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M Perretti

Publications and source records attributed to M Perretti.

At least 37 records · Page 2Linked to original sources
PubMed2001

Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression.

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.

Animals↗
PubMed2001

Involvement of the receptor for formylated peptides in the in vivo anti-migratory actions of annexin 1 and its mimetics.

An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides.

Animals↗
PubMed2001

Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation.

Transfection of the pre-monomyelocytic U937 cell line with a plasmid coding for full-length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over-expression of the N-terminal and the first domain of the protein (144 amino acids, clone ANX1-S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml(-1) tumour necrosis factor (TNF)-alpha or 1 - 40 microg ml-1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1-AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF-alpha increased ANX1 mRNA and protein expression in a time-dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1-S showed higher constitutive (more than 2 fold) and activated caspase-3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 - 100%), whereas expression of cytosolic PLA2 Bax and Bcl-2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo-monocytic lineage.

Annexin A1↗
PubMed2001

Analysis of the temporal expression of chemokines and chemokine receptors during experimental granulomatous inflammation: role and expression of MIP-1alpha and MCP-1.

1. Chemokine expression and function was monitored in an experimental model of granulomatous tissue formation after injection of croton oil in complete Freund's adjuvant (CO/CFA) into mouse dorsal air-pouches up to 28 days. 2. In the first week, mast cell degranulation and leukocyte influx (mononuclear cell, MNC, and polymorphonuclear cell, PMN) were associated with CXCR2, KC and macrophage inflammatory protein (MIP)-2 mRNA expression, as determined by TaqMan reverse transcriptase-polymerase chain reaction. KC ( approximately 400 pg x mg protein(-1), n=12) and MIP-2 (approximately 800 pg x mg protein(-1), n=12) proteins peaked at day 7, together with myeloperoxidase (MPO) activity. Highest MIP-1alpha (>1 ng x mg protein(-1), n=12) levels were measured at day 3. 3. After day 7, a gradual increase in CCR2 and CCR5 mRNA, monocyte chemoattractant protein (MCP)-1 mRNA and protein expression was measured. MCP-1 protein peaked at day 21 (approximately 150 pg x mg protein(-1), n=12) and was predominantly expressed by mast cells. A gradual increase in N-acetyl-beta-D-glucosaminidase (NAG) activity (maximal at 28 days) was also measured. 4. An antiserum against MIP-1alpha did not modify the inflammatory response measured at day 7 (except for a 50% reduction in MIP-1alpha levels), but provoked a significant increase in MPO, NAG and MCP-1 levels as measured at day 21 (n=6, P<0.05). An antiserum to MCP-1 reduced NAG activity at day 21 but increased MPO activity values (n=8, P<0.05). 5. In conclusion, we have shown that CO/CFA initiates a complex inflammatory reaction in which initial expression of MIP-1alpha serves a protective role whereas delayed expression of MCP-1 seems to have a genuine pro-inflammatory role.

Animals↗
PubMed2001

Neutrophil accumulation induced by bacterial lipopolysaccharide: effects of dexamethasone and annexin 1.

Annexin 1 (ANX-1) can reduce leucocyte migration in response to cytokines and chemokines in some rodent models of inflammation. However, its effectiveness against an inflammatory stimulus as strong as bacterial lipopolysaccharide (LPS) is unknown. Thus, we have examined whether ANX-1 can modulate LPS-induced neutrophil accumulation in the rat, as assessed by intravital microscopy and by myeloperoxidase (MPO) assay. The anti-inflammatory glucocorticoid, dexamethasone (DEX) was also studied for comparison. LPS superfusion induced adhesion of leucocytes to the endothelium and a subsequent increase in emigration from rat post-capillary venules over 2 h as assessed by intravital microscopy. Either ANX-1 or DEX was able to attenuate this adhesion and emigration of leucocytes. MPO activity in the lung, kidney and ileum was elevated after a 6-h exposure to LPS (intraperitoneal), indicating accumulation of neutrophils in these tissues. DEX attenuated the LPS-induced increase in MPO in the ileum but had no effect on MPO in the lungs or kidneys. This would suggest that the underlying mechanism by which neutrophils accumulate in the ileum, and more generally in the gastrointestinal compartment, is different from other vascular beds. ANX-1 had no effect on the LPS-induced increase in MPO activity in any of the tissues studied. Thus, from these data, ANX-1 appears to reduce leucocyte adhesion and emigration induced by a short-term (2 h), but not a longer (6 h) exposure to LPS.

