Biomedical subjects

Mauro Perretti

Publications and source records attributed to Mauro Perretti.

At least 19 recordsLinked to original sources

PubMed2006

Antiflammin-2 activates the human formyl-peptide receptor like 1.

The anti-inflammatory actions of the nonapeptide antiflammin-2, identified by homology with uteroglobin and annexin-A1 sequences, have been described in some detail, yet its mechanisms of action remain elusive. Since recent data indicate an involvement of the formyl peptide receptor (FPR)-like 1 (or FPRL-1) in the effects of annexin-A1, we have tested here the effect of antiflammin-2 with respect to this receptor family. Using HEK-293 cells expressing either human FPR and FPRL-1, and an annexin-A1 peptide as tracer ([125I-Tyr]-Ac2-26), we found that antiflammin-2 competed for binding only at FPRL-1, and not FPR, with an approximate EC50 of 1 mM. In line with data produced for the full-length protein, genuine receptor activation by antiflammin-2 was confirmed by rapid phosphorylation of extracellular-regulated kinase 1 and 2. Finally, study of the neutrophil interaction with activated endothelium under flow demonstrated an inhibitory effect of antiflammin-2, thus providing functional support to a role for the antiflammin-2/FPRL-1 anti-inflammatory axis.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Annexin-1 modulates T-cell activation and differentiation.

Annexin-1 is an anti-inflammatory protein that plays an important homeostatic role in innate immunity; however, its potential actions in the modulation of adaptive immunity have never been explored. Although inactive by itself, addition of annexin-1 to stimulated T cells augmented anti-CD3/CD28-mediated CD25 and CD69 expression and cell proliferation. This effect was paralleled by increased nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NFATs), and activator protein-1 (AP-1) activation and preceded by a rapid T-cell receptor (TCR)-induced externalization of the annexin-1 receptor. Interestingly, differentiation of naive T cells in the presence of annexin-1 increased skewing in Th1 cells; in the collagen-induced arthritis model, treatment of mice with annexin-1 during the immunization phase exacerbated signs and symptoms at disease onset. Consistent with these findings, blood CD4+ cells from patients with rheumatoid arthritis showed a marked up-regulation of annexin-1 expression. Together these results demonstrate that annexin-1 is a molecular "tuner" of TCR signaling and suggest this protein might represent a new target for the development of drugs directed to pathologies where an unbalanced Th1/Th2 response or an aberrant activation of T cells is the major etiologic factor.

Animals↗

PubMed2006

Glucocorticoid upregulation of the annexin-A1 receptor in leukocytes.

We tested here whether glucocorticoids modulated myeloid cell expression of a specific G-coupled receptor, termed formyl-peptide receptor like-1 (FPRL-1), recently shown to mediate the anti-inflammatory actions of annexin-A1. Real-time PCR and flow cytometry demonstrated rapid up-regulation of mRNA followed by the protein in HL-60 cells incubated with dexamethasone, with peaks at 2 and 24h, respectively. This effect was not restricted to dexamethasone, since reproduced by glucocorticoids. In addition, it was not restricted to the cell line, since replicated with human peripheral blood monocytes. Glucocorticoid ability to upregulate cell surface expression of FPRL-1 was specific, since no effects upon the related receptor FPR or the integrin CD11b could be detected. In view of the wide range of endogenous ligands known to interact with FPRL-1, including the anti-inflammatory protein annexin-A1, we speculate that the novel effect here described may impact on the clinical immunosuppressive and anti-inflammatory properties of glucocorticoids.

Annexin A1↗

PubMed2006

[D-Trp8]-gamma-melanocyte-stimulating hormone exhibits anti-inflammatory efficacy in mice bearing a nonfunctional MC1R (recessive yellow e/e mouse).

Two melanocortin receptors (MC1 and MC3R) have been identified as main transducers of the anti-inflammatory effects of natural and synthetic melanocortins. In this study, we have taken advantage of the recent description of the selective MC3R agonist [d-Trp(8)]-gamma-melanocyte-stimulating hormone (MSH) and of the recessive yellow (e/e) mouse, bearing a nonfunctional MC1R, thereby incrementing our knowledge on this topic. Culturing peritoneal macrophages of recessive yellow (e/e) mice with [d-Trp(8)]-gamma-MSH led to accumulation of cAMP, indicating MC3R receptor functionality: this effect was blocked by a neutralizing antibody against MC3R. Likewise, release of the chemokine KC by urate crystals was attenuated by [d-Trp(8)]-gamma-MSH, and this effect was prevented by synthetic [Ac-Nle(4)-c[Asp(5)-2'-Nal(7),Lys(10)]alpha-MSH(4-10)-NH(2) (SHU9119)] and natural [agouti-related protein (AGRP)] MC3R antagonists but not by the MC4R antagonist Ac-Cys-Nle-Arg-His-d-2-Nal-Arg-Trp-Cys-NH(2) (HS024). Systemic treatment of mice with [d-Trp(8)]-gamma-MSH inhibited KC release and polymorphonuclear cell accumulation elicited by urate crystals in the murine peritoneal cavity. SHU9119 and AGRP prevented the inhibitory actions of [d-Trp(8)]-gamma-MSH, whereas HS024 was inactive. We also demonstrate here that [d-Trp(8)]-gamma-MSH displays a dual mechanism of action by inducing the anti-inflammatory protein heme-oxygenase 1 (HO-1). Treatment with the HO-1 inhibitor zinc protoporphyrin IX exacerbated the inflammatory response elicited by urate crystals and abrogated the anti-inflammatory effects of [d-Trp(8)]-gamma-MSH. In conclusion, these data support the development of the selective MC3R agonist [d-Trp(8)]-gamma-MSH for the treatment of inflammatory pathologies, based on a dual mechanism of cytokine/chemokine inhibition and induction of the anti-inflammatory protein HO-1.

