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Functional and Ultrastructural Analysis of Annexin A1 and Its Receptor in Extravasating Neutrophils during Acute Inflammation
2008; Elsevier BV; Volume: 174; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080342
ISSN1525-2191
AutoresThaís Santana Gastardelo Bizotto, Amílcar Sabino Damazo, Jesmond Dalli, Roderick J. Flower, Mauro Perretti, Sônia Maria Oliani,
Tópico(s)Immune Response and Inflammation
ResumoThe purpose of this study was twofold: to reveal cellular events associated with the protective role of endogenous annexin A1 (AnxA1) in inflammation and to highlight the potential involvement of members of the formyl peptide receptor (Fpr) family in this process. We found that wild-type, AnxA1-null, and Fpr1-null mice all displayed an intense neutrophil recruitment into the peritoneal cavity as assessed 4 hours after carrageenin injection, and that this recruitment was most pronounced in AnxA1-null mice. In addition, this cell influx could be inhibited by the AnxA1 pharmacophore peptide, Ac2-26, in wild-type, AnxA1-null, and Fpr1-null mice, but was restored when co-treated with the pan-receptor antagonist Boc2. Using the LacZ gene reporter assay, an enhancement of AnxA1 gene promoter activity in extravasated neutrophils was evident in AnxA1-null mice; again this response was reduced after peptide treatment. The lack of functional involvement of Fpr1 prompted us to monitor the structurally related receptor Fpr2. We report, for the first time, the ultrastructural immunocytochemical co-localization of Fpr2 with AnxA1 in neutrophils that migrate into the mesenteric microcirculation and extravasate into the peritoneal fluid. Collectively, these data provide in vivo support to the hypothesis that endogenous AnxA1 is an essential effector of endogenous anti-inflammation and provide an ultrastructural indication that this mediator interacts with Fpr2 in murine neutrophils. We believe that these findings could significantly affect the development of novel therapeutics, which are modeled after the anti-migratory actions of AnxA1. The purpose of this study was twofold: to reveal cellular events associated with the protective role of endogenous annexin A1 (AnxA1) in inflammation and to highlight the potential involvement of members of the formyl peptide receptor (Fpr) family in this process. We found that wild-type, AnxA1-null, and Fpr1-null mice all displayed an intense neutrophil recruitment into the peritoneal cavity as assessed 4 hours after carrageenin injection, and that this recruitment was most pronounced in AnxA1-null mice. In addition, this cell influx could be inhibited by the AnxA1 pharmacophore peptide, Ac2-26, in wild-type, AnxA1-null, and Fpr1-null mice, but was restored when co-treated with the pan-receptor antagonist Boc2. Using the LacZ gene reporter assay, an enhancement of AnxA1 gene promoter activity in extravasated neutrophils was evident in AnxA1-null mice; again this response was reduced after peptide treatment. The lack of functional involvement of Fpr1 prompted us to monitor the structurally related receptor Fpr2. We report, for the first time, the ultrastructural immunocytochemical co-localization of Fpr2 with AnxA1 in neutrophils that migrate into the mesenteric microcirculation and extravasate into the peritoneal fluid. Collectively, these data provide in vivo support to the hypothesis that endogenous AnxA1 is an essential effector of endogenous anti-inflammation and provide an ultrastructural indication that this mediator interacts with Fpr2 in murine neutrophils. We believe that these findings could significantly affect the development of novel therapeutics, which are modeled after the anti-migratory actions of AnxA1. The annexin A1 (AnxA1) pathway in neutrophils is comprised of the ligand, receptor, and catabolic enzyme, the latter cleaving N-terminal peptides from the 346-amino acid native protein.1Perretti M Flower RJ Annexin 1 and the biology of the neutrophil.J Leukoc Biol. 2004; 76: 25-29Crossref PubMed Scopus (108) Google Scholar, 2Vong L D'Acquisito F Pederzoli-Ribeil