6N8a cells. The peak time of insulin secretion in response to Anx I treatment corresponded with the second phase insulin secretion by glucose in the perifused pseudoislets. In addition, Anx I‐stimulated insulin secretion was not affect by readily releasable pool depletion. Taken together, these findings indicate that Anx I treatment was associated with movement of the reserve pool of insulin. Furthermore, Anx I‐stimulated insulin secretion was attenuated by treatment with a microfilament inhibitor, cytochalasin B, as well as by PKC down regulation. These results indicate that Anx I may be a regulator of second phase insulin secretion.
Key Points
MPO, via its catalytic activity, inhibits the generation of adaptive immunity by suppressing DC function. MPO-mediated inhibition of adaptive immunity attenuates T cell-driven tissue inflammation.
Annexin A1 (AnxA1) is recognized as an endogenous anti-inflammatory molecule. However, its effects on the adaptive immune response and, in particular, on T cells remain unclear. In this study, we investigated the actions of AnxA1 in three distinct models of T cell-mediated inflammation. In contact hypersensitivity, collagen-induced arthritis, and inflammation induced by OT-II TCR transgenic T cells responding to OVA, AnxA1 deficiency significantly increased Ag-induced T cell proliferation and the resultant level of inflammation. In the contact hypersensitivity model, this was associated with increased adhesion of CD4(+) T cells, CD8(+) T cells, and neutrophils in the dermal microvasculature, as well as increased T cell expression of RORγt and IL-17A. In collagen-induced arthritis, deficiency of endogenous AnxA1 increased susceptibility to arthritis and Ag-specific T cell activation. Deficiency of AnxA1 also increased OVA-induced cutaneous delayed-type hypersensitivity and IFN-γ and IL-17 release. Transfer experiments using CD4(+) T cells from AnxA1(-/-) mice demonstrated that the absence of AnxA1 solely in T cells resulted in increased inflammatory responses in wild-type recipients. Similarly, experiments using AnxA1(-/-) OT-II CD4(+) T cells demonstrated that the absence of AnxA1 in T cells was sufficient to induce increased Ag-specific CD4(+) T cell proliferation in vivo, augment T cell production of IFN-γ, IL-17, TNF, and IL-6, and increase Akt, ERK, and p38 activation. Together, these findings indicate that T cell-expressed AnxA1 functions to attenuate T cell-driven inflammatory responses via T cell-intrinsic effects on intracellular signaling, proliferation, and Th1/Th17 cytokine release.
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
The annexins, are a family of calcium ion (Ca2+)-binding proteins whose physiological functions are poorly understood. Although many diverse functions have been proposed
for these proteins, such as in vesicle trafficking, this review focuses on their proposed roles as Ca2+ or other ion channels, or as intracellular ion channel regulators. Such ideas are founded mainly on in vitro and structural
analyses, but there is increasing evidence that at least some members of this protein family may indeed play a part in intracellular
Ca2+ signaling by acting both as atypical ion channels and as modulators of ion channel activity. This review first introduces
the annexin family, then discusses intracellular localization, developmental regulation, and modes of membrane association
of annexins, which suggest roles in Ca2+ homeostasis. Finally, it examines the structural and electrophysiological data that argue for key roles for annexins in the
control of ion fluxes.
Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes.
Annexin II is a calcium and phospholipid binding protein and a substrate for protein-tyrosine kinases. Increased levels of annexin II are observed in various cancer cells and tissues, and the molecule has been proposed as a marker of malignancy in vivo. Annexin II was expressed in four glioma cell lines (D-54MG, D-37MG, U251MG and GaMG), as determined by Western blot analyses, immunofluorescence staining and flow cytometric measurements. In addition, annexin II expression was also found in cryostat sections obtained from 15 consecutive brain tumor biopsies: Ten were histologically classified as glioblastomas, one as an astrocytoma, two as meningiomas and two as brain metastases. Cultured spheroids from the glioma cell lines and from three of the glioblastoma biopsies showed lower levels of annexin II, than found in the monolayers of the cell lines and in the freshly cut biopsies. The annexin II expression of the cell lines were not found to be related to their proliferative, migratory or invasive properties. These findings indicate that although annexin II may serve as a marker of malignancy in vivo, its expression can be reduced in vitro, and appear unrelated to malignant features of glioma cell lines.
