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Summary Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe−/− mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4+ T cells, generated CD4+, CD8+, T regulatory (Treg) effector and central memory cells, converted naive CD4+ T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin β receptors (VSMC-LTβRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTβRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe−/−Ltbr−/− and to a similar extent in aged Apoe−/−Ltbrfl/flTagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTβRs participate in atherosclerosis protection via ATLOs., Graphical Abstract, Highlights • Artery tertiary lymphoid organs control atherosclerosis T cell immunity • Artery tertiary lymphoid organs generate effector memory T cells • Artery tertiary lymphoid organs convert naive CD4+ T cells into induced Treg cells • Artery tertiary lymphoid organs protect from atherosclerosis, Tertiary lymphoid organs emerge during nonresolving inflammation. Habenicht and colleagues find that atherosclerosis immune responses are controlled by artery tertiary lymphoid organs in the adventitial connective tissue adjoining arteries. These lymphocyte aggregates arise through vascular smooth muscle cell lymphotoxin β receptor signaling and act as powerhouses of protective atherosclerosis immunity.

The first direct activator of BAX, a pro-apoptotic member of the BCL-2 family, has been recently identified. Herein, a structure-based lead optimization turned out into a small series of analogues, where 8 is the most potent compound published so far. 8 was used as pharmacological tool to ascertain, for the first time, the anticancer potential of BAX direct activators and the obtained results would suggest that BAX direct activators are potential future anticancer drugs rather than venoms.

Ecto-5′-nucleotidase (CD73), the ectoenzyme that together with CD39 is responsible for extracellular ATP hydrolysis and adenosine accumulation, regulates immune/inflammatory processes by controlling innate and acquired immunity cell functions. We previously demonstrated that CD73 is required for the assessment of a controlled allergic sensitization, in mice. Here, we evaluated the response to aerosolized allergen of female-sensitized mice lacking CD73 in comparison with their wild type counterpart. Results obtained show, in mice lacking CD73, the absence of airway hyperreactivity in response to an allergen challenge, paralleled by reduced airway CD23+B cells and IL4+T cells pulmonary accumulation together with reduced mast cells accumulation and degranulation. Our findings indicate CD73 as a potential therapeutic target for allergic asthma.

Benefits for vitamin E intake in diseases with inflammatory components have been described and related in part, to endogenously formed metabolites (long-chain metabolites, LCM). Here, we have evaluated the role of LCM in relieving asthma features. To this aim, the endogenous vitamin E metabolite α-13'-carboxychromanol (α-T-13'-COOH) that acts as potent 5-lipoxygenase inhibitor has been administered either intraperitoneally or by oral gavage to BALB/c mice sensitized by subcutaneous injection of ovalbumin (OVA). We also have taken advantage of the metabolically stable α-T-13'-COOH derivative α-amplexichromanol (α-AC). Intraperitoneal treatment with α-T-13'-COOH reduced OVA-induced airway hyperreactivity (AHR) as well as peri-bronchial inflammatory cell infiltration. α-AC was more efficacious than α-T-13'-COOH, as demonstrated by better control of AHR and in reducing subepithelial thickening. Both compounds exerted their protective function by reducing pulmonary leukotriene C4 levels. Beneficial effects of α-AC were coupled to inhibition of the sensitization process, as indicated by a reduction of IgE plasma levels, lung mast cell infiltration and Th2 immune response. Metabololipidomics analysis revealed that α-AC raises the pulmonary levels of prostanoids, their degradation products, and 12/15-lipoxygenase metabolites. Following oral administration, the pharmacodynamically different profile in α-T-13'-COOH and α-AC was abrogated as demonstrated by a similar and improved efficacy in controlling asthma features as well as by metabololipidomics analysis. In conclusion, this study highlights a role for LCM and of vitamin E derivatives as pharmacologically active compounds that ameliorate asthmatic features and defines an important role for endogenous vitamin E metabolites in regulating immune response underlying the sensitization process.

Lipid mediators (LM) encompass pro-inflammatory prostaglandins (PG) and leukotrienes (LT) but also specialized pro-resolving mediators (SPM) which display pivotal bioactivities in health and disease. Pharmacological intervention with inflammatory disorders such as osteoarthritis and rheumatoid arthritis commonly employs anti-inflammatory drugs that can suppress PG and LT formation, which however, possess limited effectiveness and side effects. Here, we report on the discovery and characterization of the two novel benzoxanthene lignans 1 and 2 that modulate select LM biosynthetic enzymes enabling the switch from pro-inflammatory LT to SPM biosynthesis as potential pharmacological strategy to intervene with inflammation. In cell-free assays, compound 1 and 2 inhibit microsomal prostaglandin E2 synthase-1 and leukotriene C4 synthase (IC50 ∼ 0.6–3.4 µM) and potently interfere with 5-lipoxygenase (5-LOX), the key enzyme in LT biosynthesis (IC50 = 0.04 and 0.09 µM). In human neutrophils, monocytes and M1 and M2 macrophages, compound 1 and 2 efficiently suppress LT biosynthesis (IC50

Nimesulide is a relatively selective cyclooxygenase (COX) -2 inhibitor, non-steroidal anti-inflammatory drug; it has been discovered in 1971 and firstly commercialized in Italy in 1985. There is much evidence that the pharmacological profile of nimesulide is peculiar and not shared with the other COX-2 selective inhibitors, suggesting that other molecular mechanisms besides inhibition of COX-2 derived prostaglandins are involved. Similarly, experimental data suggest that the gastrointestinal safety of nimesulide cannot be ascribed only to a COX-1 sparing effect. On the inflammatory process, the efficacy of nimesulide is dependent upon a wide spectrum of actions, due to the combination of effects on immune and non–immune cells. Early data demonstrated a central role for cyclic AMP (cAMP) in the anti-inflammatory effect of nimesulide; more recently, we have shown the involvement of the pathway ecto-5’-nucleotidase/adenosine A2A receptor. To date, the molecular mechanism(s) that confers uniqueness to nimesulide have not yet been defined. To go inside the mechanism of action of an existing drug, such as nimesulide, would be helpful to refine its therapeutic use but also to identify new targets for novel therapeutic anti-inflammatory approach. Here, we focus on accumulated evidence for a peculiar pharmacological profile of nimesulide.