M Lavagno L Flower RJ Witko-Sarsat V Perretti M Annexin 1 cleavage in activated neutrophils: a pivotal role for proteinase 3.J Biol Chem. 2007; 282: 29998-30004Crossref PubMed Scopus (97) Google Scholar We and others have demonstrated that the AnxA1 pathway is part of the complex response the host activates during inflammation to effect modulatory and homeostatic actions to keep cell activation and extravasation under a tonic inhibitory control. This model has been mostly based on pharmacological studies conducted with full-length AnxA1 (37-kDa protein), bioactive peptides derived from its N-terminal sequence (unique in the annexin superfamily of proteins)3Gerke V Creutz CE Moss SE Annexins: linking Ca2+ signaling to membrane dynamics.Nat Rev Mol Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1106) Google Scholar and passive immunization strategies.4Perretti M Flower RJ Modulation of IL-1-induced neutrophil migration by dexamethasone and lipocortin 1.J Immunol. 1993; 150: 992-999PubMed Google Scholar, 5Perretti M Ahluwalia A Harris JG Goulding NJ Flower RJ Lipocortin-1 fragments inhibit neutrophil accumulation and neutrophil-dependent edema in the mouse. A qualitative comparison with an anti-CD11b monoclonal antibody.J Immunol. 1993; 151: 4306-4314PubMed Google Scholar, 6Morand EF Hall P Hutchinson P Yang YH Regulation of annexin 1 in rheumatoid synovial cells by glucocorticoids and interleukin-1.Mediators Inflamm. 2006; 2006: 73835Crossref PubMed Scopus (17) Google Scholar, 7Morand EF Effects of glucocorticoids on inflammation and arthritis.Curr Opin Rheumatol. 2007; 19: 302-307Crossref PubMed Scopus (27) Google Scholar, 8Perretti M Ahluwalia A Harris JG Harris HJ Wheller SK Flower RJ Acute inflammatory response in the mouse: exacerbation by immunoneutralization of lipocortin.Br J Pharmacol. 1996; 117: 1145-1154Crossref PubMed Scopus (55) Google ScholarStudying AnxA1 expression and mobilization in human polymorphonuclear leukocytes (PMNs) has markedly contributed to understanding molecular and cellular mechanisms by which this endogenous inhibitory pathway can be activated. Resting PMNs express high levels of the protein in their cytosol, a large proportion of which (>50%) is in gelatinase granules.9Perretti M Christian H Wheller SK Aiello I Mugridge KG Morris JF Flower RJ Goulding NJ Annexin 1 is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis.Cell Biol Int. 2000; 24: 163-174Crossref PubMed Scopus (93) Google Scholar On PMN activation or adhesion, the protein is externalized to the cell surface10Perretti M Croxtall JD Wheler SK Goulding NJ Hannon R Flower RJ Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration.Nat Med. 1996; 2: 1259-1262Crossref PubMed Scopus (194) Google Scholar, 11Walther A Riehemann K Gerke V A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR.Mol Cell. 2000; 5: 831-840Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar where it activates downmodulating signals that inhibit PMN adhesion and transmigration.10Perretti M Croxtall JD Wheler SK Goulding NJ Hannon R Flower RJ Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration.Nat Med. 1996; 2: 1259-1262Crossref PubMed Scopus (194) Google Scholar, 12Zouki C Ouellet S Filep JG The anti-inflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells.FASEB J. 2000; 14: 572-580PubMed Google Scholar, 13Hayhoe RP Kamal AM Solito E Flower RJ Cooper D Perretti M Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement.Blood. 2006; 107: 2123-2130Crossref PubMed Scopus (185) Google ScholarGeneration of AnxA1-null mice has helped clarify the role of the AnxA1 pathway in inflammation: these mice are more prone to both acute and chronic inflammatory reactions.14Hannon R Croxtall JD Getting SJ Roviezzo F Yona S Paul-Clark MJ Gavins FN Perretti M Morris JF Buchingham JC Flower RJ Aberrant inflammation and resistance to glucocorticoids in annexin 1−/− mouse.FASEB J. 2003; 17: 253-255PubMed Google Scholar, 15Yang YH Morand EF Getting SJ Paul-Clark M Liu DL Yona S Hannon R Buckingham JC Perretti M Flower RJ Modulation of inflammation and response to dexamethasone by annexin 1 in antigen-induced arthritis.Arthritis Rheum. 