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Lipocortin-1 fragments inhibit neutrophil accumulation and neutrophil-dependent edema in the mouse. A qualitative comparison with an anti-CD11b monoclonal antibody
Abstract
The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (αCD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2- 26 exhibited powerful inhibitory effects (ED50 ≃ 5.2, 38 and 88 μg/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the αCD11b mAb. Peptide Ac2-26 and the mAb αCD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 μg/mouse) but less so than the αCD11b antibody (ED50 ≃ 0.5 mg/mouse). Both LC1 (10 μg) and Ac2-26 (200 μg) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a β2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
... into an inflamed airpouch (Perretti, Ahluwalia et al. 1993). Apart from its antiinflammatory effects, the Ac1-188 fragment has also been shown to display antipyretic properties when administered into the rat brain stimulated with INFγ and IL1-β . ...
... In vivo the Ac2-26 peptide has been shown to exert an anti-inflammatory effect in models of myocardial ischaemia reperfusion (I/R) (La, D'Amico et al. 2001), mesentery I/R (Gavins, Yona et al. 2003), glycogen peritonitis (Teixeira, Das et al. 1998) and IL1β airpouch (Perretti, Ahluwalia et al. 1993), where it was reported to significantly reduce the recruitment of neutrophils to the site of injury/inflammation (Perretti and Flower 1993). In other more complex acute models such as zymosan peritonitis apart from a reduction in PMN recruitment at an early time point (4h) a reduction of monocyte recruitment into the peritoneal cavity after 24h was also reported (Getting, Flower et al. 1997). ...
... Furthermore, in models of tissue injury such as glacial acetic acid induced gastric ulcers (Martin, Perretti et al. 2008), contusive spinal chord injury (Liu, Zhang et al. 2007), septic shock (Ritchie, Sun et al. 2003) and mouse skin oedema (Perretti, Ahluwalia et al. 1993) the administration of the Ac2-26 peptide has been shown to partially -and in some cases completely -reverse the damaged. This effect is, at least in part, mediated through down regulation of pro-inflammatory factors such as TNF-α, IL1β and Cox2. ...
... 9,10 The end point is reduced recruitment of blood-borne cells to the site of inflammation. 11,12 The mechanism(s) of action of annexin 1 and its mimetic has long remained elusive. 13 A recent study by Walther et al 10 has indicated involvement of the receptor for the tripeptide formyl-Met-Leu-Phe (fMLP), termed formyl-peptide receptor or FPR. ...
... The annexin 1 mimetic peptide Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK) and the doses used were chosen from previous studies. 11,26 The FPR antagonist Boc2 (N-tbutyloxycarbonyl-Phe-D Leu-Phe-D Leu-Phe) 27 was obtained from ICN Pharmaceuticals, Basingstoke, United Kingdom. Animals were injected with saline (100 L), peptide Ac2-26 alone (20 to 100 g per mouse), Boc2 (10 g) alone, or Boc2 and peptide Ac2-26 at the beginning of reperfusion phase. ...
... The antimigratory actions of annexin 1 and its N-terminal-ved peptide Ac2-26 have been reported nearly a decade ago. 11 Subsequently, techniques of intravital microscopy have been used to monitor potential effects of these compounds on the initial interactions between an extravasating leukocyte and the activated endothelium, highlighting an effect on the events that follow leukocyte firm adhesion. 12 A series of in vitro and in vivo studies suggested that the neutrophil was the target for annexin 1 and its peptides. ...
... Escherichia coli LPS (serotype 0111:B4, specific activity Ͼ500,000 U/mg) was purchased from Sigma-Aldrich. AnxA1 mimetic N-terminal peptide (AnxA1 Ac2-26 ; synthesized to order from Cambridge Research Biochemicals, Cleveland, UK) and the FPR antagonist N-t-butyloxycarbonyl-Phe-DLeu-Phe-DLeu-Phe (Boc2; ICN Pharmaceuticals, Basingstoke, UK) were used in doses based on previous studies (13,14). Animals were injected with saline (100 l/mouse), peptide AnxA1 Ac2-26 alone (100 g/mouse), Boc2 alone (10 g/ mouse), or Boc2 and peptide AnxA1 Ac2-26 (10 and 100 g/mouse, respectively) at the time of LPS or saline treatment. ...
... In our experimental conditions, AnxA1 Ac2-26 displayed a potent protective influence on the cerebral microvascular events induced by LPS, being effective in abrogating both leukocyte adhesion and plasma pro-tein extravasation (PPE). Leukocyte-endothelial and PPE interactions both occur in the early stages of endotoxemia but are coexisting events, the latter already reported in several different models, such as the inflamed skin and ischemia and reperfusion (I/R) of the mesentery (13,14). The beneficial effect of AnxA1 Ac2-26 on PPE reported here concurs with that observed in rodent models of I/R (13) and following the administration of other inflammogens (14,27). ...
... Leukocyte-endothelial and PPE interactions both occur in the early stages of endotoxemia but are coexisting events, the latter already reported in several different models, such as the inflamed skin and ischemia and reperfusion (I/R) of the mesentery (13,14). The beneficial effect of AnxA1 Ac2-26 on PPE reported here concurs with that observed in rodent models of I/R (13) and following the administration of other inflammogens (14,27). ...
Unregulated inflammation underlies many diseases, including sepsis. Much interest lies in targeting anti-inflammatory mechanisms to develop new treatments. One such target is the anti-inflammatory protein annexin A1 (AnxA1) and its receptor, FPR2/ALX. Using intravital videomicroscopy, we investigated the role of AnxA1 and FPR2/ALX in a murine model of endotoxin-induced cerebral inflammation [intraperitoneal injection of lipopolysaccharide (LPS)]. An inflammatory response was confirmed by elevations in proinflammatory serum cytokines, increased cerebrovascular permeability, elevation in brain myeloperoxidase, and increased leukocyte rolling and adhesion in cerebral venules of wild-type (WT) mice, which were further exacerbated in AnxA1-null mice. mRNA expression of TLR2, TLR4, MyD-88, and Ly96 was also assessed. The AnxA1-mimetic peptide, AnxA1(Ac2-26) (100 μg/mouse, ∼33 μmol) mitigated LPS-induced leukocyte adhesion in WT and AnxA1-null animals without affecting leukocyte rolling, in comparison to saline control. AnxA1(Ac2-26) effects were attenuated by Boc2 (pan-FPR antagonist, 10 μg/mouse, ∼12 nmol), and by minocycline (2.25 mg/mouse, ∼6.3 nmol). The nonselective Fpr agonists, fMLP (6 μg/mouse, ∼17 nmol) and AnxA1(Ac2-26), and the Fpr2-selective agonist ATLa (5 μg/mouse, ∼11 nmol) were without effect in Fpr2/3(-/-) mice. In summary, our novel results demonstrate that the AnxA1/FPR2 system has an important role in effecting the resolution of cerebral inflammation in sepsis and may, therefore, provide a novel therapeutic target.-Gavins, F. N. E., Hughes, E. L., Buss, N. A. P. S., Holloway, P. M., Getting, S. J., Buckingham, J. C. Leukocyte recruitment in the brain in sepsis: involvement of the annexin 1-FPR2/ALX anti-inflammatory system.
... In contrast, SHLP6 triggered ERK1/2 activation solely at FPR1. Additionally, we included the annexin peptide Ac2-26, a recognized anti-inflammatory mediator acting via FPRs 49,50 . ...
... Therefore, the signaling outcome was defined by the specific pairing of agonist and receptor rather than being linked to a particular receptor isoform, signaling texture, or a specific class of agonists, such as formylated peptides. Even the anti-inflammatory peptide Ac2-26 49 induced a similar signaling pattern at both receptors. In our analysis, we therefore did not identify a receptor-specific signaling texture that could discriminate receptor functions in the immune response. ...
Pattern Recognition Receptors are key in identifying pathogenic or damaged cell-related patterns or molecules. Among these, the closely linked formyl peptide receptors FPR1 and FPR2 are believed to hold pivotal yet differing functions in immune regulation. To address the intriguing question of how these highly related receptors with a shared agonist spectrum play differing roles in modulating inflammation, we analyzed the signaling profile for a panel of FPR agonists in vivo and ex vivo settings. Our analysis uncovered a shared core signature for both FPRs across signaling pathways. Whereas formylated peptides generally acted as potent agonists at FPR1, FPR2 agonists, irrespective of N-terminal formylation, displayed consistently low activity ratios, suggesting an underutilized signaling potential of this receptor. Signaling outcomes were defined by specific agonist receptor pairings and no receptor-specific signaling texture was identified. Activation of the FPR signaling axis by fMLF in human neutrophils did impact neutrophil survival. Overall, the distinct characteristics underlying inflammatory, anti-inflammatory, or pro-resolving profiles could not be attributed to a specific receptor isoform, signaling pattern, or a particular class of agonists, challenging assumptions about distinct inflammatory profiles linked to specific receptors, signaling patterns, or agonist classes.