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.

Airway hyperreactivity is characterized by increased responsiveness to bronchoconstrictor stimuli and it is hallmark of asthma. Adenosine is an ubiquitous signaling nucleoside resulting from ATP catabolism, whose extracellular levels increase following cellular damage or stress. Adenosine plays a role in asthma; asthmatics, but not normal subjects, present bronchoconstriction following inhalation of adenosine or of its precursor, adenosine-5'-monophosphate, most likely via adenosine A(2B) receptor on mast cells. However, the mechanism underling the increased airway smooth muscle sensitivity to adenosine in asthmatics remains to be elucidated. Early experimental studies suggested the involvement of A(1) receptor; this hypothesis has been confirmed by more recent studies on guinea pigs and is corroborated by the finding of an increased adenosine A(1) expression on asthmatic bronchial tissues. Brown Norway rats, the strain usually used to assess asthma models, develop hyperresponsiveness to adenosine 3h following allergen challenge, but not 24h thereafter, without involvement of A(1) receptor. Here, we investigated the role of adenosine A(1) receptor in sensitized Wistar rats showing airway hyperresponsiveness 24h following allergen challenge. We found that on bronchi of sensitized Wistar rats challenged with allergen there is an increased adenosine A(1) receptor expression on smooth muscle that is responsible for hyperresponsiveness to adenosine and ovalbumin.

The aim of this work was to investigate the potential of small nanoparticles (NPs) made of a poly(ethylene glycol)-poly(ε-caprolactone)-amphiphilic diblock copolymer (PEG-. b-PCL, PEG. =. 2. kDa and PCL. =. 4.2. kDa) as drug carrier system through the skin. Zinc(II) phthalocyanine (ZnPc), selected as lipophilic and fluorescent model molecule, was loaded inside NPs by a melting/sonication procedure. Loaded NPs with a hydrodynamic diameter around 60. nm, a slightly negative zeta potential and a ZnPc entrapment dependent on polymer/ZnPc ratio were obtained. Spectroscopic investigations evidenced that ZnPc was entrapped in monomeric form maintaining its emission properties. The transport of ZnPc through porcine ear skin was evaluated on Franz-type diffusion cells after treatment with different vehicles (water or PEG 0.4. kDa) containing free ZnPc or ZnPc-loaded NPs without and with (2-hydroxypropyl)-β-cyclodextrin (HPβCD) as permeation enhancer. Independently of the sample tested, ZnPc was transported in the skin without reaching receptor compartment. On the other hand, ZnPc was found in the skin in large amount and also in the viable epidermis when delivered through NPs associated with HPβCD, especially in conditions limiting water evaporation. Fluorescence images of skin samples after 24. h of permeation were in line with ZnPc dosage in the skin and demonstrated the ability of NPs covalently tagged with rhodamine to penetrate the skin and to locate in the intercellular spaces. Insight into skin chemical properties upon application of NPs by confocal Raman spectroscopy demonstrated that HPβCD caused an alteration of water profile in the skin, highly reducing the degree of hydration at stratum corneum/viable epidermis interface which can promote NP transport. Taken together, these results highlight PEG-. b-PCL NPs coupled with HPβCD as a novel vehicle for the skin delivery of highly lipophilic compounds paving the way to several applications.

The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease.

One of the major problems related to the percutaneous transluminal coronary angioplasty technique is the renarrowing of the vessel, a phenomenon known as restenosis. NO and nonsteroidal anti-inflammatory drugs have been shown to play a role in this pathology. The main problem with the use of conventional NO donors is that they affect blood pressure and flow, and for these reasons, they cannot be used safely in clinical practice. The aim of this study was to evaluate, with the use of a rat model of balloon angioplasty, whether a structural derivative of flurbiprofen, containing an added NO-releasing moiety (HCT-1026), is able to reduce or prevent neointimal formation. Rats were treated for 14 days with equimolar doses of flurbiprofen (2, 7, and 21 mg/kg) or HCT-1026 (3, 10, and 30 mg/kg). After this 14-day treatment, HCT-1026 but not flurbiprofen significantly modified the neointima/media ratio. The reduction in the neointimal proliferation obtained with HCT-1026 was well correlated with an increase in nitrite/nitrate plasma levels and a reduced cell proliferation. Neither HCT-1026 nor flurbiprofen affected inducible NO synthase induction in injured vessels. In conclusion, HCT-1026 caused a significant reduction in restenosis that appears to be directly related to NO release. HCT-1026 may prove to be beneficial in preventing or delaying restenosis in humans.

In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eαpeptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ(100 ng/mL) for 72 h caused a significant(P<0.01)increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant(P<0.05)increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323–339peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4+T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-kappaB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.