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.
A Two endemic groundwater arthropod crustacean species, Crangonyx islandicus and Crymostygius thingvallensis, were recently discovered on the mid-Atlantic volcanic island of Iceland. The extent of morphological differences from closest relatives, endemism, along with the geographic isolation of Iceland and its complete coverage by glaciers 21,000 years ago, suggests that these two species have survived glaciation periods in sub-glacial refugia. Here we provide strong support for this hypothesis by an analysis of mitochondrial genetic variation within Crangonyx islandicus. Our results show that the species is divided into several distinct monophyletic groups that are found along the volcanic zone in Iceland, which have been separated by 0.5 to around 5 million years. The genetic divergence between groups reflects geographic distances between sampling sites, indicating that divergence occurred after the colonization of Iceland. The genetic patterns, as well as the dependency of genetic variation on distances from the tectonic plate boundary and altitude, points to recent expansion from several refugia within Iceland. This presents the first genetic evidence of multicellular organisms as complex as crustacean amphipods which have survived glaciations beneath an ice sheet. This survival may be explained by geothermal heat linked to volcanic activities, which may have maintained favourable habitats in fissures along the tectonic plate boundary in Iceland during glaciations.
The systemic inflammatory activity in patients with stable coronary artery disease (CAD) is associated with a dysregulated cortisol response. Moreover, an aberrant activation status of neutrophils in CAD has been discussed; and the question of glucocorticoid resistance has been raised. The anti-inflammatory actions of glucocorticoids are mediated by annexin-1 (ANXA1). We investigated the expression of glucocorticoid receptors (GR) and ANXA1, as well as the exogenous effects of ANXA1 on neutrophils in CAD patients and related the data to diurnal salivary cortisol. Salivary cortisol levels were measured in the morning and evening during 3 consecutive days in 30 CAD patients and 30 healthy individuals. The neutrophil expression of GR and ANXA1 was determined by flow cytometry. The effect of exogenous ANXA1 was determined in a neutrophil stimulation assay. The patients showed a flattened diurnal cortisol pattern compared with healthy subjects, involving higher levels in the evening. The neutrophil expression of GR-total and GR-alpha was decreased, whereas the GR-beta expression did not differ compared with controls. The neutrophil expression of ANXA1 was significantly increased in patients. Ex vivo, ANXA1 impaired the leukotriene B(4)-induced neutrophil production of reactive oxygen species in patients but not in controls. Our findings indicate a persistent overactivation of the hypothalamic-pituitary-adrenal axis in CAD patients but do not give any evidence for glucocorticoid resistance, as assessed by the neutrophil expression of GR and ANXA1. The altered neutrophil phenotype in CAD may thus represent a long-term response to disease-related activation.
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.
Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.
The role of glucocorticoids in the regulation of lipocortin 1 (LC1) mRNA/protein expression in the brain is uncertain. This study has examined the influence of dexamethasone on the disposition of LC1 protein in various central and peripheral tissues in the rat. LC1 was readily detectable in all tissues studied by Western blot analysis. Exposure to dexamethasone in vitro (0.1 microM, 3 h) or in vivo (20 micrograms/100 g i.p., 24 h before autopsy) had no discernible effects on intracellular LC1 levels but increased the amount of LC1 attached to the outer surface of cells (pericellular LC1) in cortex, hippocampus, hypothalamus, pituitary gland and peritoneal macrophages (in vitro only). The results suggest that in central tissues, as in the periphery, glucocorticoids promote the translocation of LC1 from intracellular to pericellular sites.
The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.
