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Living in isolation is considered an emerging societal problem that negatively affects the physical wellbeing of its sufferers in ways that we are just starting to appreciate. This study investigates the immunomodulatory effects of social isolation in mice, utilising a two-week program of sole cage occupancy followed by the testing of immune-inflammatory resilience to bacterial sepsis. Our results revealed that mice housed in social isolation showed an increased ability to clear bacterial infection compared to control socially housed animals. These effects were associated with specific changes in whole blood gene expression profile and an increased production of classical pro-inflammatory cytokines. Interestingly, equipping socially isolated mice with artificial nests as a substitute for their natural huddling behaviour reversed the increased resistance to bacterial sepsis. Together these results suggest that the control of body temperature through social housing and huddling behaviour are important factors in the regulation of the host immune response to infection in mice and might provide another example of the many ways by which living conditions influence immunity.

The termination of inflammation is governed by endogenous molecules collectively referred to as 'mediators of resolution' of inflammation. There is now strong evidence to suggest that failed resolution may underpin autoimmune and inflammatory diseases and could thus be targeted to decrease inflammation. There are many molecules that have been described as mediators of resolution, and new players are still being continuously discovered. To support the emerging field of 'resolution pharmacology', here we discuss the scientific strategies required to qualify a molecule as a resolution mediator. Systematic definition of the players of resolution, their receptors, and downstream mechanisms remains a necessary knowledge to move the field forward and suggest new targets for the development of novel therapies to treat inflammatory diseases.

Microvesicles (MVs) are emerging as a novel means to enact cell-to-cell communication in inflammation. Here, we aimed to ascertain the ability of neutrophil-derived MVs to modulate target cell behaviour, the focus being the macrophage. MVs were generated in response to tumour necrosis factor-α, from healthy control neutrophils or those from rheumatoid arthritis patients. MVs were used to stimulate human monocyte-derived macrophages in vitro, or administered intra-articularly in the K/BxN mouse model of arthritis. A macrophage/fibroblast-like synoviocyte co-culture system was used to study the effects of vesicles on the crosstalk between these cells. We demonstrate a direct role for phosphatidylserine and annexin-A1 exposed by the MVs to counteract classical activation of the macrophages, and promote the release of transforming growth factor-β, respectively. Classically-activated macrophages exposed to neutrophil MVs no longer activated fibroblast-like synoviocytes in subsequent co-culture settings. Finally, intra-articular administration of neutrophil MVs from rheumatoid arthritis patients in arthritic mice affected the phenotype of joint macrophages. Altogether these data, with the identification of specific MV determinants, open new opportunities to modulate on-going inflammation in the synovia – mainly by affecting macrophage polarization and potentially also fibroblast-like synoviocytes - through the delivery of autologous or heterologous MVs produced from neutrophils., Highlights • Neutrophil microvesicles restrict the inflammatory activation of macrophages by presenting phosphatidylserine to Mer. • Annexin A1 expressed on neutrophil microvesicles induces macrophage release of TGFβ by activating FPR2. • Neutrophil microvesicles restrict the ability of macrophages to activate fibroblast-like synoviocytes. All cells release small, spherical parcels of information, called vesicles, that they use to send signals between each other. One immune cell type, the neutrophil, has been shown to release vesicles which prevents other immune cells from becoming hyperactive: herein we focus on rheumatoid arthritis. This paper demonstrates that vesicles released from neutrophils are able to prevent another immune cell type, the macrophage, from becoming activated. The importance of this is that this mechanism is occurring naturally, and if we can understand it, we may be able to use our own bodies' mechanisms to control chronic inflammation.

Chagas disease is an important disease of the heart. Lipoxins have important regulatory functions in host immune response (IR). Herein, we examined whether the receptor for lipoxin A4, the formyl peptide receptor (FPR) 2, had an effect on Trypanosoma cruzi infection. In vitro, FPR2 deficiency or inhibition improved the activity of macrophages against T. cruzi. In vivo, during the acute phase, the absence of FPR2 reduced parasitemia and increased type 2 macrophages, type 2 neutrophils, and IL-10-producing dendritic cells. Moreover, the acquired IR was characterized by greater proportions of Th1/Th2/Treg, and IFNγ-producing CD8+T cells, and reductions in Th17 and IL-17-producing CD8+T cells. However, during the chronic phase, FPR2 deficient mice presented and increased inflammatory profile regarding innate and acquired IR cells (Th1/IFN-γ-producing CD8+T cells). Notably, FPR2 deficiency resulted in increased myocarditis and impaired heart function. Collectively, our data suggested that FPR2 is important for the orchestration of IR and prevention of severe T. cruzi-induced disease.

Summary The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation., Graphical abstract, Highlights • Inflamed venular ECs exhibit an autophagic response that localizes to EC contacts • EC ATG5 deficiency promotes excessive and faster neutrophil TEM • Ablation of EC autophagy increases cell surface expression of adhesion molecules • Non-canonical autophagy operates in inflamed ECs and controls neutrophil migration, Transendothelial cell migration (TEM) is a vital step in neutrophil infiltration of tissues, but the molecular basis of its cessation is unclear. Reglero-Real et al. show that in inflamed tissues, endothelial cell autophagy remodels junctional architecture and acts as a negative regulator of neutrophil TEM.