In this study we investigated the activation of nuclear factor-kappaB in the carrageenin-induced rat pleurisy. We found that nuclear factor-kappaB DNA binding activity, measured in inflammatory cells which migrated into the pleural cavity, was detectable at 3 and 6 h, markedly increased at 24 h and decreased at 48 h after induction of the inflammation. The increase in nuclear factor-kappaB DNA binding activity paralleled both exudate formation and leukocyte infiltration. Treatment of animals with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-kappaB activation, inhibited the nuclear factor-kappaB DNA binding activity as well as exudate formation and leukocyte infiltration. These results indicate that nuclear factor-kappaB is activated in the carrageenin-induced pleurisy and suggest that its inhibition may represent a novel strategy for the modulation of inflammatory response.

We studied the role of nuclear factor-kappaB (NF-kappaB) on the tone and on the expression of inducible nitric oxide (NO) synthase, both evaluated in aortas from lipopolysaccharide-treated rats. Thoracic aorta rings from lipopolysaccharide-treated rats (4 mg/kg, i.p.), compared to those from naive animals, showed: (i) reduced contractility to phenylephrine, (ii) progressive loss in tone when contracted with phenylephrine, (iii) increased inducible NO synthase protein expression and NF-kappaB activation. Pyrrolidine dithiocarbamate (10, 30, 100 mg/kg, i.p.), an antioxidant inhibitor of NF-kappaB activation, dose dependently suppressed all these lipopolysaccharide-induced effects. These results demonstrate that in vivo inhibition of NF-kappaB activation prevented the lipopolysaccharide-induced loss of vascular tone, an effect which was correlated to reduced expression of inducible NO synthase protein.

Glucocorticoids inhibit inducible-type NO synthase activity in a variety of cell types. We report here that proteins recovered from the medium of dexamethasone-treated J774 macrophages (1, 10, 100 microg/ml) inhibited lipopolysaccharide-stimulated nitrite generation by 10.0 +/- 3.0\%, 32.3 +/- 5.3\% and 55.0 +/- 6.0\%, respectively, and inducible NO synthase mRNA expression in these cells. Immunoblotting analysis of crude and partially purified glucocorticoid-induced proteins with an anti-lipocortin-1 polyclonal antiserum revealed the presence of lipocortin-1-like immunoreactive species with a molecular mass of 35-37 kDa. Furthermore, inhibition of lipopolysaccharide-induced nitrite production by glucocorticoid-induced proteins in J774 cells was reversed by addition of anti-lipocortin-1 neutralizing polyclonal antibody (1:60 dilution; 4 h before lipopolysaccharide). Comparison of glucocorticoid-induced proteins inhibition of both nitrite production and inducible NO synthase mRNA expression suggests that these effects result mainly from inhibition of lipopolysaccharide-mediated inducible NO synthase gene expression. These results indicate that negative regulation of inducible NO synthase by glucocorticoids is, at least in part, mediated by glucocorticoid-induced proteins that involve also members of the lipocortin-like superfamily.

Objective— We investigated the role of matrix metalloproteinase-8 (MMP8) in neointima formation and in vascular smooth muscle cell (VSMC) migration and proliferation. Approach and Results— After carotid artery wire injuring, MMP8 –/– /apoE –/– mice had fewer proliferating cells in neointimal lesions and smaller lesion sizes. Ex vivo assays comparing VSMCs isolated from MMP8 knockout and wild-type mice showed that MMP8 knockout decreased proliferation and migration. Proteomics analysis revealed that a disintegrin and metalloproteinase domain–containing protein 10 (ADAM10) had lower concentrations in MMP8 knockout VSMC culture media than in MMP8 wild-type VSMC culture media. Western blot, flow cytometric, and immunocytochemical analyses showed that MMP8 knockout VSMCs contained more pro-ADAM10 but less mature ADAM10, more N-cadherin, and β-catenin in the plasma membrane but less β-catenin in the nucleus and less cyclin D1. Treatment of MMP8 wild-type VSMCs with an ADAM10 inhibitor, GI254023X, or siRNA knockdown of ADAM10 in MMP8 wild-type VSMCs inhibited proliferation and migration, increased N-cadherin and β-catenin in the plasma membrane, reduced β-catenin in the nucleus, and decreased cyclin D1 expression. Incubation of MMP8 knockout VSMCs with a recombinant ADAM10 rescued the proliferative and migratory ability of MMP8 knockout VSMCs and increased cyclin D1 expression. Furthermore, immunohistochemical analyses showed colocalization of ADAM10 with VSMCs and N-cadherin, and nuclear accumulation of β-catenin in the neointima in apoE –/– /MMP8 +/+ mice. Conclusions— MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and β-catenin–mediated pathway and plays an important role in neointima formation.

Adenosine participates to asthma physiopathology by signaling through more than just one receptor subtype. Defining the role of each receptor is complicated by evidence that often results obtained on rodents do not coincide with human studies, but what emerges is that an important condition to establish hyperresponsiveness to adenosine in any species of sensitized animals is the exposure to allergen; this feature appears to be very similar to the human situation, since allergic humans regularly undergo exposure to allergen. Furthermore, A₂B in humans, but A₃ receptor in rodents, would mediate, indirectly, the bronchoconstriction in response to adenosine and would play the main role in adenosine-induced airway inflammation and airway hyperreactivity. On the other hand, A₁ receptor over-expressed on asthmatic airways would mediate a direct adenosine bronchoconstrictor effect. Antagonists and agonists to adenosine receptors have been considered as antiasthmatic drugs but often their development has been limited by unwanted effects. Preventing adenosine accumulation in airways should be considered as a novel promising antiasthmatic strategy.