... It is expressed primarily on neutrophils and monocytes, and it is activated by a variety of endogenous and exogenous ligands, most of which are nonspecific (Le et al., 2002;Chiang et al., 2006). The prominent endogenous FPRL1 ligands are derivates of lipoxin, i.e., lipoxin A 4 (LXA4) and the aspirin-triggered lipoxins (Bannenberg et al., 2004), as well as the glucocorticoid-regulated protein annexin 1 and its N-terminal-derived peptide Ac2-26 (Perretti et al., 1993). These ligands display anti-inflammatory properties via the FPRL1 pathway in various experimental animal models of acute and chronic inflammation, hence substantiating the therapeutic potential of FPRL1 agonists. ...
... Dorsal air pouches were raised by subcutaneous injection of 2.5 ml of sterile air 6 and 3 days before treatment. CGEN-855A and Ac2-26 (Perretti et al., 1993) were dissolved in sterile pyrogen-free PBS (Invitrogen, Carlsbad, CA) and administered intravenously at 200 l (n ϭ 8), followed immediately by an intrapouch challenge with 1 mg of zymosan A (Sigma-Aldrich, Steinheim, Germany). Alternatively, CGEN-855A or vehicle was administered into the pouch (in situ) in the absence of zymosan A challenge. ...
Activation of the formyl-peptide receptor-like (FPRL) 1 pathway has recently gained high recognition for its significance in therapy of inflammatory diseases. Agonism at FPRL1 affords a beneficial effect in animal models of acute inflamma-tory conditions, as well as in chronic inflammatory diseases. TIPMFVPESTSKLQKFTSWFM-amide (CGEN-855A) is a novel 21-amino acid peptide agonist for FPRL1 and also activates FPRL2. CGEN-855A was discovered using a computational platform designed to predict novel G protein-coupled receptor peptide agonists cleaved from secreted proteins by convertase proteolysis. In vivo, CGEN-855A displays anti-inflammatory activity manifested as 50% inhibition of polymorphonuclear neu-trophil (PMN) recruitment to inflamed air pouch and provides protection against ischemia-reperfusion-mediated injury to the myocardium in both murine and rat models (36 and 25% reduction in infarct size, respectively). Both these activities are accompanied by inhibition of PMN recruitment to the injured organ. The secretion of inflammatory cytokines, including interleukin (IL)-6, IL-1, and tumor necrosis factor-, was not affected upon incubation of human peripheral blood mono-nuclear cells with CGEN-855A, whereas IL-8 secretion was elevated up to 2-fold upon treatment with the highest CGEN-855A dose only. Collectively, these new data support a potential role for CGEN-855A in the treatment of reperfusion-mediated injury and in other acute and chronic inflammatory conditions.
... Several studies using animal models of inflammation have demonstrated that the administration of ANXA1 or Ac2-26 moderates neutrophil recruitment. [54][55][56][57][58] Early studies showed that both ANXA1 and Ac2-26 inhibited adhesion of human neutrophils to activated endothelial cells under static 54,59 and flow conditions 60,61 in vitro and thus impairing neutrophil recruitment. Use of an ANXA1 blocking antibody increased the ability of neutrophils to transmigrate through an endothelial monolayer indicating that ANXA1 is able to impair neutrophil transmigration. ...
... Several studies using animal models of inflammation have demonstrated that the administration of ANXA1 or Ac2-26 moderates neutrophil recruitment. [54][55][56][57][58] Early studies showed that both ANXA1 and Ac2-26 inhibited adhesion of human neutrophils to activated endothelial cells under static 54,59 and flow conditions 60,61 in vitro and thus impairing neutrophil recruitment. Use of an ANXA1 blocking antibody increased the ability of neutrophils to transmigrate through an endothelial monolayer indicating that ANXA1 is able to impair neutrophil transmigration. ...
The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.
... Accordingly, inhibition of PMN accumulation to the myocardium or regulation of PMN activation is an obvious target for the development of novel therapies for myocardial injury following ischemia-reperfusion. Lipocortin 1 (LC1; also referred to as annexin I) is a member of the annexin super-family of proteins that is endowed with a potent leukocyte antimigratory activity (15). Treatment of experimental animals with human recombinant LC1, or with peptide analogues drawn from the LC1 N-terminal region, reduces PMN extravasation in simple models of acute inflammation (16,17). Intravenous treatment with LC1 reduces the extent of cell adhesion to the inflamed endothelium by altering the fate of the adherent leukocytes resulting in a lower degree of emigration and a higher incidence of cell detachment (18,19). ...
... 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). Antibodies against LC1 prevented the antimigratory action displayed by DEX (18,20,40). ...
... The generation of AnxA1-knockout (AnxA1-KO) mice has allowed for multiple studies to better characterize the role of this protein in different contexts of inflammation [47][48][49]. AnxA1-KO mice demonstrate a prolonged and amplified inflammatory response after local stimulation (e.g., paw edema model), an increased extent of joint damage in the antigen-induced arthritis model and a greater susceptibility to nociception [50][51][52]. Additionally, AnxA1-KO neutrophils display increased endothelial transmigration and a heightened responsiveness to inflammatory stimuli, further demonstrating the importance of endogenous AnxA1 in the resolution of inflammation [53]. ...
Dysregulated inflammatory responses are often correlated with disease severity during viral infections. Annexin A1 (AnxA1) is an endogenous pro-resolving protein that timely regulates inflammation by activating signaling pathways that culminate with the termination of response, clearance of pathogen and restoration of tissue homeostasis. Harnessing the pro-resolution actions of AnxA1 holds promise as a therapeutic strategy to control the severity of the clinical presentation of viral infections. In contrast, AnxA1 signaling might also be hijacked by viruses to promote pathogen survival and replication. Therefore, the role of AnxA1 during viral infections is complex and dynamic. In this review, we provide an in-depth view of the role of AnxA1 during viral infections, from pre-clinical to clinical studies. In addition, this review discusses the therapeutic potential for AnxA1 and AnxA1 mimetics in treating viral infections.
... Ac2-26, which is the pharmacologically active center of AnxA1, was synthesized by Cirino et al (18) as early as 1993 and it has been shown to effectively inhibit the activation of the transcription factor NF-κB and the subsequent production of proinflammatory cytokines, such as TNF-α and IL-1β (19). Although previous studies (20,21) have confirmed that Ac2-26 exhibits anti-inflammatory effects and stability in the blood circulatory system, the inability to quantify it in serum or plasma is a limitation of the present study. Several studies have demonstrated that Ac2-26 exhibits protective effects against ischemia-reperfusion injury (IRI) of the heart (10), brain (22), kidney (13) and intestine (11) by inhibiting the expression of inflammatory factors such as TNF-α, IL-1β and MPO. ...
Inflammation is one of the most crucial mechanism underlying hepatic ischemia-reperfusion injury (HIRI). Several studies have shown that Ac2-26, the active N-terminal peptide of Annexin A1, could modulate anti-inflammatory processes and protect the organs from ischemia-reperfusion injury (IRI). However the effects of Ac2-26 on an HIRI model have not been reported to date. The purpose of the present study was to determine whether Ac2-26 pretreatment could protect hepatocytes against acute HIRI by inhibiting neutrophil infiltration through regulation of the high mobility group box protein 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. To this end, a total of 72 adult C57BL/6 mice were randomly divided into sham operation (sham), ischemia-reperfusion (I/R), I/R + Ac2-26 and Ac2-26 groups. The HIRI model was established by occluding the branch of the hepatic pedicle to the left and median liver lobes with an atraumatic vascular clamp for 45 min, followed by reperfusion for 24 h. The expression of HMGB1, TLR4, NF-κB, IκBα and lymphocyte antigen 6 complex locus G6D (Ly6G) was detected using reverse transcription-quantitative PCR, western blotting and immunohistochemical staining; serum levels of HMGB1 were evaluated using an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the proportion of neutrophil. The results indicated that Ac2-26 preconditioning rescued hepatocyte dysfunctions induced by HIRI. In addition, HIRI was associated with a significant increase in HMGB1 expression and release, accompanied by increased expression of TLR4, which was significantly inhibited by Ac2-26. Furthermore, the expression of phosphorylated (p)-NF-κB and the ratio of p-NF-κB to NF-κB were markedly increased, while the expression of IκBα was decreased in the I/R group compared with those in the sham group; however, these effects were reversed by Ac2-26 administration. Additionally, Ac2-26 administration significantly inhibited neutrophil infiltration and resulted in low levels of neutrophils and Ly6G as well as reduced myeloperoxidase activity. Taken together, these results indicated that Ac2-26 pretreatment serves a protective role against HIRI by regulating the HMGB1/TLR4/NF-κB signaling pathway and inhibiting neutrophil infiltration.