The discovery and identification of omega-3 fatty acid derived specialized pro-resolving mediators (SPM) provides a molecular mechanism for the beneficial effects of fish oil supplementation in patients suffering from arthritis. Here we review the plethora of bioactions of SPM in the context of joint diseases, focusing on both cellular targets and molecular mechanisms. Whenever possible, a parallel to clinical and preclinical data produced with fish oil supplementation is made to strengthen the mechanistic link between omega-3 fatty acids and SPM biosynthesis. SPM can modulate the reactivity of many cells that are pivotal to the development and/or maintenance of joint disease. Whereas work has so far focused on the actions of SPM on immune cells and therefore, within this context, macrophages, neutrophils, mast cells and T cells, we reason that more work needs to focus on the effects that these bioactive lipid mediators may have on the structural cell component of the joint, this encompassing synovial fibroblasts, chondrocytes, osteoclasts and osteoblasts. Full definition of the properties that SPM may exert on these cells can help in unveiling their ability to promote tissue restoration and regeneration, a prerequisite to repair joint damage, and as such promote the development of innovative therapeutic strategies based on the science of SPM and resolution.

In pancreatic cancer (PC) progression the protein Annexin A1 (ANXA1) has been described as oncogenic factor. Thus, the need to inhibit its action, mainly the extracellular form, has become an appealing cue for the anti-cancer research. Heparan sulfate (HS) is a glycosaminoglycan of the extracellular matrix known to bind several molecules, as growth factors and cytokines, generating a kind of reservoir in the extracellular environment. Here, we started our study by showing the physical calcium-dependent interaction between HS and ANXA1 as both full-length protein and N-terminal portion, Ac2-26 by biophysical techniques. HS is able to inhibit the migration/invasion process of human PC MIA PaCa-2 cells and partially revert their mesenchymal phenotype as reported through the expression of specific protein markers and the growth in colonies and in 3D-spheroids. Furthermore, both on MIA PaCa-2 and PANC-1 cells, HS blocks the effects of Ac2-26, which enhances the aggressive behavior of PC cells if added alone. These effects appear evident also on endothelial cells whose activation is promoted by Ac2-26 but not in presence of HS. Thus, the interference of the interaction ANXA1-HS on angiogenesis strongly emerges. Moreover, once sequestered by HS, ANXA1 is not more able to bind the formil-peptide receptors (FPRs) preventing the increase of calcium mobilization, peculiar for cell motility. These findings introduce a new important tale in the knowledge about the inhibition of the ANXA1 action in PC development. Further information will be useful to highlight the interaction of HS with the protein, focusing on the characterization of the glycosaminoglycan and on in vivo assays.

Aims Cyclooxygenase (COX)-inhibiting nitric oxide donors (CINODs) are a new class of drugs that structurally combine a COX inhibitor with a nitric oxide (NO) donating moiety. This combination reduces potential toxicity of the non-steroidal anti-inflammatory drugs (NSAIDs) whilst maintaining the analgesic and anti-inflammatory effects. The present study was undertaken to investigate the anti-inflammatory effects of NCX 429, a naproxen-based CINOD, and to assess the additional properties of NO donation beyond those related to naproxen. Main methods We evaluated the in vitro effects of NCX 429 on oxy-radical production, phagocytosis, cytokine release, MMP-9, PPARγ expression and NF-κB activation in human monocytes/MDM and compared to naproxen. Moreover, we compared the in vivo efficacy of NCX 429 and naproxen in a murine model of peritonitis. Key findings In all the experiments performed in vitro, NCX 429 reduced the inflammatory responses with equal or higher efficacy compared to naproxen. Moreover, in in vivo experiments, NCX 429, at the lowest dose tested, was able to significantly inhibit cell influx in response to IL-1β administration although naproxen was found to be more potent than NCX 429 at reducing PGE 2 in inflammatory exudates. Significance These results demonstrate that both in vitro and in vivo – in a murine model of peritonitis – NCX 429 elicits significant anti-inflammatory activity, beyond the simple COX inhibition or pure NO release. Therefore, NO donation along with COX inhibition may represent a strategy for investigating inflammatory diseases in which pain and function are not fully resolved by analgesics/anti-inflammatory drugs.

Introduction Calcitonin (CT) has recently been shown to display chondroprotective effects. Here, we investigate the putative mechanisms by which CT delivers these actions. Methods Immortalized C-28/I2 cells or primary adult human articular chondrocytes (AHAC) were cultured in high-density micromasses to investigate: (i) CT anabolic effects using qPCR and immuhistochemistry analysis; (ii) CT anti-apoptotic effects using quantitation of Bax/Bcl gene products ratio, TUNEL assay and caspase-3 expression; (iii) CT effects on CREB, COL2A1 and NFAT transcription factors. Results CT (10 −10 –10 −8 nM) induced significant up-regulation of cartilage phenotypic markers ( SOX9, COL2A1 and ACAN ), with down-regulation of catabolic ( MMP1 and MMP13 and ADAMTS5 ) gene products both in resting and inflammatory conditions. This was mirrored by an augmented production of type II collagen and accumulation of glycosaminoglycan- and proteoglycan-rich extracellular matrix in vitro . Mechanistic analyses revealed only partial involvement of cyclic AMP formation in these effects of CT. Congruently, using reporter assays for specific transcription factors, there was no indication for CREB activation, whereas the COL2A1 promoter was genuinely and directly activated by cell exposure to CT. Phenotypically, these mechanisms supported the ability of CT, whilst inactive on its own, to counteract the pro-apoptotic effects of IL-1β, demonstrated by TUNEL-positive staining of chondrocytes and ratio of BAX / BCL genes products. Conclusion These data may provide a novel lead for the development of CT-based chondroprotective strategies that rely on the engagement of mechanisms that lead to augmented chondrocyte anabolism and inhibited chondrocyte apoptosis.