Macrophages play crucial roles in atherosclerotic immune responses. Recent investigation into macrophage autophagy (AP) in atherosclerosis has demonstrated a novel pathway through which these cells contribute to vascular inflammation. AP is a cellular catabolic process involving the delivery of cytoplasmic contents to the lysosomal machinery for ultimate degradation and recycling. Basal levels of macrophage AP play an essential role in atheroprotection during early atherosclerosis. However, AP becomes dysfunctional in the more advanced stages of the pathology and its deficiency promotes vascular inflammation, oxidative stress, and plaque necrosis. In this paper, we will discuss the role of macrophages and AP in atherosclerosis and the emerging evidence demonstrating the contribution of macrophage AP to vascular pathology. Finally, we will discuss how AP could be targeted for therapeutic utility.

Nitric oxide (NO) is an inorganic free radical gas, of formula -N=O. The demonstration in 1987 that the endothelium-derived relaxing factor (EDRF) 1 was NO-3 and the subsequent discovery of L-arginine as the amino acid precursor for the formation of NO 4,5 have disclosed a novel area of interdisciplinary research. The relevance of the L-arginine:NO pathway was primarily documented by the identification of NO as a key mediator in the regulation of vascular tone, 6 neurotransmission z8 and host defence mechanisms? Moreover, the discovery of a growing number of NO-regulated functions (e.g. memory, sensory processing, endocrine system, reproduction, airway resistance) suggests that virtually every mammalian cell is actually or potentially under the control of NO) . In this light, it is not surprising that prostaglandin (PG) biosynthesis appears to be modulated by the L-arginine:NO pathway, Interestingly, other findings suggest that prostanoids may regulate NO production in a variety of cell types. This brief review will summarize the recent information dealing with the 'cross-talk' occurring between the Larginine:NO and cyclooxygenase pathways, particularly at the level of the inflammatory response.

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled\ud phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and\ud migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30\ud ng/mL) or fetal bovine serum (5%). Bindarit (100-300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These\ud effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of\ud bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the\ud expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal\ud formation.

Objective— Clinical studies have identified that reduced numbers of circulating plasmacytoid dendritic cells (pDCs) act as a predictor of cardiovascular events in coronary artery disease and that pDCs are detectable in the shoulder region of human atherosclerotic plaques, where rupture is most likely to occur. Results from animal models are controversial, with pDCs seen to inhibit or promote lesion development depending on the experimental settings. Here, we investigated the role of pDCs in atherosclerosis in apolipoprotein E−deficient mice. Methods and Results— We demonstrated that the aorta and spleen of both apolipoprotein E−deficient and C57BL/6 mice displayed similar numbers of pDCs, with similar activation status. In contrast, assessment of antigen uptake/presentation using the Eα/Y-Ae system revealed that aortic pDCs in apolipoprotein E−deficient - mice were capable of presenting in vivo systemically administered antigen. Continuous treatment of apolipoprotein E−deficient mice with anti−mouse plasmacytoid dendritic cell antigen 1 (mPDCA-1) antibody caused specific depletion of pDCs in the aorta and spleen and significantly reduced atherosclerosis formation in the aortic sinus (by 46%; P P P Conclusion— These results identify a critical role for pDCs in atherosclerosis and suggest a potential role for pDC targeting in the control of the pathology.

We have studied the effect of hyaluronic acid (HA) in two standard models of acute and chronic inflammation in the rat, i.e. carrageenin oedema and adjuvant arthritis. Our results show that HA was able to inhibit, in a dose-related fashion, both types of inflammatory reactions. These findings suggest that HA may have a modulatory role in the inflammatory process and may explain, at least in part, the protective role of HA in the inflammatory cartilage damage.

Objective— We have previously demonstrated that bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs), is effective in reducing neointimal formation in rodent models of vascular injury by reducing smooth muscle cell proliferation and migration and neointimal macrophage content, effects associated with the inhibition of MCP-1/CCL2 production. The aim of the current study was to evaluate the efficacy of bindarit on in-stent stenosis in the preclinical porcine coronary stent model. Methods and Results— One or 2 bare metal stents (Multi-Link Vision, 3.5 mm) were deployed (1:1.2 oversize ratio) in the coronary arteries of 42 pigs (20 bindarit versus 22 controls). Bindarit (50 mg/kg per day) was administered orally from 2 days before stenting until the time of euthanasia at 7 and 28 days. Bindarit caused a significant reduction in neointimal area (39.4%, P P P P P P P Conclusion— Our results show the efficacy of bindarit in the prevention of porcine in-stent stenosis and support further investigation for clinical application of this compound.

Objective— The activation of nuclear factor-κB (NF-κB) is a crucial step in the arterial wall’s response to injury. The identification and characterization of the NF-κB essential modulator–binding domain (NBD) peptide, which can block the activation of the IκB kinase complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-κB. The aim of the present study was to evaluate the effect of the NBD peptide on neointimal formation. Methods and Results— In the rat carotid artery balloon angioplasty model, local treatment with the NBD peptide (300 μg/site) significantly reduced the number of proliferating cells at day 7 (by 40%; P P −/− mice in which the NBD peptide (150 μg/site) reduced wire-induced neointimal formation at day 28 (by 47%; P Conclusion— The NBD peptide reduces neointimal formation and smooth muscle cell proliferation/migration, both effects associated with the inhibition of NF-κB activation.