... route of infection was chosen aiming to bypass the immune responses responsible for rapid virus clearance in natural peripheral infection (St John et al., 2013b;Syenina et al., 2015). For treatment with the AnxA1 mimetic peptide (Ac 2-26 ), BALB/c WT, C57BL/6 WT, AnxA1 KO, and FPR2 KO mice were injected intra-peritoneally with Ac 2-26 (150 μg/animal; phosphate-buffered saline, PBS, as the vehicle) at the time of infection and daily after infection (Damazo et al., 2006;Galvão et al., 2017;Perretti et al., 1993;Vago et al., 2012). A129 mice were treated with Ac 2-26 (150 μg/animal; i.p.) twice a day from day 2 post-infection until sacrifice (day 5). ...
Host immune responses contribute to dengue's pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, interferon (alpha and beta) receptor 1 knockout (KO), AnxA1 KO and FPR2 KO mice were infected with Dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac 2-26 for analysis. Additionally, the effect of Ac 2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo . We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. While the absence of AnxA1 or its receptor FPR2 aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestations. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease.
... The elevated levels of proinflammatory mediators correlated with exacerbation of eosinophil accumulation and subsequent airway remodelling, suggesting a beneficial role of AnxA1 or its mimetic peptide in the treatment of severe asthma. In many in vitro and in vivo systems, the anti-inflammatory effects of AnxA1 have been reproduced by compounds derived from the N-terminal region of the protein, including the peptide Ac2-26 [5,33]. Herein, we demonstrated that intranasal treatment with peptide Ac2-26 inhibited OVA-induced tissue eosinophil infiltration, tissue remodelling, and mucus production. ...
Annexin-A1 (AnxA1) and its N-terminal derived peptide Ac2-26 regulate the inflammatory response in several experimental models of disorders. This study evaluated the effect of endogenous AnxA1 and its N-terminal peptide Acetyl 2-26 (Ac2-26) on allergic asthma triggered by house dust mite (HDM) extract in mice. ANXA1−/− and wildtype (WT) mice were exposed to intranasal instillation of HDM every other day for 3 weeks, with analyses performed 24 h following the last exposure. Intranasal administration of peptide Ac2-26 was performed 1 h before HDM, beginning 1 week after the initial antigen application. ANXA1−/− mice stimulated with HDM showed marked exacerbations of airway hyperreactivity (AHR), eosinophil accumulation, subepithelial fibrosis, and mucus hypersecretion, all parameters correlating with overexpression of cytokines (IL-4, IL-13, TNF-α, and TGF-β) and chemokines (CCL11/eotaxin-1 and CCL2/MCP-1). Intranasal treatment with peptide Ac2-26 decreased eosinophil infiltration, peribronchiolar fibrosis, and mucus exacerbation caused by the allergen challenge. Ac2-26 also inhibited AHR and mediator production. Collectively, our findings show that the AnxA1-derived peptide Ac2-26 protects against several pathological changes associated with HDM allergic reaction, suggesting that this peptide or related AnxA1-mimetic Ac2-26 may represent promising therapeutic candidates for the treatment of allergic asthma.
... The N-terminal domain mediates the anti-inflammatory functions of ANXA1 including inhibition of leukocyte migration and the transendothelial passage of neutrophils [16][17][18]. In contrast, the core region facilitates several functions such as membrane fusion and aggregation [19,20] and has been shown to act in a pro-inflammatory manner by promoting clustering and migration across the endothelium [3]. ...
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.
... Synthetic peptides from the ANXA1 N-terminal domain exhibit biological activity in several models of experimental inflammation [13,14]. We tested a series of synthetic ANXA1 Nterminal peptides and found that the N-terminal 1-15 residues are sufficient for IF7 binding [9]. ...
We previously reported that IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminus, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo , we undertook mirror-image peptide phage display using a synthetic D-peptide representing the ANXA1 N-terminus as target. We then identified peptide sequences, synthesized them as D-amino acids, and designated the resulting peptide dTIT7, which we showed bound to the ANXA1 N-terminus. Whole body imaging of mouse brain tumor models injected with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered dTIT7/geldanamycin (GA) conjugates suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that ANXA1-binding D-peptide can be developed as an orally-administrable tumor vasculature-targeted therapeutic.
... ANXA1 is a member a family of proteins that binds to membrane phospholipids resulting in inhibition of phospholipase A2 and eicosanoids production [11]. In many studies the anti-inflammatory effects of ANXA1 was reproduced by it N-terminal region of 26 amino acids termed Ac2-26 [10,17]. This peptide exerts it effects by activation of the formyl peptide receptor type 1 (FPR1) and type 2 (FPR2) [18,19]. ...
The Coronavirus Diseases-2019 (COVID-19) pandemic leads many researchers around the world to study the SARS-CoV-s2 infection and pathology to find a treatment for it. This generates a massive production of papers including pre-clinical, clinical and revisions but till now no specific treatment were identified. Meanwhile, like other coronavirus infections, COVID-19 leads to the cytokine storm syndrome resulting in hyperinflammation, exacerbated immune response and multiple organ dysfunctions indicating that drugs that modulate this response, as glucocorticoids could be a treatment option. However glucocorticoids have several side effects or usage limitations. In this sense a drug with anti-inflammatory effects and capable to reduce inflammation but with less after-effects could be a powerful tool to combat COVID-19. Thus the Ac2-26 Mimetic Peptide of Annexin A1 emerges as a possible therapy. The peptide has many anti-inflammatory effects described including the reduction of interleukin (IL)-6, one of the main mediators of cytokine storm syndrome. Therefore the hypothesis to use the Ac2-26 peptide to treat severe COVID-19 will be highlighted in this paper.
... However, differences in the respective efficacies for bacterial and mitochondrial peptides were not as pronounced. Notably, the annexin A1 peptide Ac2-26 [35], which displayed the lowest potency in our assay, was still able to suppress de novo cAMP formation to a level similar to the mitochondrial agonists. Interestingly, gG2p20, an exogenous ligand-derived from herpes virus [36], and the synthetic small molecule FPRA14 [37,38] mimicked the mitochondrial peptides to some extent. ...
Ligand-based selectivity in signal transduction (biased signaling) is an emerging field of G protein-coupled receptor (GPCR) research and might allow the development of drugs with targeted activation profiles. Human formyl peptide receptor 1 (FPR1) is a GPCR that detects potentially hazardous states characterized by the appearance of N-formylated peptides that originate from either bacteria or mitochondria during tissue destruction; however, the receptor also responds to several non-formylated agonists from various sources. We hypothesized that an additional layer of FPR signaling is encoded by biased agonism, thus allowing the discrimination of the source of threat. We resorted to the comparative analysis of FPR1 agonist-evoked responses across three prototypical GPCR signaling pathways, i.e., the inhibition of cAMP formation, receptor internalization, and ERK activation, and analyzed cellular responses elicited by several bacteria-and mitochondria-derived ligands. We also included the anti-inflammatory annexinA1 peptide Ac2-26 and two synthetic ligands, the W-peptide and the small molecule FPRA14. Compared to the endogenous agonists, the bacterial agonists displayed significantly higher potencies and efficacies. Selective pathway activation was not observed, as both groups were similarly biased towards the inhibition of cAMP formation. The general agonist bias in FPR1 signaling suggests a source-independent pathway selectivity for transmission of pro-inflammatory danger signaling.
... As aforementioned many of the biological actions of ANXA1 can be mimicked using the peptide Ac2-26 (31). Hayhoe et al. demonstrate that blocking FPR2/ALXR with a monoclonal antibody prevented ANXA1/Ac2-26 induced inhibition of human neutrophil transmigration and adhesion to the endothelial-cell monolayers under flow conditions (32). ...
Annexin-A1 (ANXA1) was first discovered in the early 1980's as a protein, which mediates (some of the) anti-inflammatory effects of glucocorticoids. Subsequently, the role of ANXA1 in inflammation has been extensively studied. The biology of ANXA1 is complex and it has many different roles in both health and disease. Its effects as a potent endogenous anti-inflammatory mediator are well-described in both acute and chronic inflammation and its role in activating the pro-resolution phase receptor, FPR2, has been described and is now being exploited for therapeutic benefit. In the present mini review, we will endeavor to give an overview of ANXA1 biology in relation to inflammation and functions that mediate pro-resolution that are independent of glucocorticoid induction. We will focus on the role of ANXA1 in diseases with a large inflammatory component focusing on diabetes and microvascular disease. Finally, we will explore the possibility of exploiting ANXA1 as a novel therapeutic target in diabetes and the treatment of microvascular disease.