There is a paucity of molecules that progress through the drug development pipeline, making the drug discovery process expensive and frustrating. Innovative approaches to drug development are therefore required to maximise opportunities. Strategies such as the Connectivity Map (CMap), which compares >7000 gene expression signatures generated from more than 1000 drugs, can produce associations between currently unrelated therapeutics, unveiling new mechanisms of action and favouring drug repositioning. Here, we discuss these opportunities that could aid the drug development process and propose rigorous publication of 'omics data with open access and data sharing. We, pharmacologists of the third millennium, must aim towards maximising knowledge in an unbiased and cost-effective manner, to deliver new drugs for the global benefit of patients.

Omega-3 polyunsaturated fatty acids (PUFAs) are known to alleviate joint stiffness and pain in rheumatoid arthritis patients. However, the mechanisms by which omega-3s exert their beneficial effects has not been fully explored. Herein we discuss a novel class of bioactive lipid mediators, which are enzymatically biosynthesized in vivo from omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), termed resolvins. These lipid mediators exert anti-inflammatory and pro-resolving properties and are log-orders more potent than their precursors. We also highlight that formation of pro-resolving mediators can be enhanced by widely used anti-inflammatory and cardioprotective drugs (aspirin and statins) via the modification of cyclooxygenase-2 enzymatic activity. These bioactive pro-resolving lipid mediators provide further rationale for the beneficial effects of dietary supplementation with fish oils, and offer new avenues for developing therapeutics for inflammatory conditions such as rheumatoid arthritis.

Salivary cortisol is commonly used as a clinical biomarker of endocrine status and also as a marker of psychosocial stress. Annexin-A1 (AnxA1) is an anti-inflammatory protein whose expression is modulated by glucocorticoids. Our principal objectives were to (i) detect the presence of and (ii) measure AnxA1 protein in whole human saliva and to (iii) investigate whether salivary cortisol and AnxA1 are correlated in healthy humans. A total of 37 healthy participants (male and female) were used in the study. Saliva was collected using salivette tubes. Salivary cortisol and AnxA1 protein were sampled at between 3 and 6 time points over 24h and measured for cortisol and AnxA1 protein using specific ELISA's. The presence of salivary AnxA1 protein was confirmed by Western blotting. AnxA1 protein is detectable in whole human saliva, as detected by Western blot analysis and ELISA. A diurnal rhythm was evident in both salivary cortisol (P0.01) and AnxA1 (P0.01) and was defined as a significant difference in time 0 (waking) samples compared to 'bed' (2300 h) samples. AnxA1 protein did not exhibit an awakening response (P0.05), whereas salivary cortisol was significantly elevated between time 0 and 30 min post waking (P0.001). AnxA1 protein correlates positively with salivary cortisol, indicating that cortisol is most likely a regulator of AnxA1 in human saliva.

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.

Background: FPR2/ALX is activated by many ligands, including annexin A1 (AnxA1), which activates resolution circuits in inflammation. Results: Cells transfected with FPR2/ALX and clones with domains swapped with FPR1 afforded identification of N-terminal and extracellular loop II domains as transducers of AnxA1 signaling. Conclusion: We identified AnxA1 distinct domains of FPR2/ALX and unveiled potential specific signaling. Significance: FPR2/ALX domain identification permits development of anti-inflammatory AnxA1 mimetics., Understanding how proresolving agonists selectively activate FPR2/ALX is a crucial step in the clarification of proresolution molecular networks that can be harnessed for the design of novel therapeutics for inflammatory disease. FPR2/ALX, a G protein-coupled receptor belonging to the formyl peptide receptor (FPR) family, conveys the biological functions of a variety of ligands, including the proresolution mediators annexin A1 (AnxA1) and lipoxin A4, as well as the activating and proinflammatory protein serum amyloid A. FPR2/ALX is the focus of intense screening for novel anti-inflammatory therapeutics, and the small molecule compound 43 was identified as a receptor ligand. Here, we used chimeric FPR1 and FPR2/ALX clones (stably transfected in HEK293 cells) to identify the N-terminal region and extracellular loop II as the FPR2/ALX domain required for AnxA1-mediated signaling. Genomic responses were also assessed with domain-specific effects emerging, so the N-terminal region is required for AnxA1 induction of JAG1 and JAM3, whereas it is dispensable for modulation of SGPP2. By comparison, serum amyloid A non-genomic responses were reliant on extracellular loops I and II, whereas the small molecule compound 43 activated extracellular loop I with downstream signaling dependent on transmembrane region II. In desensitization experiments, the N-terminal region was dispensable for AnxA1-induced FPR2/ALX down-regulation in both the homologous and heterologous desensitization modes.

Synthetic and natural melanocortin (MC) peptides afford inhibitory properties in inflammation and tissue injury, but characterization of receptor involvement is still elusive. We used the agonist AP214 to test MC-dependent anti-inflammatory effects. In zymosan peritonitis, treatment of mice with AP214 (400 to 800 μg/kg) inhibited cell infiltration, an effect retained in MC receptor type 1, or MC(1), mutant mice but lost in MC(3) null mice. In vitro, cytokine release from zymosan-stimulated macrophages was affected by AP214, with approximately 80%, 30%, and 40% reduction in IL-1β, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1β release was retained in MC(1) mutant cells but was lost in MC(3) null cells. Furthermore, AP214 augmented uptake of zymosan particles and human apoptotic neutrophils by wild-type macrophages: this proresolving property was lost in MC(3) null macrophages. AP214 displayed its pro-efferocytotic effect also in vivo. Finally, in a model of inflammatory arthritis, AP214 evoked significant reductions in the clinical score. These results indicate that AP214 elicits anti-inflammatory responses, with a preferential effect on IL-1β release. Furthermore, we describe for the first time a positive modulation of an MC agonist on the process of efferocytosis. In all cases, endogenous MC(3) is the receptor that mediates these novel properties of AP214. These findings might clarify the tissue-protective properties of AP214 in clinical settings and may open further development for novel MC agonists.