Aims Bindarit is an original compound with peculiar anti-inflammatory activity due to a selective inhibition\ud of a subfamily of inflammatory chemokines, including the monocyte chemotactic proteins MCP-1/CCL2, MCP-3/CCL7, and MCP-2/CCL8. In this study, we investigated the effect of bindarit on neointima formation using two animal models of arterial injury: rat carotid artery balloon angioplasty and wireinduced carotid injury in apolipoprotein E-deficient (apoE2/2) mice.\ud Methods and results Treatment of rats with bindarit (200 mg/kg/day) significantly reduced balloon injury-induced neointima formation by 39% at day 14 without affecting re-endothelialization and reduced the number of medial and neointimal proliferating cells at day 7 by 54 and 30%, respectively. These effects were associated with a significant reduction of MCP-1 levels both in sera and in injured carotid arteries of rats treated with bindarit. In addition, in vitro data showed that bindarit (10–\ud 300 mM) reduced rat vascular smooth muscle cell (VSMC) proliferation, migration, and invasion, processes\ud contributing to the injury-induced neointima formation in vivo. Similar results were observed in hypercholesterolaemic apoE2/2 mice in which bindarit administration resulted in a 42% reduction of the number of proliferating cells at day 7 after carotid injury and in a 47% inhibition of neointima formation at day 28. Analysis of the cellular composition in neointimal lesions of apoE2/2 mice treated with bindarit showed that the relative content of macrophages and the number of VSMCs\ud were reduced by 66 and 30%, respectively, compared with the control group.\ud Conclusion This study demonstrates that bindarit is effective in reducing neointima formation in both\ud non-hyperlipidaemic and hyperlipidaemic animal models of vascular injury by a direct effect on VSMC proliferation and migration and by reducing neointimal macrophage content. All of these data were associated with the inhibition of MCP-1 production.

Background— Studies in humans and animal models suggest that interleukin-18 (IL-18) plays a crucial role in vascular pathologies. IL-18 is a predictor of cardiovascular death in angina and is involved in atherotic plaque destabilization. Higher IL-18 plasma levels also are associated with restenosis after coronary artery angioplasty performed in patients with acute myocardial infarction. We investigated the effective role of IL-18 in neointimal formation in a balloon-induced rat model of vascular injury. Methods and Results— Endothelial denudation of the left carotid artery was performed by use of a balloon embolectomy catheter. Increased expression of IL-18 and IL-18Rα/β mRNA was detectable in carotid arteries from days 2 to 14 after angioplasty. The active form of IL-18 was highly expressed in injured arteries. Strong immunoreactivity for IL-18 was detected in the medial smooth muscle cells at days 2 and 7 after balloon injury and in proliferating/migrating smooth muscle cells in neointima at day 14. Moreover, serum concentrations of IL-18 were significantly higher among rats subjected to vascular injury. Treatment with neutralizing rabbit anti-rat IL-18 immunoglobulin G significantly reduced neointimal formation (by 27%; P Conclusions— These results identify a critical role for IL-18 in neointimal formation in a rat model of vascular injury and suggest a potential role for IL-18 neutralization in the reduction of neointimal development.

From the whole plant of Euphorbia peplus L., a new diterpene based on a rare pepluane skeleton, named pepluanone (1), was isolated together with a known pepluane diterpene (2). The stereostructure of pepluanone was determined on the basis of an extensive NMR study, MS data, and chemical reaction. The ability of these compounds to act as antiinflammatory agents has been evaluated for the first time by in vivo tests on carrageenin-induced rat paw edema, an experimental model of acute inflammation. Comparison of the bioactivity of pepluanone and compound 2 in terms of chemical structure, evidenced the high efficiency of pepluanone and the absence of appreciable activity for compound 2, thus giving a first insight into the structure-activity relationship. Further in vitro experiments performed on pepluanone let us hypothesize that its activity could be explained in reducing the production of nitric oxide, prostaglandin E(2), and TNF-alpha by inhibiting the expression of inducible nitric oxide synthase, cyclooxygenase-2, and TNF-alpha mRNA through the down-regulation of NF-kappaB binding activity.

Nitric oxide (NO) plays an important role in the pathogenesis of celiac disease. We have examined the involvement of nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1), and signal transducer and activator of transcription-1alpha (STAT-1alpha) on the synergistic induction of inducible nitric oxide synthase (iNOS) gene expression by gliadin (G) in association with interferon-gamma (IFN-gamma) in RAW 264.7 macrophages. We found that IFN-gamma was efficient in enhancing the basal transcription of the iNOS promoter at 1, 6, and 24 h, whereas G had no effect. The G plus IFN-gamma association caused an increase in iNOS promoter activity which was inhibited by pyrrolidine dithiocarbammate (PDTC) at 6 and 24 h as well as by genistein (Gen) and tyrphostine B42 (TB42) at 1 h, inhibitors of NF-kappaB, IRF-1, and STAT-1alpha activation, respectively. Similarly, the IFN-gamma and G combination treatment led to a higher increase in iNOS mRNA levels at 1, 6, and 24 h compared with IFN-gamma alone. Gen and TB42 inhibited iNOS mRNA levels at 1 h, whereas PDTC inhibited iNOS mRNA levels at 6 and 24 h. In addition, the synergistic induction of iNOS gene expression by G plus IFN-gamma correlated with the induction of NF-kappaB, IRF-1, and STAT-1alpha/DNA binding activity and mRNA expression. In conclusion, our study, which provides evidence that the effect of G on iNOS gene transcription in IFN-gamma-stimulated RAW 264.7 cells can be ascribed to all three transcription factors, may contribute to lead to new insights into the molecular mechanisms governing the inflammatory process in celiac disease.