... Annexin A1 (ANXA1), which originally believed to inhibit phospholipase activity (22), has been reported to be able to modulate various cellular functions in multiple cell types (23,24). Protective roles of ANXA1 have been observed (25,26). ...
The incidence of asthma is increasing worldwide. Bronchial epithelium injury is common in asthma. The regulatory role of Annexin A1 (ANXA1) in bronchial epithelium injury is currently not well understood. The aim of the present study was to evaluate the role of ANXA1 on bronchial epithelium injury. The cell viability and levels of apoptosis were respectively tested by Cell Counting Kit‑8 and flow cytometry. Reactive oxygen species (ROS) content and the activity of oxidative indicators were assessed by commercial kits. Enzyme linked immunosorbent assay was performed to detect the activity of active caspase‑3. Reverse transcription‑quantitative polymerase chain reaction and western blot assays were used to determine the expression levels of the target factors. The results demonstrated that ANXA1 improved the viability of benzo[a]pyrene (Bap)‑treated bronchial epithelial cells. The Bap‑induced oxidative stress was mitigated by the reduction in ROS generation, and the regulation of the activity of superoxide dismutase, glutathione peroxidases, malondialdehyde and lactic dehydrogenase. In addition, apoptosis was decreased by ANXA1 via the reduction of the expression of B‑cell lymphoma 2 (Bcl‑2), and the increase in the expression of Bcl‑2‑associated X protein and cyclin D1. Furthermore, the expression of phosphatase and tensin homolog (PTEN) and focal adhesion kinase (FAK) was rescued and the phosphorylation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) was depressed by ANXA1, when compared with the Bap group. SF1670 treatment reversed the anti‑apoptotic effect of ANXA1. In conclusion, the results highlighted the protective effects of ANXA1 on bronchial epithelium injury, which most likely occurred via the PTEN/FAK/PI3K/Akt signaling pathway. Thus, the present study contributes to a potential therapeutic strategy for asthma patients.
... It was first detected in the conditioned media or perfusate of tissues or cells upon treatment with glucocorticoids, and was found to mirror the action of these drugs in numerous in vitro and in vivo systems ( Pepinsky et al., 1986). The synthesis and release of this protein were hypothesized to account for some of the anti-inflammatory actions of these drugs and this was confirmed by later experiments using the highly purified r-huAnx-A1 ( Cirino and Cicala, 1993;Perretti et al., 1993;Wu et al., 1995;D'Amico et al., 2000;Gavins et al., 2003;Bandeira-Melo et al., 2005), Anx-A1-deficient transgenic animals ( Roviezzo et al., 2002;Croxtall et al., 2003;Hannon et al., 2003), neutralizing antibodies ( Croxtall and Flower, 1992;Duncan et al., 1993;Perretti et al., 1996;Teixeira et al., 1998), and anti-sense agents ( Croxtall and Flower, 1994;Taylor et al., 1997). Anx-A1 is now known to exert a key 'anti-inflammatory and pro-resolution' role in several important host defense responses including acute inflammation ( Perretti and D'Acquisto, 2009) and T-cell signaling (D' Acquisto et al., 2008). ...
The anti-allergic cromones were originally synthesized in the 1960s by Fisons Plc, and the first drug to emerge from this program, disodium cromoglycate was subsequently marketed for the treatment of asthma and other allergic conditions. Whilst early studies demonstrated that the ability of the cromones to prevent allergic reactions was due to their ‘mast cell stabilizing’ properties, the exact pharmacological mechanism by which this occurred, remained a mystery. Here, we briefly review the history of these drugs, recount some aspects of their pharmacology, and discuss two new explanations for their unique actions. We further suggest how these findings could be used to predict further uses for the cromones.
... Another important factor might be the mechanism by which neutrophils travel to the inflammatory site. Migration in response to zymosan and E. coli is known to be dependent on b2 integrins [22], which function to allow neutrophils to adhere to and crawl on the endothelium [23]. In contrast, neutrophil trafficking to the lungs in response to S. pneumoniae is independent of b2 integrins, and it is thought that, under these circumstances, Gal-3 is able to act as an adhesion molecule to facilitate neutrophil trafficking, which likely explains the requirement for high levels of the protein extracellularly in these models. ...
Galectin-3 has been associated with a plethora of proinflammatory functions because of its ability, among others, to promote neutrophil activation and because of the reduction in neutrophil recruitment in models of infection in Gal-3-null mice. Conversely, it has also been linked to resolution of inflammation through its actions as an opsonin and its ability to promote efferocytosis of apoptotic neutrophils. Using a self-resolving model of peritonitis, we have addressed the modulation and role of Gal-3 in acute inflammation. We have shown that Gal-3 expression is increased in neutrophils that travel to the inflamed peritoneum and that cellular localization of this lectin is modulated during the course of the inflammatory response. Furthermore, neutrophil recruitment to the inflamed peritoneum is increased in Gal-3-null mice during the course of the response, and that correlates with reduced numbers of monocytes/macrophages in the cavities of those mice, as well as reduced apoptosis and efferocytosis of Gal-3-null neutrophils. These data indicate a role for endogenous Gal-3 in neutrophil clearance during acute inflammation.
... FPRL1 can be upregulated by specific inflammatory mediators (Serhan 2002). The prominent endogenous FPRL1 ligands are derivates of LX, i.e., LXA 4 and the aspirin-triggered lipoxins (Bannenberg et al. 2004), as well as the glucocorticoid-regulated protein annexin 1 and its N-terminalderived peptide Ac2-26 (Perretti et al. 1993). However, there is downregulation of FPRL1 in aspirin and/ or ω3-PUFAs treated rats compared to PTZ-kindled group that may be explained by the reduction of LXA 4 levels. ...
There is evidence for a relationship between inflammation and seizures as epilepsy can be caused by or result in inflammation. This study aimed to investigate the effect of aspirin and/or ω-3 polyunsaturated fatty acids (ω3-PUFAs) on seizure activity and neurodegeneration in pentylenetetrazole (PTZ)-kindled rats focusing on their effect on cortico-hippocampal production of lipoxin A4 (LXA4) and expression of formyl peptide receptor-like 1 (FPRL1) receptors. Male rats were injected with PTZ (35 mg/kg, i.p) trice a week for a total of fifteen doses. Rats were treated daily with aspirin (20 mg/kg, i.p), ω3-PUFAs (85 mg/kg, p.o) or a combination of them for 35 days. LXA4 level and expression of FPRL1 receptor in the cortex and hippocampi of rats’ brain was greater in PTZ-kindled rats compared to saline group. Co-treatment with aspirin and/or ω3-PUFAs reduced the convulsive behavior, reduced level of LXA4, interleukin-1β, nuclear factor-κB and showed lower percent of cortico-hippocampal degenerative cells compared to PTZ-kindled rats. The combination of the two therapeutic agents did not provide significant improvement in comparison to monotherapies. These findings suggest the use of aspirin or ω3-PUFAs with hope to retard the development of seizures and provide neuroprotection in a clinical setting.
... Alternatively, proteolysis can cause release of the N-terminal peptide [18], which then can interact with FPR1 and 2 evoking anti-inflammatory activity [19]. Pharmacological treatment of inflammation using anxA1 and its N-terminal peptide has been studied in mouse models of neutrophil-dependent edema [20], cardiac ischemiareperfusion injury [21,22] and acute peritonitis [23], however, hitherto not in atherosclerosis. ...
Objective:
To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice.
Methods:
Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.
Results:
Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P).
Conclusion:
Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.
... We chose the proresolving mediator Ac2-26 because it is a small, stable peptide and can be targeted to atherosclerotic lesions using nanotechnology. Ac2-26 is rapidly cleared from plasma, and so repeated administrations of large amounts of peptide are needed for efficacy (21). ...
Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2-26 (Ac2-26). Collagen IV (Col IV)-targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr(-/-) mice. When administered to mice with preexisting lesions, Col IV-Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy.
Copyright © 2015, American Association for the Advancement of Science.
... An important aspect of the GC anti-inflammatory effect is the significant influence GCs have on adhesion between neutrophils and endothelial cells due to the inhibition of mRNA for ELAM-1 (endothelial-leukocyte adhesion molecule-1) as well as on ELAM-1 and ICAM-1 (intercellular adhesion molecule-1) expression [47]. GCs not only weaken adhesion of polymorphonuclear neutrophils to the endothelial surface [48], as they also lengthen the transmigration of cells, in which diapedesis has started, by a factor of 3-4 [49]. ...