Animals↗

PubMed2006

Annexin I regulates SKCO-15 cell invasion by signaling through formyl peptide receptors.

Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by approximately 2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing.

Annexin A1↗

PubMed2006

Impaired phagocytic mechanism in annexin 1 null macrophages.

The role of the anti-inflammatory protein annexin-A1 (Anx-A1) in the phagocytic process has been investigated using a murine bone marrow culture-derived macrophage model from Anx-A1(+/+) and Anx-A1(-/-) mice. Macrophages prepared from Anx-A1(-/-) mice exhibited a reduced ingestion of zymosan, Neisseria meningitidis or sheep red blood cells, when compared to Anx-A1(+/+) cells and in the case of zymosan this effect was also mirrored by a reduced clearance in vivo when particles were injected into the peritoneal cavity of Anx-A1(-/-) mice. The ablation of the Anx-A1 gene did not cause any apparent cytoskeletal defects associated with particle ingestion but the cell surface expression of the key adhesion molecule CD11b was depressed in the Anx-A1(-/-) cells providing a possible explanation for the attenuated phagocytic potential of these cells. The production of the cytokines TNFalpha and IL-6 was increased in Anx-A1(-/-) macrophages following phagocytosis of all types of particle.

Animals↗

PubMed2006

Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis.

The recent appreciation of the role played by endogenous counterregulatory mechanisms in controlling the outcome of the host inflammatory response requires specific analysis of their spatial and temporal profiles. In this study, we have focused on the glucocorticoid-regulated anti-inflammatory mediator annexin 1. Induction of peritonitis in wild-type mice rapidly (4 h) produced the expected signs of inflammation, including marked activation of resident cells (e.g., mast cells), migration of blood-borne leukocytes, mirrored by blood neutrophilia. These changes subsided after 48-96 h. In annexin 1(null) mice, the peritonitis response was exaggerated ( approximately 40% at 4 h), with increased granulocyte migration and cytokine production. In blood leukocytes, annexin 1 gene expression was activated at 4, but not 24, h postzymosan, whereas protein levels were increased at both time points. Locally, endothelial and mast cell annexin 1 gene expression was not detectable in basal conditions, whereas it was switched on during the inflammatory response. The significance of annexin 1 system plasticity in the anti-inflammatory properties of dexamethasone was assessed. Clear induction of annexin 1 gene in response to dexamethasone treatment was evident in the circulating and migrated leukocytes, and in connective tissue mast cells; this was associated with the steroid failure to inhibit leukocyte trafficking, cytokine synthesis, and mast cell degranulation in the annexin 1(null) mouse. In conclusion, understanding how inflammation is brought under control will help clarify the complex interplay between pro- and anti-inflammatory pathways operating during the host response to injury and infection.

Animals↗

PubMed2006

Interaction of human neutrophils with endothelial cells regulates the expression of endogenous proteins annexin 1, galectin-1 and galectin-3.

Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators.

Annexin A1↗

PubMed2006

The melanocortin peptide HP228 displays protective effects in acute models of inflammation and organ damage.

The clinically efficacious melanocortin peptide HP228 has here been investigated for its anti-inflammatory efficacy. In this study we have investigated the efficacy of HP228 in murine acute models of inflammation and myocardial ischaemia. Systemic treatment of mice with HP228 inhibited neutrophil accumulation in zymosan; urate crystal and carrageenan induced inflammatory models. In the urate model this was due to inhibition of pro-inflammatory chemokines and cytokines, whilst different mechanisms exist for zymosan peritonitis and carrageenan-induced air-pouch inflammation. HP228 was next evaluated in a model of myocardial ischaemia, another condition where cytokines and neutrophils are thought to play a causal role. HP228 caused a 50% reduction in myocardial damage following reperfusion. HP228 therefore inhibits the most important facet of the host inflammatory response namely leukocyte migration. These data show for the first time that the clinically efficacious peptide HP228 displays protective effects in models of inflammation and organ damage.