2004; 50: 976-984Crossref PubMed Scopus (136) Google Scholar PMNs purified from AnxA1-null mouse blood exhibit greater degrees of activation and chemotaxis in response to distinct stimuli application.16Chatterjee BE Yona S Rosignoli G Young RE Nourshargh S Flower RJ Perretti M Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro.J Leukoc Biol. 2005; 78: 639-646Crossref PubMed Scopus (105) Google Scholar This behavior is reflected in a higher degree of emigration in the inflamed microcirculation, regardless of the vascular bed observed (cremaster or mesentery) or stimulus applied (platelet-activating factor or zymosan).16Chatterjee BE Yona S Rosignoli G Young RE Nourshargh S Flower RJ Perretti M Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro.J Leukoc Biol. 2005; 78: 639-646Crossref PubMed Scopus (105) Google ScholarA breakthrough in this field by Walther and colleagues11Walther A Riehemann K Gerke V A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR.Mol Cell. 2000; 5: 831-840Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar demonstrated a direct interaction between AnxA1-derived peptide and the receptor for formylated peptides, FPR. Subsequent studies have demonstrated that the situation might be more complex, because all three human receptors of the FPR family [FPR, FPRL-1 (FPR-like 1, also called ALX because it is a functional transducer of the anti-inflammatory signal of lipoxin A4) and FPRL-2 (FPR-like 2)] could be activated with the peptide Ac2-26 and other N-terminal-derived sequences of AnxA1.17Rescher U Danielczyk A Markoff A Gerke V Functional activation of the formyl peptide receptor by a new endogenous ligand in human lung A549 cells.J Immunol. 2002; 169: 1500-1504Crossref PubMed Scopus (65) Google Scholar In addition, comparison of the binding properties of full-length AnxA1 and the peptide Ac2-26 revealed distinct interactions with FPR and ALX/FPRL-1, such that the protein bound to and activated only the latter, whereas the peptide bound and activated both receptors.13Hayhoe RP Kamal AM Solito E Flower RJ Cooper D Perretti M Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement.Blood. 2006; 107: 2123-2130Crossref PubMed Scopus (185) Google Scholar There are no data with respect to AnxA1 binding to, and/or activation of, FPRL-2. However, it is currently accepted that members of the FPR family transduce the cellular activities of AnxA1, at least at the level of monomyelocytic cells.18Perretti M The annexin 1 receptor(s): is the plot unravelling?.Trends Pharmacol Sci. 2003; 24: 574-579Abstract Full Text Full Text PDF PubMed Scopus (51) Google ScholarWe have previously used immunohistochemistry and ultrastructural analyses to shed light on components of the AnxA1 pathway during on-going inflammatory reactions in rats19Oliani SM Paul-Clark MJ Christian HC Flower RJ Perretti M Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression.Am J Pathol. 2001; 158: 603-615Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar and mice.20Damazo AS Yona S D'Acquisto F Flower RJ Oliani SM Perretti M Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation.Am J Pathol. 2005; 166: 1607-1617Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Damazo AS Yona S Flower RJ Perretti M Oliani SM Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis.J Immunol. 2006; 176: 4410-4418PubMed Google Scholar Collectively, these studies have shown that i) PMN-derived AnxA1 is mobilized on the cell surface of the adherent PMNs, probably subsequent to mobilization of gelatinase granules; ii) extravasated PMNs contain higher levels of AnxA1 compared with intravascular cells; iii) the AnxA1 gene promoter activity (monitored by expression of the reporter, the LacZ gene) is augmented in extravasated PMNs; and iv) AnxA1 cleavage appears to be more pronounced in extravasated PMNs so that cleaved (deprived on the N-terminal region) AnxA1-like immunoreactivity is more abundant in these cells, at least during the early hours of the inflammatory response.One question that remains to be answered regards the identity of the receptor(s) responsible for the anti-inflammatory properties of AnxA1 and peptide Ac2-26 observed in experimental systems in rodents. To solve this problem in AnxA1 biology is not trivial, because FPR genes have undergone expansion in the mouse genome so that at least six different genes were initially described (Fpr, Fpr-related sequence 1 to 5),22Gao JL Chen H Filie JD Kozak CA Murphy PM Differential expansion of the N-formylpeptide receptor gene cluster in human and mouse.Genomics. 