Clinical and experimental observations revealed that glucocorticoid-deficient states are associated with an enhanced inflammatory response. The antiinflammatory response of pharmacological doses of glucocorticoids has been tentatively attributed to the induction of lipocortin-I. To determine whether glucocorticoid deficiency causes lipocortin-I down-regulation, the expression of lipocortin-I mRNA and protein was quantified in rats with and without adrenalectomy (ADX). The mRNA of lipocortin-I was quantified by polymerase chain reaction, using a constant amount of modified lipocortin-I cDNA transcript as an internal standard. The lipocortin-I mRNA was decreased by 56 +/- 14% in lung tissue of ADX rats. This down-regulation of lipocortin-I mRNA was not due to a nonspecific effect of ADX, since the mRNA levels of other proteins (c-fos, c-myc, c-erbA beta, and metallothionein-II) remained unchanged. The decrease in lipocortin-I mRNA in ADX rats was reflected by a corresponding decrease in tissue (lung, spleen, liver, and kidney) lipocortin-I protein content, as assessed by quantitative Western blot analysis. Thus, ADX causes a decline in lipocortin-I message and protein, an observation compatible with the increased susceptibility to inflammatory reactions in glucocorticoid deficiency.
After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12-binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu-phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense.
An important mechanism for the antiinflammatory effect of pharmacological doses of glucocorticoids is the inhibition of arachidonic acid release from phospholipids by phospholipase A2 (PLA2). As a corollary, one might predict that low endogenous concentrations of glucocorticoids favor inflammatory disease states. Indeed, clinical and experimental observations revealed an association between glucocorticoid deficiency and disease states caused by immunological and/or inflammatory mechanisms. The purpose of the present investigation was to study the regulation of PLA2 mRNA, protein, and enzyme activity in adrenalectomized (ADX) rats where glucocorticoid concentrations were below physiological levels. The mRNA of group I and II PLA2 were measured by PCR. Group II PLA2 mRNA was increased by 126 +/- 9% in lung tissue of ADX rats, whereas group I PLA2 was increased only by 27 +/- 1.5%. The increase in group II mRNA in ADX rats was reflected by a corresponding increase of group II PLA2 protein (70-100%) in lung, spleen, liver, and kidney. This increase was reversed by the administration of exogenous corticosterone. After ADX, the percentage increase in total PLA2 activity was higher than that of mRNA or PLA2 protein, suggesting that the activity of the enzyme was modulated by inhibitors or activators. The concentration of lipocortin-I, an inhibitor of PLA2 enzyme was strongly correlated with the activity of PLA2 in the tissues (lung, spleen, liver, and kidney). In all these tissues, the concentrations of lipocortin-I declined after ADX. Thus upregulation of PLA2 enzyme and downregulation of lipocortin-I might account for the enhanced inflammatory response in hypoglucocorticoid states.
A one-step procedure for purification of mononuclear and polymorphonuclear cells from human blood is described. It is a modification of the Hypaque-Ficoll method with density of 1.095 g/ml. Centrifugation at 200 X g for 20--30 min resulted in the separation of mononuclear and polymorphonuclear cells into 2 distinct bands at the interface. The mononuclear cell fraction contained 83.9 +/- 1.6% lymphocytes and 13.8 +/- 2.3% monocytes, while the other conssisted of highly purified neutrophils (96.4 +/- 1.0%). Leukocyte recovery by this method was always greater than 80% and viability exceeded 98%. Both cell fractions retained their immunological functions.