A significant role of the stress response to many different diseases prompted a search for new specialized and non-specialized anti-stress agents. This study examines the effect of the compound L17 from the group of 5-phenyl substituted-6H-1,3,4-thiadiazine-2-amines, on the manifestations of the stress response. The authors used a standard model of immobilization stress, in which an animal was immobilized on its back for 6h a day. Parameters of the morphological and functional states of the organs studied were measured and biochemical and enzyme-immunoassays were carried out on the first and second days. This study reveals that the main mechanism by which the L17 compound mediates of its anti-stress was by activation of macrophages on the second day of the experiments and the inhibition of apoptosis in the thymus. The results enable us to suggest that the compound L17 does not improve resistance to stress; however, it does lower the reaction to stress.
Copyright © 2015 Elsevier B.V. All rights reserved.
... There are a plethora of examples of these anti-inflammatory actions observed with administration of either exogenous ANX-A1 or its commonly-studied, biologically active, Nterminal peptide N-terminal-derived annexin-A1 peptide (acetyl- AMVSEFLKQAWIENEEQEYVVQTVK), Ac2-26, in a broad range of animal models of inflammation. These include inhibition of: (i) cytokineinduced leukocyte migration (mouse air-pouch model (Perretti et al., 1993; Perretti & Flower, 1993)); (ii) carrageenan-induced acute inflammation (rat paw edema, murine peritonitis and arthritis (Cirino et al., 1993; Perretti et al., 1993; Getting et al., 1997; Yang et al., 1997)); (iii) acute I–R injury (rat heart, kidney, murine mesenteric, brain, and intestinal microcirculation (La et al., 2001a,b; Gavins et al., 2003; Gavins et al., 2007; Souza et al., 2007; Facio et al., 2011)); and (iv) allergen (ovalbumin )-provoked inflammatory responses (rat pleurisy, including mast cell degranulation and plasma protein leakage (Bandeira-Melo et al., 2005)). Ac2-26 also enhances healing of acetic acid-induced gastric ulcers (Martin et al., 2008). ...
... 34 Although Ac2-26 has been shown to have similar efficacy to full-length annexin A1 in some models, 72 which might relate to the ability of this peptide to promote transportation of full-length endogenous annexin A1 from the cytoplasm to the external surface of the cell, 37 the N-terminal peptide is approximately 200 times less potent than the parent protein. 73 Another FPR2 agonist, the 'W-peptide' (Trp-Lys-Tyr-Met-Val-D-Met), has been shown to inhibit the develop ment of allergen-induced inflammation and T H 1cell/T H 17-cell cytokine production, in situ, in a mouse model of non-eosinophilic asthma. 74 However, neither of these approaches to agonistic activation of FPR signalling using peptides has been further developed to date. ...
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.
... The Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK; Invitrogen, Grand Island, NY, USA), 100 μg/mouse.day diluted in sterile phosphate-buffered saline (PBS) (Perretti et al., 1993;Damazo et al., 2006), and CsA (Fujisawa, Osaka, Japan) (de Mattos et al., 1996), 10 mg/kg.day diluted in absolute alcohol and olive oil, were administrated subcutaneously (s.c.). ...
Immunosuppressive drugs have a critical role in inhibiting tissue damage and allograft rejection. Studies have demonstrated the anti-inflammatory effects of the annexin A1 (AnxA1) in the regulation of transmigration and apoptosis of leucocytes. In the present study, an experimental skin allograft model was used to evaluate a potential protective effect of AnxA1 in transplantation survival. Mice were used for the skin allograft model and pharmacological treatments were carried out using either the AnxA1 mimetic peptide Ac2-26, with or without cyclosporine A (CsA), starting 3 days before surgery until rejection. Graft survival, skin histopathology, leucocyte transmigration and expression of AnxA1 and AnxA5 post-transplantation were analysed. Pharmacological treatment with Ac2-26 increased skin allograft survival related with inhibition of neutrophil transmigration and induction of apoptosis, thereby reducing the tissue damage compared with control animals. Moreover, AnxA1 and AnxA5 expression increased after Ac2-26 treatment in neutrophils. Interestingly, the combination of Ac2-26 and cyclosporine A showed similar survival of transplants when compared with the cyclosporine A group, which could be attributed to a synergistic effect of both drugs. Investigations in vitro revealed that cyclosporine A inhibited extracellular-signal-regulated kinase (ERK) phosphorylation induced by Ac2-26 in neutrophils. Overall, the results suggest that AnxA1 has an essential role in augmenting the survival of skin allograft, mainly owing to inhibition of neutrophil transmigration and enhancement of apoptosis. This effect may lead to the development of new therapeutic approaches relevant to transplant rejection. Copyright © 2013 John Wiley & Sons, Ltd.
... Steroids may also bring about changes in the intracellular localization of LC1 in C6 glioma (Loxley et al, 1993;Solito et al, 1994) and in differentiated U937 cells (Perretti et al, 1993). We did not observe any change in the intracellular localization of LC1 either before or after Dex treatment. ...
The signal transduction pathway through which tumour necrosis factor (TNF) induces apoptosis in leukaemic cells may involve activation of cytosolic phospholipase A2 (cPLA2). The steroids dexamethasone (Dex) and 1,25(OH)2 D3 both render U937 leukaemic cells resistant to TNF-induced apoptosis. In this study, we found that Dex inhibited both spontaneous and TNF-induced activation of cPLA2. Dex had no direct effect on cellular cPLA2 levels, but facilitated cPLA2 degradation upon subsequent stimulation of cells with TNF. In addition, Dex increased synthesis of the endogenous cPLA2 inhibitor lipocortin 1 (LC1). An antisense oligonucleotide to LC1 could completely abrogate Dex-induced resistance to the cytotoxic action of TNF. Constitutive LC1 levels were relatively higher in myeloid leukaemic blasts showing resistance to TNF than TNF-sensitive myeloid leukaemic cell lines. Our data suggest that Dex confers the resistance of U937 cells to TNF-induced apoptosis by upregulating intracellular levels of LC1 and by facilitating a negative-feedback loop, which is activated upon stimulation with TNF. High constitutive levels of LC1 in leukaemic blasts may protect them against immune-mediated killing.
... Intraperitoneal injections of 1 or 5 μg ANXA1 in 100 μl PBS twice a day were performed. This dosing schedule was chosen based on published literature (65,83,84). ...
N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1-/-IEC and AnxA1-/- mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.
... The anti-inflammatory effects of peptide Ac2-26 have been demonstrated in numerous models including ischemia/reperfusion injury in both the rat (D'Amico et al., 2000;La et al., 2001) and mouse , the mouse air-pouch and rat paw edema models of inflammation (Cirino et al., 1993;Perretti et al., 1993), and in models of neutrophil and monocyte trafficking (Szabó et al., 1997;Bandeira-Melo et al., 2005). The wide-ranging effects of peptide Ac2-26 were clearly demonstrated www.frontiersin.org in a model of pleurisy in the rat, in which the peptide inhibited mast cell degranulation, plasma protein leakage, and accumulation of both neutrophils and eosinophils (Teixeira et al., 1998). ...
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.
Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca ²⁺ levels. Through this they act as Ca ²⁺ -regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca ²⁺ -regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.
Cardiovascular diseases (CVD) remain the leading cause of mortality worldwide. The main cause underlying CVD is associated with the pathological remodeling of the vascular wall, involving several cell types, including endothelial cells, vascular smooth muscle cells, and leukocytes. Vascular remodeling is often related with the development of atherosclerotic plaques leading to narrowing of the arteries and reduced blood flow. Atherosclerosis is known to be triggered by high blood cholesterol levels, which in the presence of a dysfunctional endothelium, results in the retention of lipoproteins in the artery wall, leading to an immune-inflammatory response. Continued hypercholesterolemia and inflammation aggravate the progression of atherosclerotic plaque over time, which is often complicated by thrombus development, leading to the possibility of CV events such as myocardial infarction or stroke. Annexins are a family of proteins with high structural homology that bind phospholipids in a calcium-dependent manner. These proteins are involved in several biological functions, from cell structural organization to growth regulation and vesicle trafficking. In vitro gain- or loss-of-function experiments have demonstrated the implication of annexins with a wide variety of cellular processes independent of calcium signaling such as immune-inflammatory response, cell proliferation, migration, differentiation, apoptosis, and membrane repair. In the last years, the use of mice deficient for different annexins has provided insight into additional functions of these proteins in vivo , and their involvement in different pathologies. This review will focus in the role of annexins in CVD, highlighting the mechanisms involved and the potential therapeutic effects of these proteins.
Chronic diseases that affect our society are made more complex by comorbidities and are poorly managed by the current pharmacology. While all present inflammatory etiopathogeneses, there is an unmet need for better clinical management of these diseases and their multiple symptoms. We discuss here an innovative approach based on the biology of the resolution of inflammation. Studying endogenous pro-resolving peptide and lipid mediators, how they are formed, and which target they interact with, can offer innovative options through augmenting the expression or function of pro-resolving pathways or mimicking their actions with novel targeted molecules. In all cases, resolution offers innovation for the treatment of the primary cause of a given disease and/or for the management of its comorbidities, ultimately improving patient quality of life. By implementing resolution pharmacology, we harness the whole physiology of inflammation, with the potential to bring a marked change in the management of inflammatory conditions.
Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 63 is January 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Host immune responses contribute to dengue's pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, IFNα/βR -/- , AnxA1 -/- and FPR2/ALX -/- mice were infected with Dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac 2-26 for analysis. Additionally, the effect of Ac2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo . We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. While the absence of AnxA1 or its receptor FPR2/ALX aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestations. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease.
Peptides have served as a versatile tool in a broad range of biomedical research and have been used to regulate key biochemical pathways and treat human diseases. Since endogenous peptides and peptides derived from protein sources do not often have optimal physicochemical and biological properties, a number of methods and strategies have been developed to achieve high potency and selectivity to their targets. This chapter summarizes frequently used tactics that have been employed to accomplish this end, including defining critical binding determinants, substituting with nonnatural amino acids, modifying peptide backbone structures, forming macrocycles, and stabilizing biologically active conformations.
In more than 30 years of research annexins have been demonstrated to regulate immune responses. The prototype member of this family, annexin A1, has been widely recognized as an anti-inflammatory mediator affecting migration and cellular responses of various cell types of the innate immune system. Evidently, effects on innate immune cells also impact on the course of adaptive immune responses. Innate immune cells provide a distinct cytokine milieu during initiation of adaptive immunity which regulates the development of T cell responses. Moreover, innate immune cells such as monocytes can differentiate into dendritic cells and take an active part in T cell stimulation. Accumulating evidence shows a direct role for annexins in adaptive immunity. Annexin A1, the annexin protein studied in most detail, has been shown to influence antigen presentation as well as T cells directly. Moreover, immune modulatory roles have been described for several other annexins such as annexin A2, annexin A4, annexin A5 and annexin A13. This review will focus on the involvement of annexin A1 and other annexins in central aspects of adaptive immunity, such as recruitment and activation of antigen presenting cells, T cell differentiation and the anti-inflammatory removal of apoptotic cells.
Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintainance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent anti-inflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis.
Various approaches for the prevention of adhesion formation have been actively explored.1–6 It is well known that good surgical technique is important in reducing adhesion formation. However, adjuncts to good techniques are needed for adhesion prevention. Physical barriers have been used in an attempt to prevent adhesion formation by limiting tissue apposition during the critical period of peritoneal healing, thereby minimizing the development of fibrin matrix between tissue surfaces.2,7,8 Studies have indicated that placement of an absorbable barrier of oxidized regenerated cellulose [Interceed® (TC7) Absorbable Adhesion Barrier; Ethicon, Somerville, NJ, USA], expanded polytetrafluoroethylene (Preclude® Surgical Membrane; W.L. Gore & Associates, Flagstaff, AZ, USA), or hyaluronic acid/carboxymethyl-cellulose (Seprafilm® Bioresorbable Membrane; Genzyme, Cambridge, MA, USA)9 between injury sites, or addition of a viscous solution (Intergel® ferric crosslinked hyaluronic acid; Ethicon) 10 to the peritoneal cavity during or after surgery may reduce postoperative adhesion formation. In the case of Interceed, Preclude, or Seprafilm, the surgeon must predict potential sites of adhesion formation to determine placement site and optimize barrier benefit. Interest therefore continues in the development of an intraperitoneal device that functions more broadly as a postsurgical adhesion prophylactic.
Bradykinin (BK) and related peptides are produced at sites of tissue injury and inflammation and have an important role in mediating the responses of the tissues to damage and associated pain. BK causes pain by direct stimulation of nociceptors and by sensitising sensory fibres to previously non-noxious stimuli (hyperalgesia). BK exerts its biological activities by stimulating two receptor subtypes, B1 and B2, with the latter localised to sensory neurones. The B2 receptor is believed to mediate many of the effects attributed to kinins although the B1 receptor has now been shown to have an important role in hyperalgesia, notably when the hyperalgesia is secondary to an earlier inflammatory insult.
Glucocorticoids (GCs) exert diverse actions in the body, which are required for the maintenance of homeostasis. Their complex effects are mediated mainly by mechanisms involving the induction or repression of key target genes. Lipocortin 1 is the product of one such gene; it fulfills a prominent role in effecting the regulatory actions of the steroids in the host defense and neuroendocrine systems.
Endogenous glucocorticoids are pro-resolving mediators, an example of which is the endogenous glucocorticoid-regulated protein Annexin A1 (AnxA1). Since silicosis is an occupational lung disease characterized by unabated inflammation and fibrosis, in this study we tested the therapeutic properties of the N-terminal AnxA1-derived peptide Ac2-26 on experimental silicosis.
Swiss-Webster mice received silica particles intranasally and were subsequently treated with intranasal peptide Ac2-26 (200 μg.mouse(-1) ) or dexamethasone (25 μg.mouse(-1) ) for 7 days, starting 6 h post-challenge. Peptide Ac2-26 abolished leukocyte infiltration, collagen deposition, granuloma formation and the generation of pro-inflammatory cytokines following silica provocation, readouts only partially inhibited by dexamethasone.
A clear exacerbation of these pathological changes was observed in AnxA1 knockout mice as compared to the wild-type littermate controls. Lung fibroblasts from WT mice, but not from formyl peptide receptor (Fpr) type 1 knockout, had the IL-13 or TGFβ-induced production of CCL2/MCP-1 and collagen reduced after incubation with peptide Ac2-26 in vitro. This compound also inhibited the production of CCL2/MCP-1 from fibroblasts of Fpr2 knockout mice.
Collectively, our findings unveil novel protective properties of the AnxA1 derivative peptide Ac2-26 on the inflammatory and fibrotic responses promoted by silica, and suggest that AnxA1 mimetic agents might be a promising strategy in innovative anti-fibrotic approaches for treatment of silicosis.
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1Lipocortin-1 and its N-terminal derivatives exert potent inhibitory actions in various models of acute inflammation. The present study examined the ability of lipocortin (LC)-1 to suppress the release of the acute pro-inflammatory mediators, tumour necrosis factor (TNFα) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin-1β (rhIL-1β).2LPS (10 μg ml−1)-stimulated release of TNFα and PGE2 from PBMC was significantly inhibited by (4 h) co-incubation of the cells with 10−6m dexamethasone (Dex), but not with 10−9m to 10−7m of a N-terminal fragment (amino acids 1–188) of recombinant human LC-1 (LC-1 fragment). However, Dex suppression of LPS-stimulated TNFα and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC-1 fragment (1:10,000 dilution) was included in the medium. rhIL-1β (5times10−8m)-stimulated release of TNFα and PGE2 from PBMC (after 18 h) was abolished by co-incubation of the cells with 10−7m LC-1 fragment.3After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti-LC-1 fragment antibody (which showed no cross-reactivity with human annexins 2 to 6). Dex caused no increase in immunoreactive (ir)LC-1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC-1-like immunoreactivity. This was accompanied by the appearance of irLC-1 in the extracellular medium.4The results of the present study implicate endogenous LC-1 in glucocorticoid suppression of TNFα and PGE2 release from human PBMC and suggest an extracellular site of action for LC-1. LC-1 may also inhibit rhIL-1β-stimulated TNFα and PGE2 secretion from PBMC.
There is a growing appreciation of the important role of resolution mediators in the successful termination of the inflammatory response. Here, we discuss the potential importance of the lipid and peptide proresolving mediators, in particular the resolvins and chemerin-derived peptides, which mediate their effects through specific G protein-coupled receptors (GPCRs).
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I–mediated antiinflammatory effects.
During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (−/−) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (−/−) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.
Galectin-3 is a β-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 μM) but chemokinetic at low concentrations (10–100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and stromal cell-derived factor-1α. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.
Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription.
Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antilipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.
Mechanisms involved in neutrophil accumulation induced by intradermal injection of interleukin‐1 (IL‐1) in the rabbit were investigated using intravenously‐injected ¹¹¹ In‐labelled neutrophils. C5a des Arg, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and leukotriene B 4 (LTB 4 ) were included for comparison.
Local inhibition of protein biosynthesis in the skin using actinomycin‐D or cycloheximide blocked ¹¹¹ In‐neutrophil accumulation induced by IL‐1, but not that induced by the other mediators.
Actinomycin‐D and cycloheximide had no effect on local plasma protein leakage induced by intradermally‐injected C5a des Arg, or that induced by zymosan. ¹¹¹ In‐neutrophil accumulation induced by zymosan was, however, partially suppressed.