Summary. Background: Although inflammation and thrombosis are now recognized to be interdependent processes that activate and perpetuate each other, the signaling molecules that link these two processes remain poorly understood. Objectives: The objective of this study was to assess the contribution of the CD40/CD40L signaling system to the enhanced microvascular thrombosis that accompanies two distinct experimental models of inflammation, that is, endotoxemia (lipopolysaccharide [LPS]) and dextran sodium sulfate (DSS)-induced colitis. Methods: Thrombosis was induced in cerebral (LPS model) and cremaster muscle (DSS model) arterioles and venules of wild-type (WT) mice and mice deficient in either CD40 (CD40−/−) or CD40L (CD40L−/−), using the light/dye (photoactivation) method. Results and conclusions: A comparison of thrombus formation between WT and mutant mice revealed a role for CD40 and/or CD40L in the inflammation-enhanced thrombosis responses in both of the cerebral and muscle vasculatures. However, the relative contributions of CD40 and its ligand to thrombus formation differed between vascular beds (brain vs. muscle) and vessel types (arterioles vs. venules). The protective effect of CD40L deficiency in cerebral arterioles exposed to LPS was significantly blunted by administration of soluble CD40L. These findings implicate CD40 and its ligand in the enhanced thrombus formation that is associated with acute and chronic inflammation.

The role of endogenous galectin-1 (Gal-1) in acute inflammation has been poorly investigated. We therefore performed the carrageenan-induced paw edema model in wild-type and Gal-1−/− mice. On subplantar injection of carrageenan, Gal-1−/− mice displayed a similar first phase of edema (≤24 hours) to wild-type mice; however, a much less pronounced second phase (48 to 96 hours) was evident in this genotype. This reduced inflammation was associated with lower paw expression of inflammatory genes and cell infiltrates. Analysis of galectin protein and mRNA expression revealed high expression of Gal-1 in wild-type paws during resolution (≥48 hours), with some expression of galectin-9 (Gal-9). Administration of stable Gal-1 to wild-type mice completely ablated the first phase of edema but was ineffective when administered therapeutically at the 24-hour time point. Conversely, Gal-9 administration did not alter the first phase of edema but significantly reduced the second phase when administered therapeutically. This suggests anti-inflammatory actions for both proteins in this model albeit at different phases of the inflammatory response. Collectively, these data indicate that the absence of endogenous Gal-1 results in an abrogated response during the second phase of the edema reaction.

In guinea-pig ileum (GPI), the chemotactic peptide N-formyl-Met-Leu-Phe-OH (fMLF) possesses spasmogenic properties through the activation of formyl peptide receptors (FPRs). Despite this, the mediators involved remain to be elucidated. fMLF (1nM-1μM) induced a dose-dependent contraction of GPI (EC(50)=24nM), that is blocked by pre-treatment with the FPRs antagonist Boc(2). The pre-treatment with tetrodotoxin (TTX) atropine or with SR140333 reduced the fMLF-induced contraction, whereas with hexamethonium, MEN10627, SB222200, mepyramine, cimetidine, thioperamide or methysergide did not produce any effect. With DuP697 pre-treatment, but not with piroxicam, reduced the fMLF-induced contraction. After stimulation with 24nM fMLF, a strong increase in the PGE(2) levels was observed. Finally, the concomitant blocking of the NK(1) receptor, the muscarinic receptors and COX-2 abolished the GPI contractions induced by fMLF. fMLF induced a concentration-dependent contraction of guinea-pig jejunum (EC(50)=11nM), proximal colon (EC(50)=3.5nM) and distal colon (EC(50)=2.2nM), with a time-course similar to that observed in GPI. In these preparations as well, the co-administration of atropine, SR140333 and DuP697 abolished the contractions induced by fMLF. Intraperitoneal injection of fMLF (0.1 or 1μmol/kg) enhanced the gastrointestinal motility in mice, abolished by the co-administration of atropine, SR140333 and DuP697. In conclusion, we showed that fMLF exerts spasmogenic actions on guinea-pig intestine both in vitro and in vivo through the release of acetylcholine and substance P from myenteric motorneurons and through prostanoids, probably from the inflammatory cells of the enteric immune system.

Recent studies have associated the dysregulated expression of Annexin-A1/Formyl peptide receptor 2 (FPR2/ALX) system with the development of autoimmune diseases. In this study we systematically scanned human leukocyte subsets for the presence of this pathway aiming to provide a roadmap that will help investigators to explore possible links between the development of immune related disorders and the expression of this system. Our results show that neutrophils, monocytes and NK cells express higher levels of both AnxA1 and FPR2/ALX compared to T or B cells. Further analysis of specific T cell subsets revealed higher levels in activated CD25(+) and memory CD45RO CD4 T cells compared to resting CD25(-) or naïve CD45RA CD4 T cells. Together the results expand our knowledge of the AnxA1-FPR2/ALX system in immune cells and provide new avenues for investigation into the functions of this signalling pathway in systems other than that classically described for neutrophils.