A video system was used to investigate the inhibitory effect(s) of the glucocorticoid dexamethasone (DEX) on polymorphonuclear leukocyte (PMN) extravasation in the microcirculation of hamster cheek pouch. DEX was given intraperitoneally at 0.1 or 0.3 mg/kg body weight 2 h before induction of extravasation by topical application of leukotriene B4 (LTB4) or formyl-methionyl-leucyl-phenylalanine (fMLP) on the microvasculature. The number, time course, and behavior of PMNs were examined in the following five steps of extravasation: (1) rolling on the venular endothelium, (2) adhesion on the endothelium, (3) passage between the endothelial cells, (4) staying in the venular wall, and (5) migration from the venular wall into the interstitial space. In either the presence or absence of DEX, topical application of LTB4 (15 pmol/50 microliters) or fMLP (10 nmol/50 microliters) caused an increase in the number of PMNs that adhered to the venules. Thus, DEX did not inhibit the adhesion of PMNs on the endothelial cells. The adhered PMNs induced by these chemoattractants became gradually smaller, finally disappearing in the vascular lumen, as observed on the monitor screen. The whole process took about 10 min. This passage of PMNs between the endothelial cells was also not inhibited by DEX, as over 90% of the adhered PMNs passed through the endothelial cells and advanced to the next step of extravasation in the presence or absence of DEX. When these chemoattractants were applied to DEX-untreated animals, the PMNs that passed through the endothelial cells stayed for about 30 min in the venular wall. Thereafter, PMNs migrated into the interstitial space. The numbers of PMNs in the interstitial space were counted at 30, 60, and 90 min after the application of chemoattractants. In the DEX-untreated animals, PMNs in the interstitial space started to appear by 30 min, and thereafter the number further increased. However, in the DEX-treated animals, the number of PMNs at 60 and 90 min was significantly suppressed. Since DEX may inhibit the synthesis and/or release of a proteinase(s) in the PMN granules, which would normally degrade the basement membrane, this inhibition may be due to the inability of PMNs to penetrate the basement membrane.
As a putative mediator of inflammation interleukin-1 has been implicated in the recruitment of leukocytes during the early stages of the inflammatory reaction. In the present report we have investigated the release of endogenous IL-1 in the rat zymosan pleurisy and in the mouse zymosan peritonitis. In both cases the release of the cytokine was maximal 4 hours after zymosan injection and appeared to be time-related to neutrophil migration into the inflammatory site. The effect of in vivo treatment with dexamethasone in rat pleurisy and with polyclonal anti-murine IL-1 beta antibody in mouse peritonitis was also assessed. The steroid reduced both cell migration and the release of IL-1-like activity as well as the formation of exudate and the release of eicosanoids. The anti-IL-1 beta serum inhibited selectively the number of neutrophil that migrated to the inflamed site (approximately 40%) and the IL-1 activity recovered in (approximately 70%) the exudate. In vitro incubation of the inflammatory exudate with polyclonal anti-murine IL-1 alpha or anti-murine IL-1 beta sera allowed the identification of the IL-1 species present. In the rat pleurisy IL-1 biological activity was mainly due to the alpha species, whereas IL-1 beta was the only species apparently present in the mouse peritoneal exudate.
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.
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 I mediates, in part, the anti-inflammatory effects of corticosteroids. To determine whether alveolar epithelial cells produce lipocortin I, we used a highly specific anti-lipocortin I antibody and found by the techniques of Western blotting and immunocytochemical analysis that lipocortin I is a normal constituent of type II alveolar epithelial cells and that it constitutes approximately 0.1% of cell lysate proteins. Two separate species of lipocortin I were identified, migrating with molecular weights of 37 and 32 kD. Dexamethasone caused a dose-dependent increase in lipocortin I in rat alveolar epithelial cells, and its effect was maximal at a dose of 10(-5) M. This effect was specific for corticosteroids since it was not seen with other steroids. These studies suggest that lipocortin I may mediate, in part, the immunosuppressive effects of corticosteroids in the lower respiratory tract.
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.
The neutrophil Mac-1 and gp100MEL-14 adhesion proteins are involved in neutrophil extravasation during inflammation. Both
the expression and activity of Mac-1 are greatly increased after neutrophil activation. In contrast, neutrophils shed gp100MEL-14
from the cell surface within 4 minutes after activation with chemotactic factors or phorbol esters, releasing a 96-kilodalton
fragment of the antigen into the supernatant. Immunohistology showed that gp100MEL-14 was downregulated on neutrophils that
had extravasated into inflamed tissue. The gp100MEL-14 adhesion protein may participate in the binding of unactivated neutrophils
to the endothelium; rapid shedding of gp100MEL-14 may prevent extravasation into and damage of normal tissues by activated
neutrophils.