Acute Disease↗

PubMed2006

Melanocortin 3 receptors control crystal-induced inflammation.

In this study we have characterized the anti-inflammatory profile of a selective melanocortin type 3 receptor (MC3-R) ligand [D-Trp8]-gamma-MSH, validating in vitro results with analyses in mice deficient for this receptor subtype. In wild-type (WT) macrophages, [D-Trp8]-gamma-MSH activated MC3-R (as tested by accumulation of cyclic AMP) and inhibited (approximately 50%) the release of interleukin (IL)-1 and the chemokine KC (CXCL1), but was ineffective in cells taken from MC3-R null mice. In vivo, administration of 3-30 microg [D-Trp8]-gamma-MSH significantly inhibited leukocyte influx and cytokine production in a model of crystal-induced peritonitis, and these effects were absent in MC3-R null mice or blocked by coadministration of an MC3-R antagonist. Finally, in a model of gouty arthritis, direct injection of urate crystals into the rat joint provoked a marked inflammatory reaction that was significantly inhibited (approximately 70%) by systemic or local administration of [D-Trp8]-gamma-MSH. In conclusion, using an integrated transgenic and pharmacological approach, we provide strong proof of concept for the development of selective MC3-R agonists as novel anti-inflammatory therapeutics.

Animals↗

PubMed2006

Novel aspects of annexin 1 and glucocorticoid biology: intersection with nitric oxide and the lipoxin receptor.

This review will emphasize the concept of anti-inflammation and propose that better understanding of the resolution phase of the inflammatory response organized by the host against inflammatory insults can lead to the identification of novel targets for drug development. Under the umbrella of anti-inflammation, we discuss here recent discoveries in the biology of annexin 1 and glucocorticoids. The fact that annexin 1 and another anti-inflammatory mediator, lipoxin A4, converge onto a specific membrane receptor (termed ALX) might help understanding the resolution phase of inflammation, and strengthen the use of this target for innovative drug development. In addition, glucocorticoids (GC), historically linked to annexin 1, are widely use in the clinical control of several pathologies with an inflammatory etiology, though their use is often burdened by several side effects. The development of new GC with more specific or improved mechanisms, e.g. the nitro-steroids, would go along this wave and, potentially, could be of large applicability. The two mediators/targets here illustrated are to be taken as examples of the clues that the study of anti-inflammation might give to the pharmaceutical industry for innovative drug discovery.

Animals↗

PubMed2005

Melanocortin receptor signaling in RAW264.7 macrophage cell line.

Melanocortin peptides modulate cytokine release and adhesion molecule expression. Here we have investigated the early cell-signaling pathway responsible for the induction of interleukin-10 (IL-10) in RAW264.7 cells. Cell incubation with ACTH(1-39) or MTII (melanotan II) did not alter ERK1/2 and JNK phosphorylation, while p38 phosphorylation and intracellular cAMP accumulation occurred within minutes. ACTH(1-39) and MTII provoked a time-dependent accumulation of IL-10 that was abrogated by the PKA inhibitor H-89 and only partially blocked by the p38 MAPK inhibitor SB203580. Thus, in RAW264.7 cells, IL-10 induction by the melanocortins is via the PKA pathway, and this mechanism could contribute to their anti-inflammatory profile.

Adrenocorticotropic Hormone↗

PubMed2005

Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement.

We have tested the effects of annexin 1 (ANXA1) and its N-terminal peptide Ac2-26 on polymorphonuclear leukocyte (PMN) recruitment under flow. Differential effects of the full-length protein and its peptide were observed; ANXA1 inhibited firm adhesion of human PMNs, while Ac2-26 significantly attenuated capture and rolling without effect on firm adhesion. Analysis of the effects of ANXA1 and Ac2-26 on PMN adhesion molecule expression supported the flow chamber results, with Ac2-26 but not ANXA1 causing l-selectin and PSGL-1 shedding. ANXA1 and its peptide act via the FPR family of receptors. This was corroborated using HEK-293 cells transfected with FPR or FPRL-1/ALX (the 2 members of this family expressed by human PMNs). While Ac2-26 bound both FPR and FPRL-1/ALX, ANXA1 bound FPRL-1/ALX only. ANXA1 and Ac2-26 acted as genuine agonists; Ac2-26 binding led to ERK activation in both FPR- and FPRL-1/ALX-transfected cells, while ANXA1 caused ERK activation only in cells transfected with FPRL-1/ALX. Finally, blockade of FPRL-1/ALX with a neutralizing monoclonal antibody was found to abrogate the effects of ANXA1 in the flow chamber but was without effect on Ac2-26-mediated inhibition of rolling. These findings demonstrate for the first time distinct mechanisms of action for ANXA1 and its N-terminal peptide Ac2-26.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Ligand-specific glucocorticoid receptor activation in human platelets.