Mast cells are involved in immune disorders so that many of the proinflammatory and tissue destructive mediators produced by these cells have been implicated in the pathogenesis of rheumatoid arthritis. This scenario prompted us to investigate the correlation between mast cell degranulation and neutrophil influx within the digits and knees joints of arthritic mice assessing what could be the functional role(s) of joint mast cells in the response to collagen immunization. DBA/1J mice were submitted to collagen-induced arthritis and disease was assessed on day 21, 32 and 42 post-immunization. Pharmacological treatment with the glucocorticoid prednisolone, commonly used in the clinic, and nedocromil, a mast cell stabilizer, was performed from day 21 to 30. Arthritis develop after immunization, gradually increased up to day 42. Neutrophil infiltration peaked on day 32 and 21, in the digits and knees, respectively, showing an unequal pattern of recruitment between these tissues. This difference emerged for mast cells: they peaked in the digits on day 21, but a higher degree of degranulation could be measured in the knee joints. Uneven modulation of arthritis occurred after treatment of mice with prednisolone or nedocromil. Neutrophils migration to the tissue was reduced after both therapies, but only prednisolone augmented mast cell migration to the joints. Nedocromil exerted inhibitory properties both on mast cell proliferation and migration, more effectively on the digit joints. Thus, collagen induced an inflammatory process characterized by tissue mast cells activation and degranulation, suggesting a potential driving force in propagating inflammatory circuits yielding recruitment of neutrophils. However, the different degree of affected joint involvement suggests a time-related implication of digits and knees during collagen-induced arthritis development. These results provide evidence for local alterations whereby mast cells contribute to the initiation of inflammatory arthritis and may be targeted in intervention strategies.

The need for novel anti-inflammatory drugs justifies the search for innovative targets that could satisfy this goal. For quite some time now, we have proposed the study of endogenous anti-inflammation as a distinctive approach to the discovery of new drugs. This approach requires development of new compounds that activate specific receptor targets to downregulate the cellular and tissue pathways operative in the host during inflammation. Here we dwell on a family of G-protein coupled receptors (GPCR) termed FPRs, acronym for formyl-peptide receptors. With three and seven members in man and mouse, respectively, these receptors harness many biological functions, spanning odour perception and hair growth, to the control of multiple facets (pain; cell migration; oxidative burst; xenobiotic engulfment) of the inflammatory reaction. We focus on FPR biology with particular attention to molecules able to produce pharmacological effects by interacting with these GPCRs, describing endogenous agonists of FPRs and, more relevantly, the current development of synthetic agonists. Besides being potential leads for the development of the anti-inflammatory therapeutics of the future, these compounds could also help clarify the properties and roles that each FPR might play in the complex network of pathways that is inflammation. We conclude that FPR2 agonists could be valid warhorses for defining a novel philosophy for anti-inflammatory drug discovery programmes: mimicking - with new compounds - the way our body disposes of inflammation could be a viable approach to regulate aberrant inflammatory responses as in the case of several chronic rheumatic and cardiovascular pathologies.

Cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, is characterized by chronic bacterial infections and inflammation in the lung. Having previously shown that deletion of CFTR is associated with lower expression of the endogenous anti-inflammatory protein Annexin A1 (AnxA1), we investigated further this possible functional connection using a validated CFTR inhibitor. Treatment of mice with the CFTR inhibitor-172 (CFTR(172)) augmented the acute peritonitis promoted by zymosan, an effect associated with lower AnxA1 levels in peritoneal cells. Similar results were obtained with another, chemically distinct, CFTR inhibitor. The pro-inflammatory effect of CFTR(172) was lost in AnxA1(-/-), as well as CFTR(-/-) mice. Importantly, administration of hrAnxA1 and its peptido-mimetic to CFTR(-/-) animals or to animals treated with CFTR(172) corrected the exaggerated leukocyte migration seen in these animals. In vitro assays with human Polymorphonuclear leukocyte (PMN) demonstrated that CFTR(172) reduced cell-associated AnxA1 by promoting release of the protein in microparticles. We propose that the reduced impact of the counterregulatory properties of AnxA1 in CF cells contributes to the inflammatory phenotype characteristic of this disease. Thus, these findings provide an important insight into the mechanism underlying the inflammatory disease associated with CFTR inhibition while, at the same time, providing a novel pharmacological target for controlling the inflammatory phenotype of CF.

Interleukin (IL)17 is a novel cytokine that has been suggested to play a key role in sustaining chronic inflammation in autoimmune diseases. Since its discovery, much attention has been given to mediators and factors responsible for the development of IL-17-producing cells while very few studies have investigated the inflammatory properties of this cytokine. Here we aimed to characterize the inflammatory properties of IL-17 and to establish if this cytokine per se can initiate an inflammatory reaction or if its main role is to contribute to the exacerbation of ongoing inflammation. To this aim we used two different mouse models of inflammation: the paw oedema and the airpouch. Interestingly, injection of IL-17 in the hind paw did not cause oedema while administration in the pre-inflamed tissues of 6-day-old air pouches induced a long lasting inflammatory reaction that was sustained between 4 and 24h post-injection. Phenotypic analysis of cellular infiltrates demonstrated selective PMN recruitment in exudates and pouch lining tissues. This event was accompanied by induction of a distinct set of pro-inflammatory cytokines including IL-1beta, IL-6, TNF-alpha and chemokines KC and MCP-1. Co-administration of a neutralizing anti-KC antibody with IL-17 significantly reduced its inflammatory response suggesting a key role for this chemokine in mediating the inflammatory effects of IL-17. In conclusion these results demonstrate that IL-17 does not initiate an inflammatory reaction while, if injected in pre-inflamed tissues, is able to further amplify biochemical and cellular events characteristic of the early stages of the inflammatory reaction.