1998; 51: 270-276Crossref PubMed Scopus (110) Google Scholar with two others being added later (Fpr-rs6 and Fpr-rs7).23Wang ZG Ye RD Characterization of two new members of the formyl peptide receptor gene family from 129S6 mice.Gene. 2002; 299: 57-63Crossref PubMed Scopus (37) Google Scholar Here we have begun unraveling this question using the tools available, namely: i) the Fpr1-null mouse colony24Gao JL Lee EJ Murphy PM Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor.J Exp Med. 1999; 189: 657-662Crossref PubMed Scopus (240) Google Scholar; ii) a new antiserum to Fpr-rs2 (now known officially as Fpr2); iii) the pan-Fpr antagonist Boc2. In addition, we have extended our detailed analysis of the AnxA1 pathway in early inflammatory responses to the carrageenin-induced mouse peritonitis model, monitoring protein expression (and its cellular localization) as well as AnxA1 gene promoter activity (using the LacZ gene reporter borne by the AnxA1-null mice).20Damazo AS Yona S D'Acquisto F Flower RJ Oliani SM Perretti M Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation.Am J Pathol. 2005; 166: 1607-1617Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Damazo AS Yona S Flower RJ Perretti M Oliani SM Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis.J Immunol. 2006; 176: 4410-4418PubMed Google ScholarMaterials and MethodsAnimalsMale wild-type (WT) littermate, AnxA1-null,14Hannon R Croxtall JD Getting SJ Roviezzo F Yona S Paul-Clark MJ Gavins FN Perretti M Morris JF Buchingham JC Flower RJ Aberrant inflammation and resistance to glucocorticoids in annexin 1−/− mouse.FASEB J. 2003; 17: 253-255PubMed Google Scholar and Fpr1-null mice22Gao JL Chen H Filie JD Kozak CA Murphy PM Differential expansion of the N-formylpeptide receptor gene cluster in human and mouse.Genomics. 1998; 51: 270-276Crossref PubMed Scopus (110) Google Scholar (20 to 25 g of body weight) were used for all experiments. Founders of the Fpr-null colony were kindly donated by Ji Liang Gao and Philip Murphy (National Institutes of Health, Bethesda, MD).24Gao JL Lee EJ Murphy PM Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor.J Exp Med. 1999; 189: 657-662Crossref PubMed Scopus (240) Google Scholar Animals were maintained on a standard chow pellet diet with tap water ad libitum and housed at a density of five animals per cage in a room with controlled lighting (lights on from 8:00 AM to 8:00 PM) in which the temperature was maintained at 21 to 23°C. Animal work was performed according to U.K. Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act 1986).Model of InflammationExperimental peritonitis was induced by an intraperitoneal injection of 1.5 mg/kg carrageenin (type λ; Sigma Chemical Co., Poole, UK) in phosphate-buffered saline (PBS). Vehicle animals were injected intraperitoneally with PBS rather than carrageenin. In all cases, mice (n = 5 per group) were sacrificed 1 and 4 hours later by CO2 exposure.Drug TreatmentFor this set of pharmacological experiments, mice (n = 5) were administered intravenously with 100 μg per mouse of the annexin 1 mimetic peptide Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK)19Oliani SM Paul-Clark MJ Christian HC Flower RJ Perretti M Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression.Am J Pathol. 