Few studies have addressed the effects of classical anti-inflammatory glucocorticoids on platelet function. Here, we report for the first time that human platelets contain the glucocorticoid receptor (GR) as identified by a combination of biochemical and functional techniques. Ligand-binding studies revealed the presence of a high- and low-affinity binding site for [3H]-dexamethasone in platelets. The 2 GR ligands prednisolone and dexamethasone competed for [3H]-dexamethasone binding, as did the mineralocorticoid aldosterone. However, while prednisolone (1-10 microM) reduced adenosine diphosphate (ADP, 4 microM) and thromboxane A2 receptor agonist U46619 induced platelet aggregation (up to 75%), dexamethasone had no effect. The inhibition produced by prednisolone was reversed by preincubation with the GR antagonist mifepristone (10 microM; RU486), suggesting the functional importance of the ligand-receptor complex. In addition, prednisolone caused a marked (approximately 50%) reduction in thromboxane B2 levels, whereas dexamethasone was without effect. The apparently anomalous binding data were clarified by the fact that washed platelets (1) contained mineralocorticoid receptor and that (2) it was associated with GR. Taken together, our data suggest that platelet GR forms a heterodimeric complex with the mineralocorticoid receptor that is susceptible to differential activation by specific receptor ligands.

Adenosine Diphosphate↗

PubMed2005

Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients.

Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftr(tm1Unc), cftr-/-) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 6-11, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr-/- mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr-/- mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A2. More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.

Adolescent↗

PubMed2005

Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro.

The role of the endogenous anti-inflammatory mediator annexin 1 (AnxA1) in controlling polymorphonuclear leukocyte (PMN) trafficking and activation was addressed using the recently generated AnxA1 null mouse. In the zymosan peritonitis model, AnxA1 null mice displayed a higher degree (50-70%) of PMN recruitment compared with wild-type littermate mice, and this was associated with reduced numbers of F4/80+ cells. Intravital microscopy analysis of the cremaster microcirculation inflamed by zymosan (6 h time-point) indicated a greater extent of leukocyte emigration, but not rolling or adhesion, in AnxA1 null mice. Real-time analysis of the cremaster microcirculation did not show spontaneous activation in the absence of AnxA1; however, superfusion with a direct-acting PMN activator (1 nM platelet-activating factor) revealed a subtle yet significant increase in leukocyte emigration, but not rolling or adhesion, in this genotype. Changes in the microcirculation were not secondary to alterations in hemodynamic parameters. The phenotype of the AnxA1 null PMN was investigated in two in vitro assays of cell activation (CD11b membrane expression and chemotaxis): the data obtained indicated a higher degree of cellular responses irrespective of the stimulus used. In conclusion, we have used a combination of inflammatory protocols and in vitro assays to address the specific counter-regulatory role of endogenous AnxA1, demonstrating its inhibitory control on PMN activation and the consequent impact on the inflamed microcirculation.

Animals↗

PubMed2005

An overview of the effects of annexin 1 on cells involved in the inflammatory process.

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.

Annexin A1↗

PubMed2005

A novel effect for annexin 1-derived peptide ac2-26: reduction of allergic inflammation in the rat.

Previous investigations have provided evidence that the N-terminal peptide of annexin 1 (peptide Ac2-26) has the capacity of reproducing the anti-inflammatory actions of the full-length protein in many systems. In the current study, we report the effectiveness of the peptide Ac2-26 as an antiallergic tool in a model of rat pleurisy and provide indication for some of the mechanisms involved. In rats inflamed by injection of ovalbumin into the pleural cavity 14 days postsensitization, peptide Ac2-26 (50-200 microg/cavity) inhibited mast cell degranulation, plasma protein leakage, and the accumulation of both neutrophils and eosinophils. Treatment with either peptide Ac2-26 (200 microg/cavity) or dexamethasone (1 mg/kg i.p.) inhibited ovalbumin-induced eotaxin release in the pleural effluents. In vitro, peptide Ac2-26 inhibited ovalbumin-evoked histamine release from subcutaneous tissue fragments obtained from sensitized rats (33-66 microM) and interleukin-13-evoked eotaxin generation from cultured rat mesothelial cells (16-33 microM) but not eosinophil chemotaxis. This work demonstrates that the annexin 1 mimetic peptide Ac2-26 prevents allergen-evoked eosinophilic inflammatory response in rats. Combined analysis of the in vivo and in vitro experiments presented herein suggests that the blockade of secretion of pivotal mediators for the allergic response, such as histamine and eotaxin, could be responsible for the inhibitory actions displayed by peptide Ac2-26.

Animals↗