Barbara Palazzetti

Galectin-1 suppresses experimental colitis in mice

BACKGROUND & AIMSnUncontrolled T-cell activation plays a critical role in the pathogenesis of inflammatory bowel diseases. Therefore, pharmacologic strategies directed to restore the normal responsiveness of the immune system by deleting inappropriately activated T cells could be efficacious in the treatment of these pathologic conditions. Galectin-1 is an endogenous lectin expressed in lymphoid organs that plays a role in the maintenance of central and peripheral tolerance. The aim of the present study was to evaluate the therapeutic effects of galectin-1 on T-helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice.nnnMETHODSnCells and tissues from mice with TNBS colitis receiving treatment with several doses of human recombinant galectin-1 (hrGAL-1) were analyzed for morphology, cytokine production, and apoptosis.nnnRESULTSnProphylactic and therapeutic administration of rhGAL-1 resulted in a striking improvement in the clinical and histopathologic aspects of the disease. hrGAL-1 reduced the number of hapten-activated spleen T cells, decreased inflammatory cytokine production, and profoundly reduced the ability of lamina propria T cells to produce IFN gamma in vitro. Moreover, hrGAL-1 led to the appearance of apoptotic mononuclear cells in colon tissue when administered in vivo and induced selective apoptosis of TNBS-activated lamina propria T cells in vitro.nnnCONCLUSIONnCollectively, these data show that hrGAL-1 exerts protective and immunomodulatory activity in TNBS-induced colitis and it might be effective in the treatment of inflammatory bowel diseases.

Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis.

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH2, a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH2, whereas the scramble control peptide, LSIGRL-NH2, was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH2, but not the scramble peptide, directly inhibited IFN-γ secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.

NCX-1000, a NO-releasing derivative of ursodeoxycholic acid, selectively delivers NO to the liver and protects against development of portal hypertension

Portal hypertension resulting from increased intrahepatic resistance is a common complication of chronic liver diseases and a leading cause of death in patients with liver cirrhosis, a scarring process of the liver that includes components of both increased fibrogenesis and wound contraction. A reduced production of nitric oxide (NO) resulting from an impaired enzymatic function of endothelial NO synthase and an increased contraction of hepatic stellate cells (HSCs) have been demonstrated to contribute to high intrahepatic resistance in the cirrhotic liver. 2-(Acetyloxy) benzoic acid 3-(nitrooxymethyl) phenyl ester (NCX-1000) is a chemical entity obtained by adding an NO-releasing moiety to ursodeoxycholic acid (UDCA), a compound that is selectively metabolized by hepatocytes. In this study we have examined the effect of NCX-1000 and UDCA on liver fibrosis and portal hypertension induced by i.p. injection of carbon tetrachloride in rats. Our results demonstrated that although both treatments reduced liver collagen deposition, NCX-1000, but not UDCA, prevented ascite formation and reduced intrahepatic resistance in carbon tetrachloride-treated rats as measured by assessing portal perfusion pressure. In contrast to UDCA, NCX-1000 inhibited HSC contraction and exerted a relaxing effect similar to the NO donor S-nitroso-N-acetylpenicillamine. HSCs were able to metabolize NCX-1000 and release nitrite/nitrate in cell supernatants. In aggregate these data indicate that NCX-1000, releasing NO into the liver microcirculation, may provide a novel therapy for the treatment of patients with portal hypertension.

Importance of Innate Immunity and Collagen Binding Integrin α1β1 in TNBS-Induced Colitis

Inflammation occurs in the context of integrin-mediated adhesive interactions of cells with their extracellular matrix environment. We investigated the role of the collagen binding integrin alpha1beta1 in a model of colitis. alpha1beta1 was expressed on lamina propria T cells and monocytes during disease. Both alpha1 deficiency and anti-alpha1 mAb treatment (prophylactic and therapeutic) protected against colitis. In vivo alpha1beta1 blockade improved macroscopic and histologic scores, decreased inflammatory cytokine production, and profoundly affected the ability of lamina propria mononuclear cells to proliferate and produce IFN-gamma in vitro. Development and alpha1-mediated inhibition of colitis can be lymphocyte independent, suggesting that activated monocytes also represent a key alpha1beta1-expressing cell type involved in colitis. These results underscore the importance of innate immunity and, specifically, of leukocyte/matrix interactions in regulating local inflammatory responses.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4+ T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1β, IL-18, and IFN-γ release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.

CRYPTOCOCCUS NEOFORMANS DIFFERENTLY REGULATES B7-1 (CD80) AND B7-2 (CD86) EXPRESSION ON HUMAN MONOCYTES

To induce a specific response in primary resting T cells, two signals must be provided by antigen‐presenting cells (APC). The first antigen‐specific signal is mediated by formation of the T cell receptor major histocompatibility complex molecule ternary complexes. The second signal is delivered by interaction of either B7‐1 or B7‐2 expressed by APC with CD28 or CTLA‐4 on T cells. In this study, we examined the modulation of B7‐1 and B7‐2 molecules on human monocytes exposed to encapsulated or acapsular Cryptococcus neoformans or Candida albicans. In our experimental system, C. albicans or acapsular C. neoformans are able to induce B7‐1 expression while the encapsulated yeast is a poor stimulator. A modest increase of B7‐2 expression was also observed after monocyte treatment with acapsular C. neoformans or C. albicans, while the encapsulated yeast was ineffective in inducing B7‐2 molecules. Kinetic analysis showed the maximum expression of B7‐1 after 24 to 48 h. Addition of the opsonic IgG1 mAb 2H1 to monocytes and C. neoformans significantly increased B7‐1, but not B7‐2, expression. The contribution of B7‐1 and B7‐2 co‐stimulatory (CS) molecules to cryptococcal‐specific T cell activation was analyzed and a substantial inhibition of T cell proliferation was observed. In this study we provide the first demonstration of fungal interference in the regulation of CS molecules. Our results suggest a potential mechanism for poor inflammatory responses observed in C. neoformans infections.