2001; 158: 603-615Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar in PBS, 15 minutes before experimental peritonitis. As a control to Fpr receptors, the WT mice received intravenously 10 μg per mouse of the FPR antagonist Boc2 (N-t-butyloxycarbonyl-Phe-Dleu-Phe-Dleu-Phe) (ICN Pharmaceuticals, Basingstoke, UK) followed by peptide treatment.25La M D'Amico M Bandiera S Di Filippo C Oliani SM Gavins FN Flower RJ Perretti M Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action.FASEB J. 2001; 15: 2247-2256Crossref PubMed Scopus (109) Google ScholarReal-Time Polymerase Chain Reaction (PCR) Analysis for Formyl Peptide Receptors and AnxA1 ProteinTo quantify specific mRNA levels, RNA was isolated from total peritoneal neutrophils using spin column methodology according to the manufacturer's guidance (RNeasy kit; Qiagen, Crawley, UK). Contaminating genomic DNA was removed by on-column pre-DNase digestion as per the manufacturer's instructions (Qiagen). RNA was reverse-transcribed using 2 μg of oligo(dT) 15 primer (Promega, Southampton, UK), 10 U avian myeloblastosis virus (AMV) reverse transcriptase, 40 U ribonuclease inhibitor (Promega), and 1.25 mmol/L each deoxyribonucleoside triphosphate (dNTP) for 60 minutes at 42°C. The synthesized cDNA was used for real-time PCR. An equal amount of first-strand cDNA was amplified by PCR using platinum TaqDNA polymerase (Invitrogen, Carlsbad, California, USA). The forward and reverse primers for mouse Fpr1, Fpr-rs1, and Fpr2 are described by Wang and Ye.23Wang ZG Ye RD Characterization of two new members of the formyl peptide receptor gene family from 129S6 mice.Gene. 2002; 299: 57-63Crossref PubMed Scopus (37) Google Scholar, 26Hartt JK Barish G Murphy PM Gao JL N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR.J Exp Med. 1999; 190: 741-747Crossref PubMed Scopus (97) Google Scholar The PCR reaction was performed at 94°C for 5 minutes followed by 40 cycles of 94°C for 30 seconds, 61°C for 30 seconds, and 72°C for 30 seconds with a final extension of 72°C for 7 minutes. Amplification data are expressed as number of cycles whereby GAPDH is used as endogenous control.Assessment of Cell ExtravasationNeutrophil migration was assessed both into the mesenteric tissue and into the peritoneal cavity. After sacrifice, 3 ml of PBS were injected into peritoneal cavity; then 100 μl of peritoneal fluid were collected and diluted 1/10 in Turk's solution (0.1 crystal violet in 3% acetic acid). Quantification of neutrophils in the peritoneal fluids was performed with a Neubauer chamber (BOECO, Hamburg, Germany) using a ×40 objective.Fixation, Processing, and Embedding for Light and Electron MicroscopyPeritoneal cells and fragments of mesentery were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde, and 0.1 mol/L sodium cacodylate buffer (pH 7.4) for 24 hours at 4°C, washed in sodium cacodylate, and dehydrated through graded percentages of ethanol, and embedded in LRGold (London Resin Co., Reading, UK). Sections (0.5 μm thick) were stained with 1% toluidine blue in 1% borax solution (TAAB Laboratories, Aldermaston, UK). Sections of mesentery were analyzed on an Axioskop 2-Mot Plus Zeiss microscope (Carl Zeiss, Jena, Germany) for quantification of neutrophils. Values are shown as mean ± SEM of cells number per mm2 of four sections (0.5 μm) per sample (five samples per animal). For electron microscopy, sections (∼90 nm) were cut on an ultramicrotome (Reichert Ultracut; Leica, Viena Austria) and placed on nickel grids for immunogold labeling.AnxA1 Gene Expression by X-Gal StainAnxA1-null mice have a LacZ gene inserted into the targeting construct to facilitate measurement of gene expression. Cells and tissues from AnxA1-null mice were therefore stained with the histochemical X-Gal technique. In the presence of β-galactosidase, this staining produces a characteristic Prussian blue color. Samples were fixed in 4% paraformaldehyde, 0.1 mol/L phosphate buffer (pH 7.3) for 1 hour at 4°C, and washed with a rinse solution (0.1 mol/L phosphate buffer, pH 8, 2 mmol/L magnesium chloride, 0.1% Triton X-100), three times for 30 minutes each. Samples were stained overnight at 37°C using X-Gal staining solution (5 mmol/L potassium ferrocyanide in rinse buffer plus 1 mg/ml β-galactosidase in dimethylformamide). Fragments were then washed in PBS at room temperature and postfixed in 4% paraformaldehyde before embedding in LRGold resin. The embedded cellular and tissue samples