A monoclonal antibody, MoAb 60.3, recognising neutrophil surface CD18 antigen, was preincubated with ¹¹¹ In‐neutrophils before intravenous injection. This pretreatment did not affect circulating numbers of radiolabelled cells, but it inhibited their accumulation in response to IL‐1, C5a des Arg and the other mediators.
The results suggest that neutrophil accumulation induced by IL‐1, but not the other mediators, requires local protein biosynthesis, probably in the microvascular endothelium. Neutrophil accumulation to IL‐1 and the other mediators appears to require neutrophil surface antigen, CD18. The inflammatory response to zymosan may be mediated by both endogenous C5a des Arg and IL‐1.
Previous in vitro findings suggest a critical role for the polymorphonuclear leukocyte (PMN) membrane glycoprotein complex CD18 in PMN adherence and chemotaxis. We examined the effect of the murine monoclonal antibody (MoAb) 60.3, recognizing CD18, on induced PMN accumulation in vivo. Rabbits were pretreated with MoAb 60.3, and the chemotactic factors fMLP, leukotriene (LT)B4, and C5a, as well as histamine, were injected intradermally; 4 hours later, plasma leakage (125I-albumin) and the PMN accumulation (myeloperoxidase) were determined. Both PMN accumulation and PMN-dependent plasma leakage were abolished in the inflammatory skin lesions of rabbits pretreated with MoAb 60.3 as compared with control animals, whereas histamine-induced PMN-independent plasma leakage was unaffected. Intravital microscopy of the rabbit tenuissimus muscle revealed that MoAb 60.3 inhibited both PMN adherence in the venules and migration into the tissue following application of LTB4 and zymosan-activated serum (ZAS). Rolling of PMNs along the venular endothelium was unaffected. Thus, these experiments confirm and extend earlier in vitro findings of the critical role of the membrane glycoprotein complex, CD18, in PMN adherence and chemotaxis.
Intradermal injection of zymosan into nonsensitized rabbits induces plasma exudation, which is dependent on two mediators: C5a generated in extravascular tissue fluid and a vasodilator prostaglandin generated from substrates localized in cell membranes. This relationship between the complement system and the prostaglandin synthesis system had not previously been explored, and complement activation has generally been associated with increased vascular permeability via histamine release. We report that C5a increases vascular permeability by a mechanism that is not dependent on histamine release; however plasma exudation is virtually undetectable in the absence of a vasodilator substance. Because the permeability-increasing activity is stable in plasma, analogy with other species suggests that the activity is a result of C5a devoid of its carboxyl terminal arginine (C5a des Arg). This relates the observed permeability-increasing activity with effects on leukocytes rather than effects as an anaphylatoxin.
The intradermal administration of endothelial IL‐8 (IL‐8 1–77 ) or monocyte derived IL‐8 (IL‐8 1–72 ) to rabbits produced a concentration‐dependent increase in plasma extravasation and an accumulation of polymorphonuclear leukocytes (PMNs) when measured over a 3 h time period. When plasma extravasation and PMN accumulation were measured over a 30 min time period no significant increases in PMN accumulation or plasma extravasation were observed in response to IL‐8 alone. However, under these conditions, the addition of prostaglandin E 2 (100 pmol) produced a significant potentiation of IL‐8‐induced plasma extravasation. There was no significant difference between the biological activities of IL‐8 177 and IL‐8 1–72 .
Plasma extravasation and PMN accumulation induced by IL‐8 were inhibited in rabbits pretreated with the monoclonal antibody designated IB4 (1 mg kg ⁻¹ , i.v.) directed against the common β chain (CD 18) of the leukocyte integrins.
The intra‐articular administration to rabbits of IL‐8 1–77 (1 nmol) resulted 24 h later in the appearance of a mixed population of leukocytes (PMNs and mononuclear cells) in synovial lavage fluid. Biochemical analyses revealed the presence of an increased level of sulphated proteoglycans (sPG) and of the metalloproteinase stromelysin. Pretreatment of rabbits with IB4 (3 mg kg ⁻¹ , i.v.) inhibited the accumulation of PMNs but had no effect on the mononuclear infiltrate nor on the levels of sPG or stromelysin.
The intradermal or intra‐articular injection of E. coli ‐derived endotoxin induced similar inflammatory changes to those observed with IL‐8. The possibility that the biological activities of IL‐8 were attributable to minor contamination with endotoxin is unlikely for two reasons. Firstly, biological effects of endotoxin were observed at levels greater than that contained in the IL‐8 preparation. Secondly, reduction of the endotoxin content of the IL‐8 preparation by a factor of 10 did not produce a concomitant reduction in the observed biological activity of the IL‐8.
Recently the critical requirement for the CD18 family of adhesion molecules on leucocytes for their adhesion and migration to inflammatory reactions has been recognized in humans and several animal models. The in vivo studies have mostly utilized antibodies to CD18, the common beta-subunit of CD11a,b,c/CD18 molecules and thus have blocked the function of all three family members, making evaluation of the role of individual subunits impossible. Furthermore, none of the reagents used were suitable for studies in rats. Here we report the effects on polymorphonuclear leucocyte (PMNL) adhesion and in vivo migration of a new monoclonal antibody (mAb) TA3, which recognizes and blocks rat CD11a/CD18 (LFA-1). These studies also evaluated mAb MRC OX42, which reacts with rat CD11b/CD18 (CR3, MAC-1). Neither antibody alone inhibited rat PMNL adhesion to interleukin-1 (IL-1)-activated rat endothelium, but the combination inhibited adhesion by 44%. OX42 treatment of rat PMNL inhibited phorbol myristate acetate (PMA) activated adhesion by 88%, while TA3 only inhibited this adhesion in combination with OX42, resulting in 99% inhibition of PMA-induced PMNL adhesion. Treatment of rats with TA3 alone partially inhibited 51Cr-labelled rat blood PMNL migration into zymosan-activated serum (C5adesArg; ZAS), but not IL-1, or endotoxin [lipopolysaccharide (LPS)] induced dermal inflammatory reactions. MAb OX42 had no such effect in vivo. However, treatment with both antibodies virtually eliminated any PMNL accumulation in all three types of inflammatory reactions. Ex vivo treatment of the 51Cr-labelled PMNL, prior to i.v. infusion showed that mAb TA3 again preferentially inhibited PMNL migration to ZAS. These results suggest that in the rat, CD11a/CD18 plays a major role in PMNL migration to C5a and that either CD11a or CD11b/CD18 can function to maintain normal PMNL migration to IL-1 or LPS dermal inflammatory reactions. More than one member of this adhesion family or their ligands may need to be targeted for effective modulation of PMNL infiltration, at least in this species.
Although a bewildering array of cell surface carbohydrate structures have been described, the physiological relevance of any
of these complex molecules has often eluded biologists. A family of cell surface glycoproteins, the "selectins," has a characteristic
ability to use some of these carbohydrate structures in adhesive mechanisms that help localize leukocytes to regions of inflammation.
This article will review the biology of these carbohydrate-binding adhesive proteins and discuss the potential for developing
anti-inflammatory antagonists that could inhibit binding events that are selectin-mediated.
1. A model has been developed to compare the inhibitory effects of the topical steroid, betamethasone-17-valerate, to those of systemically administered betamethasone upon oedema responses induced by 5-hydroxytryptamine (5-HT), platelet activating factor (PAF) and zymosan-activated serum (ZAS) +/- prostaglandin E1 (PGE1), measured in the rat skin by use of 125I-labelled human serum albumin. 2. Systemic betamethasone had a selective, time- and dose-dependent inhibitory effect upon oedema treatment, with 1 mg kg-1 and a 3 h pretreatment having the greatest effect of the doses and times employed. 3. Topical betamethasone inhibited the oedema responses to all of the stimuli showing no apparent selectivity. 4. Topical betamethasone inhibits inflammatory stimuli in a different manner from systemic betamethasone. The broad spectrum of inhibition suggests that topical betamethasone acts by affecting a fundamental feature of the inflammatory response common to all of the stimuli.
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 human recombinant lipocortin-1 (hrLC-1) on the pyrogenic actions of the synthetic polyribonucleotide polyinosinic:polycytidylic acid (poly I:C) has been studied in conscious rabbits. Poly I:C (2.5 micrograms kg-1) given i.v. produced a biphasic fever with a first peak after 90-105 min and a second peak between 225-240 min. hrLC-1 (50 micrograms kg-1) given i.v. simultaneously with the poly I:C produced a significant reduction in the febrile response but without complete suppression. The thermal response index over 5 h (TRI5) was 4.69 +/- 0.51 for poly I:C given with saline and the TRI5 for poly I:C given with hrLC-1 was 2.66 +/- 0.45 (values are for n = 5 +/- s.e. mean, P less than 0.05). hrLC-1 administered alone had no effect on body temperature and its antipyretic activity was lost on heating. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mg kg-1) given i.v. reduced the pyrogenic response (TRI5) to poly I:C (2.5 micrograms kg-1) from 4.87 +/- 0.54 without dexamethasone to 2.00 +/- 0.25 (n = 5, P less than 0.05) and dexamethasone given alone had no effect on body temperature. These data demonstrate that LC-1 possesses antipyretic actions and raises the possibility that the antipyretic actions of dexamethasone are mediated through the induction of LC-1.