The glucocorticoid receptor (GR) and peroxisome proliferator-activated receptors (PPARs) play important roles in both physiological and pathological conditions such as cell differentiation, lipolysis, control of glucose metabolism, immunity, and inflammation. In fact, recent studies suggest that the thiazolidinedione (TZD) class of PPAR-gamma ligands, like glucocorticoids, may also be clinically beneficial in several inflammatory diseases, even if the molecular mechanisms responsible for these activities have not yet been clarified. In this study, by using a murine model of inflammation, the carrageenin-induced paw edema in mouse, we show that the anti-inflammatory activity exhibited by the PPAR-gamma agonists rosiglitazone and ciglitazone is reversed by the GR antagonist RU486 (17 beta-hydroxy-11 beta-[4-dimethylamino phenyl]-17 alpha-[1-propynyl]estra-4,9-dien-3-one). Moreover, by using a conditional GR null cell line, we demonstrate, for the first time to our knowledge, that one of the possible mechanisms explaining the anti-inflammatory activity of TZDs is their ability to activate GR nuclear translocation. In addition, by using J774 cell line lacking PPAR-gamma, we demonstrate that PPAR-gamma expression could not be essential for TZD-mediated GR nuclear translocation, thus explaining, at least in part, the molecular mechanism underlying their anti-inflammatory activity.

The heat shock response is a highly conserved mechanism of protection elicited in the cell by various kinds of stimuli, such as heat, sodium arsenite, oxidants and inflammation. Among the mechanisms potentially involved in mediating the protective effects of hsp, one of the most investigated is the inhibition of pro-inflammatory gene expression such as inducible nitric oxide synthase (iNOS) and inflammatory cytokines. Nevertheless, data about the effects of heat shock response on cyclooxygenase-2 expression in activated macrophages are so far not available in literature. The aim of this study was to investigate the changes in cyclooxygenase-2 expression following lipopolysaccharide stimulation of heat shocked J774 murine macrophages. We found, by Western blotting analysis and reverse transcription-polymerase chain reaction analysis (RT-PCR), that the lipopolysaccharide-induced cyclooxygenase-2 gene expression was reduced in heat shocked cells. Such a reduction was associated to activation of heat shock factor, increased levels of heat shock protein 72 and inhibition of lipopolysaccharide-induced nuclear factor-kappaB binding activity. These data suggest that the heat shock response inhibits cyclooxygenase-2 gene expression at transcriptional level, i.e. by preventing the activation of nuclear factor-kappaB, and provide additional information about mechanism(s) underlying the anti-inflammatory effect of the heat shock proteins.

Two ethanolic propolis extracts (EPE) with and without the caffeic acid phenethyl ester (CAPE), CAPE and galangin (major components of propolis) were investigated for anti-inflammatory activity in rats using carrageenin foot oedema, carrageenin pleurisy and adjuvant arthritis. In our experiments, EPE with CAPE and CAPE alone significantly inhibited carrageenin oedema, carrageenin pleurisy and adjuvant arthritis. In contrast EPE without CAPE and galangin did not exhibit anti-inflammatory effects in acute and chronic inflammation. These results suggest that the anti-inflammatory activity of propolis is due to CAPE.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, antiinflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure- function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.

In this study, using rat carrageenin-induced pleurisy, we found that treatment of rats with either indomethacin or NS-398 suppressed the pleurisy at 2 h but significantly exacerbated this reaction at 48 h. Exacerbated inflammation was associated with reduced prostaglandin D(2) levels, decreased heat shock factor 1 (HSF1) activation, reduced hsp72 expression and increased activation of nuclear factor kappaB (NF-kappaB). Replacement of cyclopentenone prostaglandins by treating rats with either prostaglandin J(2) or prostaglandin D(2) reversed the exacerbating effects of cyclooxygenase inhibitors leading to the resolution of the reaction. In conclusion, we demonstrate that cyclopentenone prostaglandins may act as anti-inflammatory mediators by inducing in inflammatory cells HSF1-dependent hsp72 expression and NF-kappaB inhibition, two crucial events for the remission of inflammation.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the inflammatory process. In the present study we investigated in an acute model of inflammation, the carrageenin-induced hind paw edema, the anti-inflammatory effect of double stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type, but not mutant-ODN decoy, dose-dependently inhibited edema formation induced by carrageenin in rat paw. Molecular analysis performed on soft tissue obtained from inflamed paw demonstrated: (1) an inhibition of NF-kappaB DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, exhibited a similar profile of activity of ODN decoy. Our results indicate for the first time that ODN decoy, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate acute inflammation.

Objective and Design: The role of nitric oxide (NO) on leucocyte migration has been investigated in rat carrageenin-induced pleurisy. Material: Male Wistar rats. Treatment: L-arginine, NOC-18 and aminoguanidine were administered subcutaneously 1 h prior to carrageenin injection. Methods: Leucocyte accumulation into the pleural cavity was measured 4h after carrageenin challenge. Statistical significance was calculated by Bonferroni test. Results: L-arginine (10 mg/kg) or the NO donor NOC-18 (10 mg/kg), significantly inhibited leucocyte infiltration by 31% and 20% respectively (P < 0.01). On the contrary, when these compounds were given at high doses (L-arginine 300 mg/kg; NOC-18 30 mg/kg), leucocyte accumulation was increased by 22% and 33% respectively (P < 0.01). Aminoguanidine, a relatively selective inhibitor of the inducible NO synthase, depending on the dose, showed a biphasic effect on cell migration. Thus, at low doses (30 and 100 mg/kg), aminoguanidine increased (by 40% and 74% respectively, P < 0.01) leucocyte infiltration which was inhibited by 41% (P < 0.01) when the drug was given at high dose (300 mg/kg). Conclusions: These results suggest that in rat carrageenin induced pleurisy NO primarily inhibits leucocyte migration.