were then cut (∼1 μm) on a microtome (RM2265; Leica, Wetzlar, Germany) and counterstained with hematoxylin for subsequent analysis. Densitometric analysis for X-Gal staining was done according to an arbitrary scale ranging from 0 to 255 by Axiovision software acopled on Zeiss Axioskop 2 light microscope.Postembedding Immunogold LabelingTo detect the co-localization of endogenous AnxA1 protein with the Fpr2 receptor in neutrophils, ultrathin sections (∼90 nm) of mesentery and peritoneal fluid cell pellet were incubated in a step by step manner with the following reagents at room temperature: i) distilled water; ii) 0.1 mol/L phosphate buffer containing 1% egg albumin (PBEA); iii) 0.1 mol/L PBS containing 5% egg albumin (PBEA) for 30 minutes; iv) sheep polyclonal antibody termed LCS3, raised against intact and cleaved isoforms of AnxA1 (1:200 in PBEA) and rabbit polyclonal antibody Fpr2β (1:100 in PBEA) for 2 hours, normal sheep and rabbit sera were used as control (1:200); v) three washes (5 minutes each) in PBEA containing 0.01% Tween 20. To detect AnxA1, vi) donkey anti-sheep IgG (Fc fragment-specific) antibody (1:100 in PBEA) conjugated to 10-nm colloidal gold (British Biocell, Cardiff, UK) was added, and to detect Fpr2 receptor, goat anti-rabbit IgG (Fc fragment-specific) antibody (1:100 in PBEA) conjugated to 20 nm colloidal gold (British Biocell, Cardiff, UK) was added; vii) after 1 hour, sections were washed extensively in PBEA containing 0.01% Tween 20, then in distilled water. Sections were stained with uranyl acetate and lead citrate before examination using a Zeiss Leo 906 electron microscope. The density of immunogold, conjugated to AnxA1 and Fpr2, was calculated and expressed for plasma membrane of neutrophils. Values are reported as mean ± SEM of number of gold particles per μm.Statistical AnalysisAll data are mean ± SEM of n ≥ 5 mice per groups. In vitro analyses were repeated at least twice. Statistical differences between means were determined by analysis of variance followed, if significant, by the Bonferroni post-hoc test (on selected experimental groups). A probability value less than 0.05 was taken as significant.ResultsEffects of Peptide Ac2-26 and Boc2 on Carrageenin-Induced Neutrophil MigrationInitially, we used the carrageenin-induced mouse peritonitis model to study the function of the AnxA1 pathway. Figure 1 shows that carrageenin injection produced marked neutrophil infiltration into the mesenteric tissues. AnxA1-null mice displayed larger numbers of intravascular and extravasated neutrophils when compared with WT (Figure 1, A and B). Fpr1-null mice displayed attenuated cell recruitment in relation to WT mice; however, the difference was not significant (Figure 1C). Figure 1H reports the quantitative values, with a near doubling of neutrophils adherent in the vasculature in AnxA1-null mice, and a fourfold increase in the number of extravasated cells.Treatment of mice with the peptide Ac2-26 exerted significant inhibitory properties on this inflammatory response, with a selective effect on extravasated cell numbers. This AnxA1 mimetic was equally effective in both WT and Fpr1-null mice, and produced a remarkable reduction of this inflammatory readout especially in the AnxA1-null mice, in which the response was very pronounced (Figure 1, D, F, and G). The antimigratory property of the peptide on intravascular adherent neutrophils was observed only in AnxA1-null mice (Figure 1H).Finally, co-injection of the pan-receptor antagonist Boc2 prevented the inhibitory effect of peptide Ac2-26 on neutrophil extravasation, restoring values close to those measured in carrageenin-treated WT mice (Figure 1, E and H). Table 1 shows that carrageenin injection produced a marked neutrophil-infiltration, and details the antimigratory property of peptide Ac2-26 and Boc2 antagonist effect on neutrophil influx into the peritoneal fluid. These results justified the use of this model for monitoring Anxa1 gene promoter activity and the expression of AnxA1 protein in the neutrophils.Table 1Effects of the Peptide Ac2-26 and the Fpr Antagonist Boc2 Treatment on Neutrophil Influx of Peritoneal FluidNeutrophils × 105/ml in peritoneal fluidWTAnxa1-nullFpr1-nullControl0.00 ± 0.000.00 ± 0.000.03 ± 0.03CG4h14.33 ± 1.76**P < 0.01 and30.67 ± 1.2***P < 0.001 vs. respective vehicle group;16.33 ± 2.02**P < 0.01 andAc2-26 + CG4h8.0 ± 0.57§P < 0.05 and6.33 ± 2.18P < 0.01 vs. respective CG4h group;9.0 ± 0.57§P < 0.05 andBoc2 + Ac2-26 + CG4h14.0 ± 1.15#P < 0.05 vs. respective Ac2-26 + CG4h group. ANOVA plus Bonferroni test.CG, Carrageenin. Data are mean ± SEM of n = 5 mice per group.