An in vivo experimental peritonitis model was investigated in the rabbit using zymosan as the inflammatory stimulus. After an i.p. injection of zymosan, exudate was removed at intervals and tested in the back skin of assay rabbits. Assay rabbits received i.v. injections of 125I-albumin and 111In-neutrophils, and the local accumulation of each label was measured in response to intradermal injections of exudate samples mixed with a potentiating dose of PGE2. When peritoneal exudate samples were tested in the presence of a specific anti-C5a antibody, virtually all the edema-inducing and neutrophil chemoattractant activity was abolished in samples taken up to 2 h after the zymosan injection. Later samples, however, contained increasing levels of a non-C5a component. In C5a-depleted 6-h exudate two peaks of inflammatory activity were separated using cation exchange HPLC. Evidence is presented that C5a itself is unable to stimulate the production of these activities. Both peaks of activity appear related to IL-8/NAP-1 as they inhibited the binding of 125I-IL-8/NAP-1 to human neutrophils.
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.
Lipocortins form a group of proteins that have been proposed as mediators of the anti-inflammatory actions of glucocorticoids. Intracerebroventricular injection of a recombinant fragment of lipocortin 1 (NH2-terminal 1-188) caused dose-dependent (0.4-1.2 micrograms) reductions in the acute increases in colonic temperature and oxygen consumption, which occurred in response to central injections of recombinant interleukin 1 beta and gamma-interferon in conscious rats. In contrast the lipocortin fragment did not affect the response to prostaglandin E2, and its activity was prevented by heat treatment or by pretreatment of animals with polyclonal antiserum raised to the fragment. Central injection of antiserum significantly enhanced the thermogenic responses to interleukin 1 beta in rats treated with dexamethasone without affecting the responses in normal animals. These results support a physiological role for lipocortin in the central effects of glucocorticoids.
Article synthese sur les proteines de surface cellulaire impliquees dans l'adherence des phagocytes au niveau de l'endothelium de la veine ombilicale dans les mecanismes inflammatoires. Le role d'antigenes tels que l'antigene cD18 dans l'emigration in vivo des phagocytes est etudie
Macrophage interactions with extracellular matrix and other cells are important in phagocytosis, inflammation, and immunity. To learn more about the surface molecules involved in adhesion we compared the binding of murine macrophages and polymorphonuclear leukocytes (PMN) with artificial substrate in vitro. A distinctive type of adhesion of thioglycollate-elicited peritoneal macrophages (TPM) to bacteriologic plastic (BP) was defined, which was pronase-sensitive, Mg2+-dependent, and required cytoskeletal stabilization. A rat mAb designated 5C6 was isolated because it inhibited TPM attachment to BP, as well as mediating detachment of TPM adherent to that substratum. In addition, it inhibited the attachment of PMN to tissue culture plastic. This antiadhesive property of 5C6 mAb required intact IgG; the F(ab')2 fragment was partially effective and Fab was ineffective. 5C6 recognized the type 3 complement receptor, inhibiting rosetting of EAC3bi to TPM and immunoprecipitating a heterodimer of 160 and 95 kD that comigrated with the M1/70 immunoprecipitate. 5C6 recognized a pronase-stable epitope distinct from that of M1/70. Other mAbs, including M1/70 (CR3) and 2.4G2 (FcR), failed to have any antiadhesive effect in vitro. The inhibitory activity of 5C6 in short-term adhesion assays correlated with its inhibition of recruitment of myelomonocytic cells to a thioglycollate-elicited peritoneal exudate in vivo, after intravenous injection of mAb. 5C6 IgG inhibited recruitment of myelomonocytic cells by 84 +/- 3% at 1 d compared with saline-injected controls. The F(ab')2 fragment and a class-matched control IgG had little effect. Recruitment of TPM at 4 d was also efficiently inhibited by 5C6 IgG. 5C6 IgG was not cytotoxic, had no effect on marrow egress, did not cause increased phagocytic clearance of circulating neutrophils, and had no adverse effect on chemotaxis in vitro. We show that CR3 alone of the LFA-family is necessary for the recruitment of myelomonocytic cells to inflammatory stimuli such as thioglycollate broth. This strategy may be of general use in isolating reagents that inhibit the adhesive function of CR3 and provides a novel approach to antiinflammatory therapy.
Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
Corticosteroids may mediate some of their anti-inflammatory effects via induction of a specific 38 kD protein, lipocortin-1. Autoantibodies to lipocortin-1 were measured by enzyme linked immunosorbent assay (ELISA) in 90 healthy subjects and in 63 patients with rheumatoid arthritis (RA), 36 with systemic lupus erythematosus (SLE), 26 with polymyalgia rheumatica, and 13 with chronic airways disease. Sixteen patients with RA receiving prolonged, high steroid doses (prednisolone greater than 7.5 mg/day) had raised IgM antilipocortin-1 levels, while 19 patients with RA untreated with steroids had normal levels. This association was independent of disease activity. In SLE, raised antilipocortin-1 levels were associated with active disease and were independent of steroid treatment. Antilipocortin-1 antibody levels were not raised in patients with polymyalgia rheumatica and chronic airways disease. Thus steroid treatment alone appears insufficient to induce antilipocortin-1 antibodies, unless an underlying autoimmune state is also present. In RA, antilipocortin-1 antibodies may impair anti-inflammatory actions of steroids and render some patients 'steroid resistant'.
The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.
The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.
The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.
Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.1.4), may provide such a dual action and recent research has concentrated on the role of PLA2-inhibitory proteins as possible anti-inflammatory agents. Blastokinin or uteroglobin is a steroid-induced rabbit secretory protein with PLA2-inhibitory activity. Its biochemical and biological properties have been extensively studied and its crystallographic structure has been resolved at 1.34 A (refs 15, 16). Lipocortins are a family of related proteins, which, it has been suggested, mediate the anti-inflammatory effects of glucocorticoids (for a review, see ref. 23). Some proteins of this group have been purified and the complementary DNA sequences of two human lipocortins are known. Lipocortins inhibit PLA2 in vitro, although their mechanism of action is still unclear. Recombinant lipocortin I inhibits eicosanoid synthesis in isolated perfused lungs from the guinea pig. Here, we report that synthetic oligopeptides corresponding to a region of high amino-acid sequence similarity between uteroglobin and lipocortin I have potent PLA2 inhibitory activity in vitro and striking anti-inflammatory effects in vivo.
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.
An acetylated polypeptide corresponding to residues 2-26 of human lipocortin 1 was synthesized and the anti-inflammatory activity assessed in three models of acute inflammation in rat and mouse. In the carrageenin rat paw oedema test, the peptide produced a maximal inhibition of approximately 41% at the 3 h time point with a 10 micrograms dose. When rat paw oedema was induced by the injection of venom phospholipase A2, the peptide produced a significant inhibition (31%) at the top dose of 20 micrograms per paw. In the mouse air-pouch model, systemic treatment with the peptide produced a dramatic reduction in cytokine-induced leukocyte migration with an ID50 of approximately 40 micrograms per mouse. The N-terminal peptide 2-26 shares the actions of lipocortin 1 in these acute models of inflammation.
The anti-inflammatory protein lipocortin 1 has been proposed as a mediator of some of the anti-inflammatory actions of glucocorticoid hormones. In view of reported glucocorticoid effects on leukocyte Fc gamma receptors, the effect of short-term lipocortin 1 pre-incubation on expression and IgG binding capacity of human Fc gamma receptors was examined in vitro. The formation of erythrocyte-antibody rosettes, binding of fluoresceinated IgG ligand and the expression of three defined types of Fc gamma receptors were observed following lipocortin 1 treatment. Maximal inhibition of EA rosetting (70%) by peripheral blood mononuclear cells and a 30-50% inhibition of binding of fluoresceinated human IgG1 to purified human monocytes occurred in the presence of 400 nM lipocortin 1 (p < 0.01). However, there was no accompanying decrease in expression of the three known Fc gamma receptor types measured by specific monoclonal antibodies. Similar observations were made for peripheral blood polymorphonuclear cells. On the other hand, IgG binding was not inhibited by lipocortin 1 in lymphocytes or in a panel of cell lines which express Fc gamma receptors and none of these cell types had the capacity to bind lipocortin.
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.
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