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.

Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery.

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01–1μM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.

We tested here the hypothesis that calcitonin and glucocorticoids, known to modulate bone metabolism, could have opposite actions on bone cells regulating expression of cytokine receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG). In the U2OS osteosarcoma cell line, calcitonin (10(-11) to 10(-9) mol/L) reduced RANKL and augmented OPG both at the mRNA and protein levels. Cell incubation with prednisolone (10(-8) to 10(-6) mol/L), the glucocorticoid chosen for this study, produced opposite results. These molecular studies prompted more functional analyses whereby osteoclast bone resorptive activity was determined. Calcitonin (10(-10) mol/L) abrogated the stimulating effect of 10 ng/ml RANKL or 10(-9) mol/L prednisolone; similar results were obtained with OPG. Assessment of calcitonin and prednisolone effects in an in vivo model of rheumatoid arthritis revealed partially surprising results. In fact, calcitonin not only preserved bone morphology (as assessed on day 18) in rats subjected to arthritis and treated with prednisolone (0.8 to 4 mg/kg daily from day 13) but also synergized with the steroid to elicit its antiarthritic effects. These results suggest that calcitonin could be used as a novel cotreatment to augment efficacy and reduce side effects associated with the prolonged use of steroids.

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.

The inflammatory response is a life-saving protective process mounted by the body to overcome pathogen infection and injury; however, in chronic inflammatory pathologies this response can become deregulated. The existence of specialized anti-inflammatory pathways/mediators that operate in the body to down-regulate inflammation have now emerged. Thus, persistence of inflammation leading to pathology could be due to malfunctioning of one or more of these counter-regulatory pathways. Here we focus on one of them, the anti-inflammatory mediator annexin 1, and provide an update on its inhibitory effects upon the leukocyte trafficking process. In particular, recent evidence that receptors of the formyl-peptide family, which includes also the lipoxin A4 receptor, could be the annexin 1 receptor(s) in the context of anti-inflammation might provide new avenues for exploiting this pathway for drug discovery.

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counterregulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages—cell types known to function in ex-perimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS.

Historical data suggested that a soluble protein, since identified as annexin-A1 (Anx-A1) was released from macrophages following glucocorticoid stimulation and could modulate eicosanoid production and other functions of these cells. Here, we review some recent findings using a line of Anx-A1(-/-) mice to explore the impact of Anx-A1 gene deletion on macrophage biology. The absence of Anx-A1 selectively alters phagocytic capacity of rodent resident peritoneal macrophages apparently through changes in surface adhesion molecule expression. Anx-A1 is also apparently important in the tonic down-regulation of other macrophage functions such as COX-2 induction, PGE(2) release and the production of reactive oxygen species.

Recent studies have proposed a functional link between annexin 1 (ANXA1), an endogenous anti-inflammatory mediator, and receptors of the formyl-peptide family. In particular, exogenous and endogenous ANXA1 and its peptidomimetics interact with one member of this family, the formyl-peptide-receptor-like 1. Further analyses of the interactions between ANXA1 and this and other members of this receptor family, and a better characterization of the ANXA1 receptor systems in models of inflammation, might clarify their mechanism of anti-inflammatory effects. This line of research will facilitate the development of ANXA1 mimetics and take advantage of >20 years of biological research into the functions of this glucocorticoid-modulated protein.

Galectin-1 (Gal-1), the prototype of a family of β-galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 μg equivalent to 20 pmol) inhibited interleukin-1β-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and postadherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.

Addition to the prednisolone structure of a chemical moiety (linker+nitric ester) that releases NO species yielded a novel glucocorticoid (nitro-prednisolone or NCX-1015) with enhanced anti-inflammatory activities. Nitro-prednisolone was much more potent than prednisolone and the derivative devoid of the nitric ester in an acute peritonitis model (higher impact on neutrophil migration and soluble mediator generation) as well as in models of chronic inflammation (air-pouch granuloma and collagen II-induced arthritis). In the collagen II-induced arthritis model, NCX-1015 abrogated the plasma levels of a catabolite of cartilage and bone metabolism, indication of a disease modifying action. In an in vitro assay of bone resorption, NCX-1015 did not activate osteoclast activity, whereas prednisolone did. This lack of effect of NCX-1015 was chiefly due to NO. We propose that NCX-1015 is the prototype of a new class of glucocorticoids, the nitro-steroids, endowed with enhanced anti-inflammatory properties and reduced side effects. These and other experimental observations here reviewed may prompt the assessment of the clinical impact of the nitro-steroids on rheumatoid arthritis and inflammatory bowel disease.

The aim of this commentary is to discuss recent data on the role of prostaglandins generated by both constitutive and inducible cyclooxygenases (COXs). According to a popular hypothesis, COX-1 generates 'good' prostaglandins for physiological 'housekeeping' functions like gastrointestinal (GI) mucosal integrity and regulation of renal blood flow, while COX-2 forms the 'bad' prostaglandins responsible for inflammatory symptoms. However, recent data show that the biological functions of prostanoids formed by the two enzymes are much more complex and interrelated than previously appreciated. Experimental evidence indicates that a full inflammatory response is likely sustained by prostanoids generated by both enzymes, and an effective anti-inflammatory effect requires the inhibition of the two enzymes. Similarly, the selective inhibition of either COX-1 or COX-2 does not elicit GI damage, but inhibition of both enzymes is necessary for GI mucosal damage to develop. Prostaglandins generated by both enzymes contribute to normal renal function by regulating the vascular tone and the normal blood flow. The synthesis of endothelial prostacyclin is mainly driven by COX-2, so that the selective COX-2 inhibition may bias vascular prostaglandin synthesis in favour of COX-1-derived thromboxane A(2) in platelets, leading to a prothrombotic outcome. Moreover, prostaglandins formed by COX-2 appear to have a major role in myocardial protection. We propose that the complexity of the situation in the field of COX-derived mediators should be borne in mind when anti-inflammatory therapy is required.