A NO-releasing derivative of acetaminophen spares the liver by acting at several checkpoints in the Fas pathway.

NCX‐701 is a nitric oxide (NO)‐releasing acetaminophen (APAP) derivative. In the present study we demonstrated that NCX‐701 is as effective as APAP in controlling body temperature in a rat model of endotoxin‐induced fever. Liver toxicity is a major complication of APAP overdosing. To investigate whether NCX‐701 is hepatotoxic, BALB/C mice were injected with 100u2003–u2003500u2003mgu2003kg−1 APAP or NCX‐701 alone or in combination (i.e. 500u2003mgu2003kg−1 of both compounds). Our results demonstrated that although APAP caused a dose‐dependent liver injury, NCX‐701 was completely devoid of liver toxicity. At the dose of 500u2003mgu2003kg−1 APAP caused an ∼40 fold increase of AST plasma levels and extensive centrilobular necrosis. APAP and NCX‐701 share the same metabolic pathway as demonstrated by the time‐course of APAP‐glucuronide concentrations in plasma and liver. NCX‐701 was safe in mice with pre‐existing chronic liver disease. Indeed, while C57BL6 transgenic mice expressing the hepatitis B virus (HBV) at the age of 8 months were significantly more susceptible to liver damage induced by APAP (500u2003mgu2003kg−1) than their congenic littermates, treating HBV‐transgenic mice with NCX‐701, 500u2003mgu2003kg−1, caused no damage. Co‐administration of NCX‐701 at the dose 500u2003mgu2003kg−1 to mice treated with APAP, 500u2003mgu2003kg−1, completely protected against liver damage induced by APAP. APAP, but not NCX‐701, upregulated liver Fas and Fas Ligand mRNA expression in vivo. Incubating mouse hepatocytes with APAP, but not with NCX‐701, increased cell surface Fas expression and sensitized hepatocytes to death induced by challenge with a Fas‐agonistic antibody. Collectively, these observations suggest that APAP toxicity is Fas mediated and that NCX‐701 spares the liver by acting at several checkpoints in the Fas pathway.

Regulatory role of exogenous IL-10 in the development of immune response versus Cryptococcus neoformans.

The most important event involved in host defence against Cryptococcus neoformans is the development of an adequate cell‐mediated immune response. IL‐10, abundantly produced during AIDS progression, could be a negative factor that affects the T cell response through its own immunosuppressive action on antigen‐presenting cells. To determine whether this cytokine affects the course of immune response against C. neoformans, we added exogenous IL‐10 to cultured Cryptococcus‐laden monocytes plus T lymphocytes. The data from this study confirmed the down‐regulatory effect of exogenous IL‐10 on monocytes and expanded the known inhibitory role to include an increase of the deleterious effect due to capsular material of C. neoformans on (i) lymphoproliferation, (ii) down‐regulation of MHC class II molecules, (iii) inhibition of IL‐2 mRNA expression and protein secretion by T lymphocytes. These results indicate that the presence of IL‐10 in AIDS patients, due to the progression of disease, could represent a pivotal problem contributing to augment the pathogenic effect of C. neoformans.

B7 COSTIMULATORY LIGAND REGULATES DEVELOPMENT OF THE T-CELL RESPONSE TO CRYPTOCOCCUS NEOFORMANS

The contribution of B7 molecules to the induction and maintenance of the T‐cell response to the human pathogenic fungus Cryptococcus neoformans was investigated. T‐cell activation by C. neoformans was regulated by B7 molecules. This costimulatory signal was necessary for initiation and maintenance of the T‐cell response, through early and late requirements for B7–CD28 interaction. Blocking B7‐2 inhibited the normal T‐cell proliferative response. This inhibition was due, in part, to a reduced capability of Tu2003cells to produce interleukin‐2 (IL‐2). In contrast, the same T‐cell population produced more interferon‐γ. Suppression of the normal lymphoproliferation and IL‐2 secretion responses to encapsulated C. neoformans by antibodies to B7 was largely reversed by addition of the monoclonal antibody 2H1, that is reactive with the major capsular polysaccharide, glucuronoxylomannan. Overall, our data indicate that B7 molecules play a critical role in T‐cell activation by C. neoformans and suggest that appropriate manipulation could drive T helper type 1 cell development.

Human Immunodeficiency Virus Type 1 Envelope Protein gp120 Impairs Intracellular Antifungal Mechanisms in Human Monocytes

The key to success of fungal opportunistic pathogens in the immunocompromised host is related to survival inside phagocytic cells, which represent the first line of defense against microorganisms. The contribution of human immunodeficiency virus-1 recombinant envelope protein gp120 on effector functions of peripheral blood monocytes (PBM) against Candida albicans was investigated. gp120 binds CD4 receptors on PBM while not affecting the access of the fungus into the lysosome compartment. However, gp120 reduces the antifungal capacity of PBM. This phenomenon correlates with impaired oxygen-dependent antimicrobial machinery and reduced ability of phagolysosome acidification. The maintenance of phagolysosomal pH at approximately 6.2 restricts antimicrobial properties of the enzyme that work at a low pH, as evidenced by reduced antifungal capability of lysosomal protein extracted from gp120-treated PBM. These findings highlight gp120 perturbation of intracellular antimicrobial mechanisms of phagocytic cells and suggest a new aspect for gp120 in impairing immune functions.