Design and characterization of a cleavage-resistant Annexin A1 mutant to control inflammation in the microvasculature

Blood ◽  
2010 ◽  
Vol 116 (20) ◽  
pp. 4288-4296 ◽  
Author(s):  
Magali Pederzoli-Ribeil ◽  
Francesco Maione ◽  
Dianne Cooper ◽  
Adam Al-Kashi ◽  
Jesmond Dalli ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Early Stage ◽  
Leukocyte Adhesion ◽  
Formyl Peptide Receptor ◽  
Annexin A1 ◽  
Formyl Peptide ◽  
Anti Inflammatory ◽  
Human Polymorphonuclear Leukocytes

Abstract Human polymorphonuclear leukocytes adhesion to endothelial cells during the early stage of inflammation leads to cell surface externalization of Annexin A1 (AnxA1), an effector of endogenous anti-inflammation. The antiadhesive properties of AnxA1 become operative to finely tune polymorphonuclear leukocytes transmigration to the site of inflammation. Membrane bound proteinase 3 (PR3) plays a key role in this microenvironment by cleaving the N terminus bioactive domain of AnxA1. In the present study, we generated a PR3-resistant human recombinant AnxA1—named superAnxA1 (SAnxA1)—and tested its in vitro and in vivo properties in comparison to the parental protein. SAnxA1 bound and activated formyl peptide receptor 2 in a similar way as the parental protein, while showing a resistance to cleavage by recombinant PR3. SAnxA1 retained anti-inflammatory activities in the murine inflamed microcirculation (leukocyte adhesion being the readout) and in skin trafficking model. When longer-lasting models of inflammation were applied, SAnxA1 displayed stronger anti-inflammatory effect over time compared with the parental protein. Together these results indicate that AnxA1 cleavage is an important process during neutrophilic inflammation and that controlling the balance between AnxA1/PR3 activities might represent a promising avenue for the discovery of novel therapeutic approaches.

scholarly journals Annexin A1 attenuates neutrophil migration and IL-6 expression through Fpr2 in a mouse model of Streptococcus suis-induced meningitis

2020 ◽  
pp. IAI.00680-20
Author(s):  
Chengpei Ni ◽  
Song Gao ◽  
Yuling Zheng ◽  
Peng Liu ◽  
Yajie Zhai ◽  
...  
Keyword(s):  
Mouse Model ◽  
Inflammatory Mediator ◽  
Therapeutic Option ◽  
Neutrophil Migration ◽  
Streptococcus Suis ◽  
Formyl Peptide Receptor ◽  
Annexin A1 ◽  
Formyl Peptide ◽  
Anti Inflammatory

Streptococcus suis (S. suis) serotype 2 is a crucial pathogenic cause of bacterial meningitis, a life-threatening disease with neurological sequelae and high rates of mortality. Inflammation triggered by S. suis infection must be precisely regulated to prevent further tissue damage. As a glucocorticoid anti-inflammatory mediator, Annexin A1 (AnxA1) mainly acts through formyl peptide receptor 2 (Fpr2) to alleviate inflammation in the peripheral system. In this research, we evaluated the roles of AnxA1 and Fpr2 in a mouse model of S. suis meningitis created via intracisternal infection in Fpr2-deficient (Fpr2−/−) and wild-type (WT) mice. We revealed that Fpr2−/− mice were highly susceptible to S. suis meningitis, displaying increased inflammatory cytokine levels, bacterial dissemination, and neutrophil migration compared with the findings in WT mice. Additionally, AnxA1 exerted anti-inflammatory effects through Fpr2, such as attenuation of leukocyte infiltration, inflammatory mediator production, and astrocyte or microglial activation in the brain. Importantly, we found that the anti-migratory function of AnxA1 decreases neutrophil adherence to the endothelium through Fpr2. Finally, an in vitro study revealed that AnxA1 potentially suppresses IL-6 expression through the Fpr2/p38/COX-2 pathway. These data demonstrated that Fpr2 is an anti-inflammatory receptor that regulates neutrophil migration in mice with S. suis meningitis and identified AnxA1 as a potential therapeutic option.

scholarly journals Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

2019 ◽  
Author(s):  
Simon Lind ◽  
Martina Sundqvist ◽  
Rikard Holmdahl ◽  
Claes Dahlgren ◽  
Huamei Forsman ◽  
...  
Keyword(s):  
Human Subjects ◽  
Formyl Peptide Receptor ◽  
Healthy Human ◽  
Formyl Peptide ◽  
Blood Neutrophils ◽  
Small Molecule Compound ◽  
Anti Inflammatory ◽  
Future Utility

AbstractDespite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by the MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gαq-independent transient rise in intracellular Ca2+ and recruitment of β-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

scholarly journals Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 228
Author(s):  
Izabella Lice ◽  
José Marcos Sanches ◽  
Rebeca D. Correia-Silva ◽  
Mab P. Corrêa ◽  
Marcelo Y. Icimoto ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Biological Effects ◽  
Experimental Models ◽  
Formyl Peptide Receptor ◽  
Saline Control ◽  
Acute Peritonitis ◽  
Formyl Peptide ◽  
Leukocyte Influx

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1β. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes

1982 ◽  
Vol 20 (2) ◽  
pp. 203-214 ◽  
Author(s):  
Richard G. Painter ◽  
Manfred Schmitt ◽  
Algirdas J. Jesaitis ◽  
Larry A. Sklar ◽  
Klaus Preissner ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Photoaffinity Labeling ◽  
Formyl Peptide Receptor ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Human Polymorphonuclear Leukocytes

scholarly journals Anti-Inflammatory Mechanisms of the Annexin A1 Protein and Its Mimetic Peptide Ac2-26 in Models of Ocular Inflammation In Vivo and In Vitro

2013 ◽  
Vol 190 (11) ◽  
pp. 5689-5701 ◽  
Author(s):  
Ana P. Girol ◽  
Kallyne K. O. Mimura ◽  
Carine C. Drewes ◽  
Simone M. Bolonheis ◽  
Egle Solito ◽  
...  
Keyword(s):  
Ocular Inflammation ◽  
Annexin A1 ◽  
Mimetic Peptide ◽  
Anti Inflammatory

L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

1989 ◽  
Vol 67 (5) ◽  
pp. 456-464 ◽  
Author(s):  
J. Gillard ◽  
A. W. Ford-Hutchinson ◽  
C. Chan ◽  
S. Charleson ◽  
D. Denis ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Potent Inhibitor ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Lipoxygenase Inhibitor ◽  
12 Lipoxygenase ◽  
Rat Paw ◽  
Human Polymorphonuclear Leukocytes

L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 μM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 μg topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.Key words: leukotriene, 5-lipoxygenase, polymorphonuclear leukocytes, leukotriene B4, leukotriene inhibitor.

Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2871-2878 ◽  
Author(s):  
Praxedis Martin ◽  
Reinhard Wallich ◽  
Julian Pardo ◽  
Arno Müllbacher ◽  
Markus Munder ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Human Neutrophils ◽  
Enzymatic Assays ◽  
Effector Functions ◽  
Healthy Control ◽  
Polymerase Chain ◽  
Human Polymorphonuclear Leukocytes ◽  
Cytotoxic Effector

AbstractPolymorphonuclear leukocytes have been shown to use a multitude of effector functions to combat pathogens and tumors, including enzymes, defensins, and toxic products such as oxygen radicals and nitrogen oxides. Recent studies provided evidence for the expression of granzymes (gzms) and perforin (perf) within the cytotoxic arsenal of human neutrophils, the validity of which was questioned by 2 subsequent studies. We have now used cytology, intracellular flow cytometry, enzymatic assays, immunoelectron microscopy, and quantitative reverse transcriptase-polymerase chain reaction to obtain evidence of the presence of gzms and/or perf in mouse Gr-1+ granulocyte populations. The data obtained clearly demonstrate that neither in vitro- nor in vivo-derived mouse granulocytes synthesize gzmA and gzmB or perf, even following infection/immunization with pathogens or pathogen-derived material. A parallel comparable analysis on the expression of gzmB in human neutrophils from 3 healthy control subjects and 4 patients with diverse diseases failed to detect gzmB expression. The data indicate that polymorphonuclear leukocytes from mice and humans lack the 3 cytotoxic effector molecules, gzmA, gzmB, and perf, generally associated with natural killer and cytotoxic T lymphocytes. (Blood. 2005;106:2871-2878)

scholarly journals Annexin A1 Mimetic Peptide and Piperlongumine: Anti-Inflammatory Profiles in Endotoxin-Induced Uveitis

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3170
Author(s):  
Ana Paula Girol ◽  
Caroline de Freitas Zanon ◽  
Ícaro Putinhon Caruso ◽  
Sara de Souza Costa ◽  
Helena Ribeiro Souza ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Mononuclear Cells ◽  
Formyl Peptide Receptor ◽  
Annexin A1 ◽  
Protective Effects ◽  
Mimetic Peptide ◽  
Formyl Peptide ◽  
Leukocyte Influx ◽  
Cox 2 ◽  
Anti Inflammatory

Uveitis is one of the main causes of blindness worldwide, and therapeutic alternatives are worthy of study. We investigated the effects of piperlongumine (PL) and/or annexin A1 (AnxA1) mimetic peptide Ac2-26 on endotoxin-induced uveitis (EIU). Rats were inoculated with lipopolysaccharide (LPS) and intraperitoneally treated with Ac2-26 (200 µg), PL (200 and 400 µg), or Ac2-26 + PL after 15 min. Then, 24 h after LPS inoculation, leukocytes in aqueous humor, mononuclear cells, AnxA1, formyl peptide receptor (fpr)1, fpr2, and cyclooxygenase (COX)-2 were evaluated in the ocular tissues, along with inflammatory mediators in the blood and macerated supernatant. Decreased leukocyte influx, levels of inflammatory mediators, and COX-2 expression confirmed the anti-inflammatory actions of the peptide and pointed to the protective effects of PL at higher dosage. However, when PL and Ac2-26 were administered in combination, the inflammatory potential was lost. AnxA1 expression was elevated among groups treated with PL or Ac2-26 + PL but reduced after treatment with Ac2-26. Fpr2 expression was increased only in untreated EIU and Ac2-26 groups. The interaction between Ac2-26 and PL negatively affected the anti-inflammatory action of Ac2-26 or PL. We emphasize that the anti-inflammatory effects of PL can be used as a therapeutic strategy to protect against uveitis.

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