The glucocorticoids inhibit our 'defence reactions' at many levels. One way in which they achieve this is by inhibiting the synthesis of chemicals involved in the promotion of the inflammatory response. The production of many mediators involved in the response to infection, injury, haemorrhage or metabolic disturbances are under glucocorticoid control such that elevated levels of hormone in the blood suppresses their formation. In many cases the action of these mediators is blocked as well. It might be thought that the glucocorticoids act simply by decreasing the synthesis rate of these protein regulators of inflammation such as the lymphokines, or of the enzymes which make prostaglandins. Whilst this undoubtedly does occur, another mechanism is also employed: that is, the glucocorticoid-induced synthesis of inhitibory proteins. Lipocortin (and possibly other related proteins) then is a sort of 'second messenger' of the glucocorticoids. It is only one of many such regulatory proteins but it is an important one, controlling as it does the mediators which promote development of the symptoms of the inflammatory response. It is undoubtedly the significant component of the inbuilt mechanism for terminating the inflammatory response which the physician exploits, for when he gives his patients relatively large doses of steroids to control an inflammatory response, he is in reality increasing the synthesis of these 'second mesenger' proteins such as lipocortin to a near maximum. All the early studies on lipocortin were performed in vitro, that is under conditions in which steroids were not normally present. Under these circumstances the generation and appearance of lipocortin seemed absolutely dependent upon the presence of glucocorticoids in the perfusing medium. These findings have led some to the erroneous notion that lipocortin was only present following treatment with exogenous steroids. Of course, all healthy mammals have circulating glucocorticoids and thus it is more rational to expect that lipocortin is a normal constituent of plasma and tissues (as indeed it appears to be), although the amount present in the cells can be increased by raising the concentration of endogenous or exogenous steroids. There has been a corresponding change in our appreciation of the function of lipocortin. Originally, it was regarded mainly as an 'anti-inflammatory protein' but today it seems more likely that this protein is present in most cells, and that its function is to control phospholipase A2 activity and to allow lipid hydrolysis only under strictly defined circumstances. This reversible inhibitory function of lipocortin could well be controlled by the phosphorylation and dephosphorylation cycle described above. Naturally, during inflammation, phospholipase is substantially activated and thus there is a requirement for a greater than normal supply of the inhibitory protein, hence the relationship between the rate of synthesis and the release of steroid hormones. The discovery, characterisation, islation, sequencing and cloning of lipocortin has opened up an entirely new and exciting chapter in cell biology and also holds out a strong promise for the future of anti-inflammatory therapy. In addition to their beneficial clinical effects, steroids produce a wide spectrum of side effects which preclude the use of these drugs for long periods of time except in the very seriously ill. These side effects are caused by changes in the transcription of specific genes in the same way as the anti-inflammatory effects. It has long been an article of a faith of scientists working in this are that if we could identify and isolate the 'second messengers' of steroid action that are responsible for the anti-inflammatory effects, it should be possible to produce drugs which possess many of the beneficial action of steroids without incurring the heavy penalty of side effects. The real value of this work is that it enables us to take our first stpes in that direction.
A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).
When injected into a 6‐day‐old mouse air‐pouch, human recombinant interleukin‐8 (IL‐8; 0.03–3 μg) induced, in a dose‐dependent fashion, an accumulation of neutrophils which could be reliably assessed 4 h after the injection. No protein extravasation was measured above the values obtained with the vehicle alone (carboxymethylcellulose, CMC, 0.5% w/v in phosphate‐buffered solution, PBS).
The IL‐8 effect (routinely evaluated at 1 μg dose) was inhibited neither by local administration of actinomycin D (1 μg) nor by systemic treatment with indomethacin (1 mg kg ⁻¹ , i.v.), BWA4C (5 mg kg ⁻¹ , p.o.), methysergide (6 mg kg ⁻¹ , i.p.) and RP67580 (2 mg kg ⁻¹ , i.p.).