Animals↗
PubMed2001

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action.

Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.

Animals↗
PubMed2001

Natural and synthetic agonists of the melanocortin receptor type 3 possess anti-inflammatory properties.

The effects of the natural and synthetic ligands for the melanocortin receptor type 3 (MC3-R) have been evaluated in a murine model of experimental gout. Systemic treatment of mice with gamma2-melanocyte-stimulating hormone (gamma2-MSH) and the synthetic agonist MTII inhibited accumulation of KC, interleukin-1 beta (IL-1beta), and PMN elicited by urate crystals in the peritoneal cavity. In vitro, macrophage (Mø) activation, determined as release of KC and IL-1beta, was inhibited by gamma2-MSH and MTII. The mixed MC3/4-R antagonist SHU9119 prevented the inhibitory actions of gamma2-MSH and MTII in vitro and in vivo, whereas the selective MC4-R antagonist HS024 was without effect. Western blotting also showed the presence of MC3-R protein on murine peritoneal Mø. Furthermore, agonism at the MC3-R evoked accumulation of cAMP within the Mø, which was inhibited by SHU9119. Thus, naturally occurring melanocortins, as well as the synthetic long-acting compound MTII, activate MC3-R on peritoneal Mø to inhibit the experimental inflammatory response.

Animals↗
PubMed2001

Cell localization of the anti-inflammatory protein annexin 1 during experimental inflammatory response.

The localization of the glucocorticoid-inducible protein annexin 1 (ANX-1) in leukocytes during the process of experimental inflammation has been studied using immunocytochemistry. ANX- 1 immunoreactivity was detected in extravasated neutrophils and eosinophils as well as in resident tissue mast cells. Following injection of carrageenin, the mesenteric tissue was highly inflamed with large presence of leukocytes (predominantly neutrophils with a small percentage of eosinophils) adherent to post-capillary venules and extravasated in the perivascular tissue. ANX-1 immunoreactivity was detected in the cytosol of neutrophils and eosinophils mainly associated with granules and/or vesicles. A good degree of localization in the endosomes was observed in the neutrophils. In both cell types, some ANX-1 immunoreactivity in the nucleus and in the plasma membrane was also detected. Resident mast cells were also activated. Mast cells were positive for ANX-1, without apparent changes in protein content in relation to their activation status. Degranulated mast cells still presented ANX-1 associated with the granule matrix. In conclusion, this study demonstrated the presence of ANX-1 in leukocytes that play a central role in the host inflammatory response. These are the extravasating polymorphonuclear cells, or the resident mast cells. These data provide morphological support to the notion that endogenous and exogenous ANX-1 are able to modulate the reactivity of these cell types, and more generally, of the experimental inflammatory reaction.

Animals↗
PubMed2001

Arthritic diseases: melanocortin type 3 receptor agonists as potential therapeutics.

Gouty arthritis is currently treated with drugs that have an array of side effects. Therefore, identification of novel endogenous targets for drug development may have beneficial properties ACTH4-10, a heptapeptide fragment derived from the hormone adrenocorticotrophin (ACTH) modulates the inflammatory response in a corticosterone-independent manner, via agonism at melanocortin type 3 receptors (MC3-R) expressed on peritoneal macrophages. MC3-R agonists inhibit cytokine formation and subsequent neutrophil migration, while antagonists abrogate these effects. Together, these data highlight MC3-R as a potential therapeutic target and suggest that small molecule agonists directed at MC3-R with more specific actions, may be potentially novel therapeutics for treating this pathology.