Ischaemia-reperfusion injury underlies many of the most important cardiovascular diseases such as myocardial infarction, thrombotic stroke, embolic vascular occlusions and peripheral vascular insufficiency. Neutrophils feature prominently in this inflammatory component of post-ischaemic injury. Experimental therapies, shown to reduce neutrophil-mediated ischaemia-reperfusion injury include neutrophil depletion, direct inhibitors of neutrophil activators, antibodies against neutrophil adhesion molecules and the endothelial adhesion molecules. However, aside from these approaches, it is increasingly recognised that glucocorticoids are potent inhibitors of neutrophil-mediated injury. The anti-inflammatory actions of glucocorticoid include the activation of classical cytoplasmic receptors leading to changes in gene transcription as well as the induction of regulatory proteins, such as annexin 1. Annexin 1 is a potent inhibitor of neutrophil extravasation in vivo. Administration of the annexin 1 or peptides derived from its N-terminal domain, reduce neutrophil extravasation in models of acute inflammation. In addition, as reviewed by this article, annexin 1 protects against ischaemia-reperfusion in the heart and mesenteric microcirculation, as well as in multiple organ failure associated with splanchnic ischaemia-reperfusion. Such findings would suggest annexin 1 is a novel anti-inflammatory agent with a potential for the treatment of cardiovascular pathologies associated with neutrophil activation and recruitment.

Administration of ovalbumin by aerosol to sensitised rats produced a rapid (15 min) protein exudation in different airway tissues, as determined by Evans blue staining. This was associated with marked mast cell degranulation determined by histological examination, with there being no difference between mucosal and connective tissue mast cells. A 5-day administration regimen with compound 48/80 selectively depleted connective tissue mast cell (positive to berberine staining) without modifying ovalbumin-induced plasma protein extravasation. Treatment of rats with dexamethasone (1 mg/kg, −12 h) or nor-dihydroguaiaretic acid (30 mg/kg i.p., −30 min) significantly reduced ovalbumin-induced protein extravasation and preserved mucosal mast cell morphology. Indomethacin (4 mg/kg i.v., −30 min) exerted no effect on either parameter. In conclusion, we propose the mucosal mast cell as a target cell responsible at least partly for the inhibitory actions of known anti-inflammatory drugs. We suggest an involvement of endogenous leukotriene(s), but not prostanoid(s), in mucosal mast cell activation/degranulation.

Rats turned diabetic by treatment with alloxan exhibit a significant reduction in serosal mast cell numbersin parallel with decreased insulin levels in the plasma. Our aim was to investigate the putative involvement of endogenous glucocorticoid hormone in this phenomenon. The findings indicated that rats treated with alloxan responded with an increase in levels of serum corticosterone concomitantly with decreased mast cell numbers in the pleural space. We found that either surgical bilateral adrenalectomy or pretreatment with the steroid antagonist RU 486 (20 mg/kg, i.p.) impaired the drop in pleural mast cell counts in alloxinated rats. Administration of insulin (15 U/kg) prevented the increase in corticosterone levels and restored pleural mast cell levels in diabetic animals. In addition, treatment of naive rats with corticosterone (0.5 mg/kg, s.c.) or dexamethasone (0.1 mg/kg, s.c.), for 3 consecutive days, led to a reduction in the number of mast cells recovered from the pleural cavity as noted in diabetic animals. In contrast, insulin reduced serum corticosterone levels and induced a significant elevation in pleural mast cell numbers in naive rats. We conclude that there is a causative relationship between increased levels of glucocorticoids and down-regulation of mast cell numbers associated with the diabetic state, both phenomena clearly sensitive to insulin.

Recently, it was suggested that melatonin (N-acetyl-5-methoxytryptamine) is oxidized by activated neutrophils in a reaction most probably involving myeloperoxidase (Biochem. Biophys. Res. Commun. (2000) 279, 657-662). Myeloperoxidase (MPO) is the most abundant protein of neutrophils and is involved in killing invading pathogens. To clarify if melatonin is a substrate of MPO, we investigated the oxidation of melatonin by its redox intermediates compounds I and II using transient-state spectral and kinetic measurements at 25 degrees C. Spectral and kinetic analysis revealed that both compound I and compound II oxidize melatonin via one-electron processes. The second-order rate constant measured for compound I reduction at pH 7 and pH 5 are (6.1 +/- 0.2) x 10(6) M(-1) s(-1) and (1.0 +/- 0.08) x 10(7) M(-1) s(-1), respectively. The rates for the one-electron reduction of compound II back to the ferric enzyme are (9.6 +/- 0.3) x 10(2) M(-1) s(-1) (pH 7) and (2.2 +/- 0.1) x 10(3) M(-1) s(-1) (pH 5). Thus, melatonin is a much better electron donor for compound I than for compound II. Steady-state experiments showed that the rate of oxidation of melatonin is dependent on the H(2)O(2) concentration, is not affected by superoxide dismutase, and is quickly terminated by sodium cyanide. Melatonin can markedly inhibit the chlorinating activity of MPO at both pH 7 and pH 5. The implication of these findings in the activated neutrophil is discussed.