Treatment of mice with the H 1 antagonist, mepyramine (1–10 mg kg ⁻¹ , i.p.) resulted in a dose‐dependent inhibition of the cell accumulation elicited by the chemokine, with a maximal reduction of approximately 50–60%. The mepyramine effect was not due to a non specific reduction of neutrophil function, since treatment with this drug (6 mg kg ⁻¹ , i.p.) did not modify the cell infiltration measured in response to a challenge with interleukin‐1β (20 ng) or with the vehicle CMC to any extent. Moreover, treatment of mice with mepyramine did not modify cell counts in a peripheral blood film with respect to controls. Two other H 1 antagonists, chemically unrelated to mepyramine, diphenhydramine (9 mg kg ⁻¹ , i.p.) and triprolidine (0.5 mg kg ⁻¹ , i.p.), inhibited IL‐8‐induced migration to a similar extent (∼ 50–60%), whereas the H 2 antagonist, ranitidine (5 mg kg ⁻¹ , i.p.) was without effect.
The concept that endogenous histamine could be involved in the IL‐8 effect was strengthened in two ways: (i) addition of histamine (0.2–2 μg) to a small dose of IL‐8 (0.3 μg) potentiated the cell elicitation induced by the chemokine without having any effect on its own; (ii) IL‐8‐induced neutrophil accumulation was greatly impaired in animals depleted of mast cell amines by sub‐chronic (5 day) treatment with compound 48/80 according to an established protocol.
The glucocorticoid dexamethasone (Dex; 1–50 μg per mouse, i.v., corresponding approximately to 0.03–1.5 mg kg ⁻¹ , given i.v. 2 h prior to challenge with IL‐8) potently inhibited neutrophil infiltration with an approximate ED 50 of 5 μg per mouse (∼ 0.3 mg kg ⁻¹ , i.v.). Passive immunisation of mice with a polyclonal sheep serum raised against the steroid‐inducible anti‐inflammatory protein lipocortin 1 (LC1) abolished the inhibitory action of Dex whereas a control serum was without effect.
Local administration of Dex at a dose which was ineffective when given systemically (1 μg) also reduced neutrophil migration induced by IL‐8, either alone or in combination with histamine. This local inhibition (∼ 50%), also seen with hydrocortisone (30 μg), was prevented by the concomitant administration of the steroid antagonist RU38486 (10 μg) indicating the involvement of glucocorticoid receptor in the response.
These findings characterize further the mechanisms underlying PMN recruitment induced by IL‐8 in vivo , and point to a role for histamine. The anti‐inflammatory action of the glucocorticoids, as in some other models, appears to be LC1‐dependent when these drugs are given systemically and LC1‐independent when the steroids are given locally.
The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.
This study investigates the effect of dexamethasone on leukocyte extravasation in the post-capillary venules of the hamster cheek pouch, using an intravital microscopy technique, and seeks to clarify the potential involvement of the steroid-inducible protein lipocortin 1. Topical application of FMLP (10 nmol), or substance P (10 nmol), to the superfused cheek pouch induced at the level of the post-capillary venules the three characteristic phenomena of leukocyte rolling, adhesion, and transmigration. Pretreatment of hamsters with an anti-inflammatory dose of dexamethasone (1 mg/kg) increased lipocortin 1 levels in circulating leukocytes as assessed by flow cytometry, but did not modify either leukocyte rolling or the number of adherent cells; however approximately 65% of the adherent leukocytes subsequently detached and returned to the blood stream, whereas those that entered into the diapedesis process exhibited a long latency (approximately three- to fourfold longer than in control animals) before transmigration. In hamsters passively immunized with a polyclonal anti-lipocortin 1 serum, leukocyte diapedesis started at similar times in both control and dexamethasone-treated animals, whereas a significant prolongation was observed in those animals treated with a non-immune sheep serum. These observations indicate that 1) lipocortin 1 is elevated in circulating leukocytes following dexamethasone treatment; 2) the step of leukocyte extravasation affected by dexamethasone in the actual transmigration process, and 3) this specific effect upon leukocyte diapedesis is mediated by endogenous lipocortin 1.