Animals↗
PubMed2000

Modulation of the effects of osteoprotegerin (OPG) ligand in a human leukemic cell line by OPG and calcitonin.

The discovery of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), and RANK has elucidated the mechanism by which osteoblasts and stromal cells regulate osteoclastic differentiation and function and mediate the effects exerted by other hormones and cytokines. We have investigated the effects of these novel cytokines on the preosteoclastic cell line FLG 29.1. We show that OPGL alone and in combination with macrophage colony-stimulating factor (CSF-1) dramatically reduced replication and increased tartrate-resistant acid phosphatase activity. However, although FLG29.1 cells appear to adhere to the bone surface, they are not able to form resorption lacunae. OPG and calcitonin completely abolished the differentiation induced by OPGL. RANK was detectable in FLG 29.1 and the number of positive cells was increased by OPGL/CSF-1 treatment while reduced by calcitonin. We propose that calcitonin could interact with the OPG/OPGL, and its effects on RANK may explain in part the action of this hormone in suppressing bone resorption.

Acid Phosphatase↗
PubMed2000

Association between kinin B(1) receptor expression and leukocyte trafficking across mouse mesenteric postcapillary venules.

Using intravital microscopy, we examined the role played by B(1) receptors in leukocyte trafficking across mouse mesenteric postcapillary venules in vivo. B(1) receptor blockade attenuated interleukin (IL)-1beta-induced (5 ng intraperitoneally, 2 h) leukocyte-endothelial cell interactions and leukocyte emigration ( approximately 50% reduction). The B(1) receptor agonist des-Arg(9)bradykinin (DABK), although inactive in saline- or IL-8-treated mice, caused marked neutrophil rolling, adhesion, and emigration 24 h after challenge with IL-1beta (when the cellular response to IL-1beta had subsided). Reverse transcriptase polymerase chain reaction and Western blot revealed a temporal association between the DABK-induced response and upregulation of mesenteric B(1) receptor mRNA and de novo protein expression after IL-1beta treatment. DABK-induced leukocyte trafficking was antagonized by the B(1) receptor antagonist des-arg(10)HOE 140 but not by the B(2) receptor antagonist HOE 140. Similarly, DABK effects were maintained in B(2) receptor knockout mice. The DABK-induced responses involved the release of neuropeptides from C fibers, as capsaicin treatment inhibited the responses. Treatment with the neurokinin (NK)(1) and NK(3) receptor antagonists attenuated the responses, whereas NK(2), calcitonin gene-related peptide, or platelet-activating factor receptor antagonists had no effect. Substance P caused leukocyte recruitment that, similar to DABK, was inhibited by NK(1) and NK(3) receptor blockade. Mast cell depletion using compound 48/80 reduced DABK-induced leukocyte trafficking, and DABK treatment was shown histologically to induce mast cell degranulation. DABK-induced trafficking was inhibited by histamine H(1) receptor blockade. Our findings provide clear evidence that B(1) receptors play an important role in the mediation of leukocyte-endothelial cell interactions in postcapillary venules, leading to leukocyte recruitment during an inflammatory response. This involves activation of C fibers and mast cells, release of substance P and histamine, and stimulation of NK(1), NK(3), and H(1) receptors.

Animals↗
PubMed2000

Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I).

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.

Annexin A1↗
PubMed2000

Annexin I is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis.

Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.

Annexin A1↗
PubMed2000

Kinin B(1) receptors and the cardiovascular system: regulation of expression and function.