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-α: 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.

The kinins, particularly bradykinin (BK), are important mediators involved in both the initiation and progression of an inflammatory response. The pro-inflammatory effects of kinins are mediated by at least two receptors: the B 2 subtype is expressed constitutively and the B 1 receptor is induced following tissue inflammation and damage. The endogenous ligand for the B 1 receptor is des-arg 9 BK, a cleavage product of the activity of carboxypeptidases on BK. Activation of B 1 receptors produces a range of pro-inflammatory effects including oedema, pain and promotion of blood-borne leukocyte trafficking. In this article Amrita Ahluwalia and Mauro Perretti briefly describe the biology of BK and its receptors, and discuss the possible development of B 1 receptor antagonists as novel anti-inflammatory agents.

1. Migration of blood-derived leukocytes to tissue sites of inflammation is a hallmark of the response that the host organizes to counteract an insult or a trauma or an infection. A cascade of events is then activated to allow interaction between the leukocyte and the endothelium of postcapillary venule, and this cascade is finely regulated such that mechanisms of negative control are operating side by side with pathways that promote and sustain the extravasation process. Examples of both these positive and negative regulatory systems are discussed here. 2. In vivo accumulation of specific subtypes of leukocytes in response to application of selective chemokines operates through an indirect mechanism that includes the perivenular mast cell and, in particular, the mast cell-derived amines, such as histamine and serotonin. In fact, treatments of animals with (1) histamine H1 or serotonin antagonists or with (2) the mast cell stabilizer cromolyn or with (3) prior depletion of intact mast cells are maneuvers that successfully reduce eosinophil, neutrophil and monocyte extravasation in response to eotaxin, interleukin-8 or monocyte chemoattractant protein-1, respectively. A model in which histamine provides a P-selectin-dependent rolling phenomenon is then postulated. 3. The discovery that neutrophil-derived lipocortin 1 acts as an autocrine mediator with an inhibitory action on the emigration (diapedesis) process confirms the growing body of experimental data that showed that exogenously administered lipocortin 1 and lipocortin 1 mimetics (peptide Ac2-26) potently inhibit neutrophil extravasation in response to different stimuli. Externalization of lipocortin 1 on the plasma membrane of adherent neutrophils reduces their rate of passage through the endothelial gaps. Because cell-associated lipocortin 1 levels are under the partial control of corticosterone (endogenous circulating glucocorticoid hormone in rodents) and dexamethasone (a synthetic glucocorticoid hormone with a potent anti-inflammatory profile), a model is proposed in which a balance between anti-inflammatory (lipocortin 1, etc.) and pro-inflammatory (adhesion molecules, cytokines and chemokines) mediators explains the difference in the rate of leukocyte accumulation during the different stages of the host inflammatory response. 4. In conclusion, this review emphasizes the importance of in vivo experimental systems as a valid way of obtaining pertinent observations and reiterates the importance of negative regulatory mechanisms on the leukocyte extravasation process operating within the host.

Western blot and densitometric analysis of organ homogenates from lipopolysaccharide (LPS)-treated rats (1–10 mg kg−1, i.p.) exhibited a strong induction of inducible nitric oxide synthase (iNOS) expression seen at all the doses tested (1, 3, and 10 mg kg−1, n = 3). In particular, 3 hr after challenge of rats with LPS, iNOS was detectable in the liver, kidney, aorta, spleen and lung. Dexamethasone (DEX) (0.1–1 mg kg−1; −1 hr) dose-dependently reduced iNOS expression in lung homogenates after exposure to LPS (1 mg kg−1; P < 0.05). A partial reversal of DEX-induced suppression of iNOS expression in lung homogenates 3 hr after challenge with LPS was observed in rats which received a specific anti-lipocortin 1 sheep serum (LCS3; 1 mL kg−1 24 hr prior to the steroid), with an inhibition of 35 ± 8%, as compared to animals passively immunised with normal sheep serum where dexamethasone exhibited an inhibition of 60 ± 7% (n = 4). Peritoneal macrophages collected from rats treated with LPS (1 mg kg−1; 3 hr) and cultured for 16 hr, released significant amounts of nitrite (51 ± 1 μM) into the cell supernatants; this was reduced (−70 ± 6%) after pre-treatment with dexamethasone (0.3 mg kg−1) and this effect was neutralised if animals were passively immunised with LCS3 (P < 0.01; n = 4). Thus lipocortin 1 mediates, at least in part, the inhibitory action exerted by dexamethasone on both iNOS protein expression in lung and iNOS activity (as measured by nitrite release) in primary peritoneal cells of rats.

Leukocyte extravasation occurs in many pathophysiological conditions, including inflammation, neoplasia and asthma. In recent years many studies have elucidated the steps that promote the initial interaction between extravasating cells and endothelium of the post-capillary venule; the sequential role of several classes of adhesion molecules (cell-specific chemokines) and activators (multipotent cytokines) is well established. In this review, Mauro Perretti focuses on a less well investigated mechanism by which the host downregulates extravasation at the leukocyte-endothelium interface. The neutrophilic polymorphonuclear leukocyte is used as the prime example of a leukocyte that interacts with the endothelium, and particular emphasis is given to the possibility that novel anti-inflammatory therapies might be developed from a better understanding of the inhibitory mechanisms activated by endogenous mediators such as adenosine, lipocortin 1, NO, prostacyclin and cathepsin G.