** P < 0.01 and*** P < 0.001 vs. respective vehicle group;§ P < 0.05 and§§ P < 0.01 vs. respective CG4h group;# P < 0.05 vs. respective Ac2-26 + CG4h group. ANOVA plus Bonferroni test. Open table in a new tab AnxA1 Gene ExpressionActivation of the AnxA1 gene promoter was monitored in the mesentery and peritoneal fluid neutrophils from AnxA1-null mice. As expected, mesenteric neutrophils from vehicle mice displayed negative reactivity for X-Gal staining assay (Figure 2A). We did not detect neutrophils in peritoneal fluid from vehicle mice (Figure 2B). Injection of carrageenin provoked intense gene promoter activation with marked staining both in intravascular and extravasated mesenteric neutrophils as well as transmigrated neutrophils in the peritoneal fluids (Figure 2, C and D). Figure 2E reports the densitometric analysis of this set of results: there was a time-dependent AnxA1 gene promoter activation, with a rapid response as early as 1 hour after carrageenin. In addition, extravasated neutrophils were more positive than intravascular cells.Figure 2Analysis of LacZ gene expression on neutrophils from mesentery and peritoneal fluid. AnxA1 gene promoter activity was observed by LacZ gene expression as detailed in the Materials and Methods. Intravascular (arrowhead) and extravascular (arrow) neutrophils from mesentery (A) and macrophages (curved arrow) from peritoneal fluid (B) displayed LacZ-negative staining in vehicle mice. Higher staining after 4 hours of inflammation (CG4h) in intra- and extravascular neutrophils from mesentery (C) and neutrophils (open arrow) from peritoneal fluid (D). Counterstaining: hematoxylin. E: Statistical analysis. Data are mean ± SEM of n = 5 mice per group. ***P < 0.001 and *P < 0.05 versus respective vehicle group. Scale bars = 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ultrastructural Immunocytochemistry of Elements of the AnxA1 PathwayIn the last section of the study we sought to extend the data produced with the LacZ gene reporter assay by monitoring the expression of the AnxA1 protein and its putative receptor Fpr2. To do so, we tested the expression of potential receptors responsible for the effects of peptide Ac2-26 to analyze the elements of the AnxA1 pathway. Real-time PCR of peritoneal neutrophils revealed presence of mouse Fpr1 and Fpr2 (Figure 3) but not Fpr-rs1 (data not shown). AnxA1 mRNA expression is shown for comparative purposes (Figure 3). Encouraged by these findings, we then performed ultrastructural immunocytochemistry in extravasated mesenteric neutrophils. These cells, which were induced with carrageenin, were strongly positive for AnxA1 and displayed reactivity also for Fpr2 (Figure 4A); the same held true for peritoneal neutrophils (Figure 4B). We did not detect ultrastructural immunocytochemistry co-localization of AnxA1 with Fpr2 in neutrophils treated with the antagonist Boc2, demonstrating that this antagonist blocks AnxA1 co-localization with Fpr2 (data not shown). Lower AnxA1 and Fpr2 immunoreactivity was observed in plasma membrane of mesenteric (Figure 4C) and peritoneal (Figure 4D) neutrophils after treatment of mice with peptide Ac2-26. No labeling was detected in sections incubated with the control nonimmune sheep or rabbit sera (Figure 4E).Figure 3Real-time PCR analysis for mouse formyl peptide receptors and AnxA1 protein. Peritoneal neutrophils from WT mice were used to quantify specific mRNA levels. Primers for GADPH were used as controls. Data are presented as mean ± SEM of n = 5 mice per group.View Large Image
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