Kinins are important peptide mediators of a diverse range of physiological and pathological functions of the cardiovascular system. The kinin peptides exert their effects by selective activation of two distinct G-protein coupled receptors termed B(1) and B(2). The principal kinin peptides involved in the acute regulation of cardiovascular function during normal physiology are bradykinin (BK) and Lys-BK which produce their effects via activation of B(2) receptors. The B(1) receptor is activated by the des-Arg(9)kinin metabolites namely des-Arg(9)BK and Lys-des-Arg(9)BK, the synthesis of which are increased during inflammation. The B(1) receptor, which is not constitutively expressed, is induced in various pathologies relating to inflammation. Recent investigations into the molecular mechanisms of B(1) receptor induction and their distribution and function in the cardiovascular system have shown that following an inflammatory stimulus the B(1) receptor is induced and may play an important role in modulation of cardiovascular function. This review summarises recent studies on B(1) receptor expression and function in the cardiovascular system and discusses the role of these receptors in regulation of circulatory homeostasis and their potential as therapeutic targets.

Animals↗
PubMed2000

An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: modulation by inflammation and dexamethasone.

The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of clusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.

Animals↗
PubMed2000

21-NO-prednisolone is a novel nitric oxide-releasing derivative of prednisolone with enhanced anti-inflammatory properties.

1. Anti-inflammatory effects of a novel derivative of the glucocorticoid prednisolone were investigated. NCX-1015 (prednisolone 21-[(4'-nitrooxymethyl)benzoate]) incubation in human platelet-rich plasma produced a time (0 - 60 min) and concentration (3 - 300 microM) dependent release of nitrite, that was mirrored by accumulation of cyclic guanosine monophosphate in the human platelets. Intraperitoneal injection of NCX-1015 to mice (up to 27.7 micromol kg(-1)) produced nitrite accumulation in the peritoneal cavity maximal at 60 min. 2. NCX-1015 dose-dependently induced the steroid sensitive cell surface marker CD163 in human peripheral blood mononuclear cells (PBMCs). NCX-1015 was more potent than prednisolone in inducing CD163. Similarly, lipopolysaccharide induced interleukin-1 beta release from these cells was inhibited by NCX-1015 with higher potency than prednisolone. 3. In the zymosan peritonitis model, NCX-1015 was more active than prednisolone in suppressing neutrophil extravasation (ED(50) of 5.5 and 25.8 micromol kg(-1), respectively), nitrite accumulation (ED(50) of 1.38 and 22.2 micromol kg(-1), respectively) and release of the chemokine KC (ED(50) of 5.5 and 27.7 micromol kg(-1), respectively) as determined at the 4 h time-point. No differences were measured for the levels of interleukin-1 beta or prostaglandin E(2). NCX-1015 administered orally was also found to be equally active. Co-administration of the nitric oxide donors NOC-18 ((z)-1-[(2-aminoethyl)-N-(2-aminoethyl)amino] diazen-1-ium-1, 2-diolate; 7.9 micromol kg(-1)) or sodium nitroprusside (13.8 micromol kg(-1)) with prednisolone resulted in an additive anti-migratory action. 4. In a chronic model of granulomatous tissue inflammation, administration of NCX-1015 (13.9 micromol kg(-1)) from day 1 (i.e. after induction of inflammation) was more effective than prednisolone in reducing the granuloma dry weight, and this was associated to a lower anti-angiogenic effect. 5. In conclusion we show that NCX-1015 is more potent than prednisolone in controlling several, though not all, parameters of acute and chronic inflammation, and propose that this effect may be due to a co-operation between the steroid moiety and nitric oxide or related species released in biological fluids. Whereas this aspect needs to be further clarified, we propose NCX-1015 as the first member of a novel class of anti-inflammatory compounds, the nitro-steroids.

Administration, Oral↗
PubMed2000

Lipocortin 1 reduces myocardial ischemia-reperfusion injury by affecting local leukocyte recruitment.

We assessed here the effect of the glucocorticoid-regulated protein lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25-min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with approximately 50% inhibition at the highest dose tested of 50 microg per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose-dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor a, and macrophage inflammatory protein-1a. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 microg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte-dependent myocardial damage associated with an ischemia-reperfusion procedure.

Animals↗