Lipocortin 1, a putative mediator of the anti-inflammatory actions of glucocorticoids, is present intracellularly in a variety of tissues including human peripheral blood leukocytes. We investigated the presence of lipocortin 1 in human leukocyte subsets using permeabilization flow cytometry. Constitutive lipocortin 1 was detected in U937 myelomonocytic leukemia cells, and lipocortin 1 was increased by treatment with PMA or PMA+IFN-gamma (P < 0.05) but not by dexamethasone. Lipocortin 1 was present in all leukocyte subsets except B lymphocytes (CD19/20+, P < 0.001). Lipocortin 1 content was maximal in monocytes and polymorphonuclear neutrophils and least in lymphocytes (P < 0.001). Monocyte lipocortin 1 was strongly associated with surface expression of CD14 and HLA-DR. Among non-B lymphocytes, a range of lipocortin 1 fluorescence was observed. Lipocortin 1 fluorescence was greatest in natural killer cells (CD56+, P < 0.001) and CD57+ cells, but T cell subset markers did not otherwise discriminate variations in lipocortin 1. Induction of lymphocyte proliferation by PHA, anti-CD3, Con A, superantigen, and SAC was not associated with significant shifts in lipocortin 1 content. Dexamethasone (10(-10)-10(-6) M) did not induce increases in PB leukocyte lipocortin 1. We conclude that lipocortin 1 content in human leukocytes varies significantly among phenotypic subsets. This has significance for the investigation of inflammatory disease where certain cell types predominate.
Cytokines orchestrate the complex network of cellular interactions that regulate effector cell functions of natural and immune resistance. Although T cells, natural killer (NK) cells and monocytes/macrophages are the main producers of cytokines, a number of reports in the last few years have demonstrated that polymorphonuclear neutrophils (PMN) also have the ability to synthesize and release immunoregulatory cytokines. Here, Marco Cassatella describes novel facets of the regulation of cytokine production by PMN that highlight the involvement of of PMN in cell-cytokine crosstalk.
To study the clinical significance of the decrease of glucocorticoid receptor (GR) in stress and shock, GR was blocked about 80% by mifepristone (RU38486), and the effects of the blockade on the pathological changes of endotoxemia were studied in rats. The results revealed that GR blockade may exacerbate the pathological and pathophysiological changes of endotoxemia: (1) the more rapid drop in arterial blood pressure, (2) the more severe pathological changes involving multiple organs, especially the lung and small intestine, (3) the increase of leukocyte adherence in venules and more pronounced rheological changes in the mesenteric microcirculation, and (4) the striking elevation of serum acid phosphatase (ACP), phospholipase A2 (PLA2) activity, and lipoperoxide (LPO). The changes of serum ACP, PLA2, and LPO in the rats with 80% GR blockade were more marked than in those with 50% GR blockade. Based on these findings, we propose that the decrease in GR during stress and shock might be a contributing factor in the pathogenesis of shock and multiple organ failure (MOF). The possible mechanisms of the above noted findings are discussed.
Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U-937 cells were incubated with or without 10 nM phorbol 12-myristate 13-acetate (PMA) to induce cell differentiation. Then 1 microM dexamethasone was added and incubation carried out for increasing times (1-24 h). Dexamethasone caused a time-dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U-937 cells the steroid-induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U-937 cells. Since extracellular lipocortin 1 has anti-inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.