The role of nitric oxide (NO) in the reverse passive Arthus reaction elicited in the rat skin has been studied. The reverse passive Arthus reaction was modulated by test compounds given by intradermal injection in combination with anti-bovine serum albumin antibody. L-arginine (1.5-15 micromol/site) and the NO donor [1-hydroxy-2-oxo-3,3-bis (3-amonoethyl)- 1-triazenel (NOC-18; 1-10 micromol/site) both significantly reduced neutrophil infiltration and increased plasma leakage. The NO scavenger haemoglobin (30 and 100 micromol/site) did not affect oedema formation but increased neutrophil infiltration and attenuated the effects of L-arginine. The non-selective nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl ester (0.3-100 nmol/site) and N(G)-monomethyl-L-arginine (0.3-100 nmol/site) or the relatively selective inhibitors of the inducible NO synthase aminoguanidine (30-1000 nmol/site) and S-methylthiourea (3-1000 nmol/site) significantly reduced plasma leakage when given at high doses. Furthermore all these inhibitors exhibited a dose-related biphasic effect on neutrophil infiltration which was significantly increased by low doses and reduced by high doses, while intermediate doses had no effect. Phenylpropanolamine, a sympathomimetic vasoconstrictor (15-60 micromol/site), dose-dependently reduced both oedema formation and neutrophil infiltration. These results provide evidence for a relevant role of NO as a modulator of rat dermal reverse passive Arthus reaction and suggest that at the vascular level NO controls primarily the interaction between leucocyte and endothelial cell rather than the vascular permeability.

The correlation between endogenous nitric oxide (NO) generation and prostaglandin biosynthesis was studied in rat carrageenin pleurisy induced by the injection of 0.2 mL of 1\% lambda-carrageenin into the pleural cavity. The pleural exudate was collected at 4 hr and the amounts of NO2- + NO3- (NOx) and prostaglandin E2 (PGE2) measured. The NOx present in the inflammatory exudate was determined by measuring the NO2- with the Griess reaction, after the reduction of NO3- to NO2- using acid-washed cadmium powder. PGE2 was measured by radioimmunoassay. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1-3-10 mg/kg subcutaneously) reduced NOx by 20 +/- 7\%, 41 +/- 6\% and 55 +/- 9\% (P < 0.01) and PGE2 by 9 +/- 6\%, 41 +/- 11\% and 74 +/- 9\% (P < 0.001). Conversely, L-arginine (300 mg/kg SC) increasedNOx by 39 +/- 7\% (P < 0.01) and PGE2 by 78 +/- 6\% (P < 0.001). The NO scavenger haemoglobin (Hb), coinjected into the pleural cavity (3 mg/site) with carrageenin, produced a parallel inhibition of NOx (65 +/- 16\%, P < 0.001) and PGE2 (71 +/- 18\%, P < 0.001). The soluble guanylate cyclase inhibitor methylene blue (Mb; 2 mg/site) had no effect. Moreover haemoglobin, but not methylene blue, was able to significantly suppress the L-arginine-induced increase of both NOx and PGE2. In each pleural exudate, independently from the animal treatment, the amount of NOx was highly correlated to the amount of PGE2 (r = 0.93, P < 0.001). These results suggest that in rat carrageenin pleurisy the modulation of the L-arginine:NO pathway results in a parallel modulation of prostaglandin biosynthesis. The interaction between cyclooxygenase and the NO pathway may represent an important mechanism for the modulation of the inflammatory response.

The influence of endogenous nitric oxide (NO) on prostaglandin biosynthesis in rat carrageenin oedema was studied. Oedema formation and prostaglandin E 2 generation in the inflamed paw were both increased by l - but not d -arginine and reduced by the NO synthase inhibitor, l - N G -nitro arginine methyl ester, and the NO scavenger, haemoglobin. Methylene blue, an inhibitor of the soluble guanylate cyclase, had no effect. These results suggest that endogenous NO modulates carrageenin oedema by increasing prostaglandin biosynthesis at the inflammatory site through a cGMP-independent mechanism.

1. Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)-induced paw oedema and measuring both the foot volume and the amount of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2. Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor L-NG-nitro arginine methyl ester (L-NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 mumol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 mumol/paw) and L-arginine (15 mumol/paw). 3. The NO-donors (10 mumol/paw) 3-morpholino-sydnonimine-hydrochloride (SIN-1), S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4. SIN-1 (2.5, 5 and 10 mumol/paw) produced a significant dose-dependent increase of the oedema induced by AA which was correlated with increased amounts of 6-keto-PGF1 alpha in the fluid recovered from inflamed paws. 5. Both oedema and prostaglandin biosynthesis induced by the combination AA+SIN-1 were greatly suppressed by either Hb (30 mumol/paw) or indomethacin (3 mumol/paw or 5 mg kg-1 s.c.) but unaffected by Mb (3 mumol/paw). 6. In LPS-treated rats (6 mg kg-1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by L-NAME or Hb, unaffected by Mb and increased by L-arginine.7. These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3': 5'-cyclic monophosphate (cyclic GMP)-independent mechanism and suggest that the interaction between NO synthase and cyclo-oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.