It has been suggested that the induction of lipocortin-1, a phospholipase A2-inhibitory protein, may mediate the anti-inflammatory action of glucocorticoids. We assessed the production of prostaglandin E2, thromboxane B2, and leukotriene B4 and the expression of lipocortin-1 in different populations of blood leukocytes and in alveolar macrophages (obtained by bronchoalveolar lavage) from patients with inflammatory lung diseases (bronchial asthma, n = 21; interstitial lung disease, n = 6) undergoing glucocorticoid treatment at clinically effective doses. No inhibition of eicosanoid production was observed in either whole blood or single populations of blood leukocytes (granulocytes and monocytes) stimulated with ionophore A-23187, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or zymosan. Conversely, eicosanoid production from alveolar macrophages (assessed in 9 cases) was significantly inhibited by glucocorticoids. After ionophore stimulation, eicosanoid production was as follows (in ng/ml): prostaglandin E2, 0.19 +/- 0.06 and 0.06 +/- 0.01; thromboxane B2, 2.9 +/- 0.9 and 0.5 +/- 0.1; leukotriene B4, 6.6 +/- 1.1 and 3.6 +/- 1.0, before and after treatment, respectively (P < 0.05 for all differences). Lipocortin-1 expression, determined by Western blot and enzyme immunoassay, was significantly (P < 0.05) stimulated in alveolar macrophages, but not in blood leukocytes, by glucocorticoid treatment. These results indicate that alveolar macrophages, at variance from blood leukocytes, are the most likely cell target for glucocorticoid-induced eicosanoid inhibition and lipocortin expression. We suggest that cell responsiveness to glucocorticoids is acquired during differentiation from monocyte to tissue macrophage.
The anti‐inflammatory actions of dexamethasone on vascular and leukocyte responses in rabbit skin were investigated.
Neutrophil accumulation and oedema formation were simultaneously measured as the local accumulation of i.v. administered ¹¹¹ In‐labelled neutrophils and ¹²⁵ I‐labelled albumin. Systemically administered dexamethasone (3 mg kg ⁻¹ ) inhibited neutrophil accumulation induced by i.d. zymosan activated plasma (ZAP), N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and leukotriene B 4 (LTB 4 ) when co‐injected with prostaglandin E 2 (PGE 2 ). Dexamethasone also inhibited oedema formation elicited by these stimuli and the responses induced by i.d. platelet activating factor (PAF) + PGE 2 and bradykinin (BK) + PGE 2 .
Intradermal dexamethasone (2 × 10 ⁻¹⁰ mol per site) but not indomethacin (10 ⁻⁸ mol per site) inhibited oedema formation induced by i.d. ZAP + PGE 2 and BK + PGE 2 . This inhibitory effect of dexamethasone was significant only with pretreatment periods of 4 h, shorter pretreatment periods resulting in greatly reduced effects. Intradermal dexamethasone had no effect on neutrophil accumulation induced by ZAP + PGE 2 .
Intradermal dexamethasone (2 × 10 ⁻¹⁰ mol per site) had no effect on increase in blood flow induced by PGE 2 as measured by ¹³³ Xenon clearance.
The accumulation of neutrophils isolated from donor rabbits pretreated with i.v. saline or dexamethasone (3 mg kg ⁻¹ ) was investigated in untreated recipient rabbits. The accumulation of neutrophils, induced by ZAP + PGE 2 , FMLP + PGE 2 and LTB 4 + PGE 2 , from dexamethasone‐pretreated donors was significantly smaller than the accumulation of neutrophils from saline‐pretreated donors.
The results of this study suggest that dexamethasone can have a direct effect on vascular endothelial cells resulting in an inhibition of oedema formation.
Neutrophil accumulation can be inhibited by an effect of dexamethasone on the neutrophil itself or on the vascular endothelium. These results indicate that at least part of the inhibitory effect is on the circulating neutrophil induced by dexamethasone or a dexamethasone‐induced product.
IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.
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.
Lipocortin 1, a member of the annexin superfamily of calcium and phospholipid binding proteins, mediates some of the anti-inflammatory actions of the glucocorticoid hormones. Lipocortin 1 binds to the surface of murine peripheral blood monocytes and polymorphonuclear leukocytes (Kd estimate 2 x 10(-8) M) but not lymphocytes. Resident peritoneal macrophages exhibit binding (Kd estimate 1.3 x 10(-8) M) but lymphocytes do not. A 95-98% reduction in lipocortin 1 binding was observed to leukocytes obtained from air pouch or peritonitis models of inflammation. When given intravenously, lipocortin 1 binds rapidly to murine leukocytes within 5 min but disappears before 10 min, leaving the binding capacity of the cells unaltered. Modulation of lipocortin 1 binding sites could be an important step in regulating the function of inflammatory cells.