1. The role of endogenous nitric oxide (NO) in adjuvant arthritis in Lewis rats has been studied by use of L-arginine, the amino acid from which NO is synthesized, and NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Prolonged modulation (35 days) of the L-arginine: NO pathway in rats was achieved by dissolving test compounds in the drinking water (L-arginine: 3, 10 and 30 mg ml-1; L-NAME: 0.1, 1 and 10 mg ml-1). 2. Arthritis was exacerbated by L-arginine and suppressed by L-NAME in a dose-related fashion. Combined treatment with L-NAME (1 mg ml-1) and L-arginine (30 mg ml-1) did not modify the arthritis. 3. Reduced weight gain, which is a feature of adjuvant arthritis, was modified by these compounds so that L-arginine reduced weight gain whereas L-NAME increased weight gain compared with that in control animals. 4. D-Arginine (30 mg ml-1), NG-nitro-D-arginine methyl ester (D-NAME: 1 mg ml-1) and L-lysine (30 mg ml-1), an amino acid not involved in the generation of NO, were without effect on either arthritis or body weight gain. 5. Antigen-stimulated proliferation of T-lymphocytes as well as generation of nitrite (NO2-) and release of acid phosphatase from macrophages were all enhanced in L-arginine-treated arthritic rats and reduced in L-NAME-treated animals. 6. These results suggest that endogenous NO modulates adjuvant arthritis, possibly by interfering with the activation of T-lymphocytes and/or macrophages.

The role of endogenous nitric oxide (NO) in acute inflammation was investigated using two inhibitors of NO synthase (NG-nitro-L-arginine methyl ester(L-NAME) and NG-monomethyl-L-arginine (L-NMMA)) as well as L- or D-arginine. The effect of test compounds was studied on the carrageenin-induced increase in vascular permeability in rat skin and in dextran- and carrageenin-induced paw oedema. Both L-NAME and L-NMMA dose dependently inhibited the increase in vascular permeability and oedema formation. L- but not D-arginine increased these inflammatory responses and reversed the inhibitory effects of L-NAME and L-NMMA. In dexamethasone-treated rats L-arginine enhanced the dextran-induced oedema and the early phase of carrageenin-induced oedema but did not modify the inhibition by dexamethasone of the late phase of carrageenin-induced oedema. These results suggest that endogenous NO is released at the site of acute inflammation and modulates oedema formation. Depending on the time course or on the type of inflammation, NO may be predominantly generated by the constitutive or by the inducible NO synthase.

The anti-inflammatory effect of glucocorticoids depends, at least in part, on the induction of two regulatory proteins, lipocortin and vasocortin, both preventing the release of inflammatory mediators. Lipocortin inhibits phospholipase A2 (PLA2) and therefore reduces arachidonic acid metabolites formation. Vasocortin inhibits histamine release from mast cells. Lipocortin and vasocortin may be regarded as the first two identified members of the (perhaps greater) family of glucocorticoid-induced proteins.

We investigated the occurrence and the role of HSF1 activation and inducible hsp72 expression in the carrageenin pleurisy in the rat. Molecular analysis performed on pleural cells collected from rat pleural cavity after carrageenin challenge revealed increased HSF1 activation and hsp72 expression. Moreover, local injection of a double-stranded oligodeoxynucleotide (ODN) containing the heat shock element sequence, acting as transcription factor decoy, exacerbated the inflammatory reaction. The exacerbation, induced by wild-type, but not by mutant ODN decoy, was associated to both inhibition of HSF1/DNA binding activity and reduction of hsp72 expression. In conclusion, this study shows that HSF1 activation and hsp72 expression both actually occur in acute inflammation and that the remission of the inflammatory reaction is tightly associated to the HSF1-dependent hsp72 expression, suggesting a a relevant role for the HSF1/hsp72 pathway as an endogenous anti-inflammatory system.

Vasocortin, a glucocorticoid-induced anti-inflammatory protein, has been purified from the peritoneal lavage fluid of dexamethasone-treated rats. Vasocortin inhibited the release of histamine from rat peritoneal cells stimulated by dextran or concanavalin A but did not alter the release induced by calcium ionophore A23187 or compound 48/80. This selective effect exhibited by vasocortin mimics the glucocorticoid inhibition of histamine release from rat mast cells.

The anti-inflammatory effect of glucocorticoids has been investigated in two standard models of experimental inflammation, i.e. rat paw oedema induced by carrageenin or dextran. Both types of oedema are suppressed by dexamethasone while indomethacin and BW755C only suppress carrageenin oedema. Dexamethasone inhibits dextran oedema according to the accepted mode of action of steroid hormones since the inhibition occurs after a 2-3 h time lag and is abolished by pretreating animals with actinomycin D. Dextran oedema and carrageenin oedema are also controlled by endogenous corticoids since adrenalectomy potentiates the paw oedema formation induced by low concentrations of phlogogenic agents. It has been shown that glucocorticoids induce both in vitro and in vivo the formation and release of antiphospholipase proteins which are anti-inflammatory in that they greatly suppress carrageenin oedema. However, these proteins have no effect on dextran oedema. We conclude that the inhibition of dextran oedema by glucocorticoids depends on the formation of another type of anti-inflammatory protein.