Daniel Closa

Oxygen Free Radicals and the Systemic Inflammatory Response

The generation of oxygen free radicals is known to be involved in the development of the systemic inflammatory response syndrome. In addition to their actions as noxious mediators generated by inflammatory cells, these molecules play also a crucial role contributing to the onset and progression of inflammation in distant organs. In the early stages of the process, free radicals exert their actions via activation of nuclear factors, as NFκB or AP‐1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion molecules. Finally, free radicals exert their toxic effects at the site of inflammation by reacting with different cell components, inducing loss of function and cell death. This review focuses on progress in the understanding the different actions of free radicals at the sequential stages of the development of the systemic inflammatory response. IUBMB Life, 56: 185‐191, 2004

Effect of simultaneous inhibition of TNF-α production and xanthine oxidase in experimental acute pancreatitis: The role of mitogen activated protein kinases

Objective:To assess the effects of inhibiting both tumor necrosis factor (TNF)-α production and xanthine oxidase activity on the inflammatory response, mitogen-activated protein kinase (MAPK) activation and mortality in necrotizing acute pancreatitis in rats. Summary Background Data:Pancreatic injury triggers 2 major pathways involved in the systemic effects of severe acute pancreatitis: pro-inflammatory cytokines and oxidative stress. Methods:Pancreatitis was induced by intraductal infusion of 3.5% sodium taurocholate. We examined whether treatment with oxypurinol, a specific inhibitor of xanthine oxidase, and/or pentoxifylline, an inhibitor of TNF-α production, affects pancreatic damage, ascites, lung inflammation, and MAPK phosphorylation. Results:Oxypurinol prevented p38 phosphorylation in the pancreas and partially avoided the rise in lung myeloperoxidase activity. Pentoxifylline prevented erk 1/2 and JNK phosphorylation in the pancreas, and it partially reduced ascites and the rise in lung myeloperoxidase activity. Combined treatment with oxypurinol and pentoxifylline almost completely abolished ascites, MAPK phosphorylation in the pancreas, and the increase in lung myeloperoxidase activity. Histology revealed a reduction in pancreatic and lung damage. These changes were associated with a significant improvement of survival. Conclusions:Simultaneous inhibition of TNF-α production and xanthine oxidase activity greatly reduced local and systemic inflammatory response in acute pancreatitis and decreased mortality rate. These effects were associated with blockade of the 3 major MAPKs.

In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats

This study examines the activity of the antioxidant N‐acetylcysteine on bleomycin‐induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. Rats receiving N‐acetylcysteine (300 mg kg−1 day−1, intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N‐acetylcysteine. Alveolar macrophages from bleomycin‐exposed rats released augmented amounts of superoxide anion and nitric oxide. N‐Acetylcysteine did not modify superoxide anion generation but reduced the increased production of nitric oxide. N‐Acetylcysteine suppressed the bleomycin‐induced increased activation of lung NF‐κB (shift assay and immunohistochemistry), and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor‐α, interleukin‐β, interleukin‐6 and macrophage inflammatory protein‐2 observed in bronchoalveolar lavage fluid at 1 and 3 days postbleomycin exposure. At 15 days postbleomycin, N‐acetylcysteine decreased collagen deposition in bleomycin‐exposed rats (hydroxyproline content: 6351±669 and 4626±288 μg per lung in drug vehicle‐ and N‐acetylcysteine‐treated rats, respectively; P<0.05). Semiquantitative histological assessment at this stage showed less collagen deposition in N‐acetylcysteine‐treated rats compared to those receiving bleomycin alone. These results indicate that N‐acetylcysteine reduces the primary inflammatory events, thus preventing cellular damage and the subsequent development of pulmonary fibrosis in the bleomycin rat model.

Activation of alveolar macrophages in lung injury associated with experimental acute pancreatitis is mediated by the liver.

OBJECTIVE To evaluate (1) whether alveolar macrophages are activated as a consequence of acute pancreatitis (AP), (2) the implication of inflammatory factors released by these macrophages in the process of neutrophil migration into the lungs observed in lung injury induced by AP, and (3) the role of the liver in the activation of alveolar macrophages. SUMMARY BACKGROUND DATA Acute lung injury is the extrapancreatic complication most frequently associated with death and complications in severe AP. Neutrophil infiltration into the lungs seems to be related to the release of systemic and local mediators. The liver and alveolar macrophages are sources of mediators that have been suggested to participate in the lung damage associated with AP. METHODS Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. The inflammatory process in the lung and the activation of alveolar macrophages were investigated in animals with and without portocaval shunting 3 hours after AP induction. Alveolar macrophages were obtained by bronchoalveolar lavage. The generation of nitric oxide, leukotriene B4, tumor necrosis factor-alpha, and MIP-2 by alveolar macrophages and the chemotactic activity of supernatants of cultured macrophages were evaluated. RESULTS Pancreatitis was associated with increased infiltration of neutrophils into the lungs 3 hours after induction. This effect was prevented by the portocaval shunt. Alveolar macrophages obtained after induction of pancreatitis generated increased levels of nitric oxide, tumor necrosis factor-alpha, and MIP-2, but not leukotriene B4. In addition, supernatants of these macrophages exhibited a chemotactic activity for neutrophils when instilled into the lungs of unmanipulated animals. All these effects were abolished when portocaval shunting was carried out before induction of pancreatitis. CONCLUSION Lung damage induced by experimental AP is associated with alveolar macrophage activation. The liver mediates the alveolar macrophage activation in this experimental model.

Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease

Background and aims: Increased pancreatitis associated protein (PAP) mRNA has been reported in active inflammatory bowel disease (IBD). The aims of the current study were to characterise PAP production in IBD and the effects of PAP on inflammation. Patients and methods: Serum PAP levels were determined in healthy controls (n = 29), inflammatory controls (n = 14), and IBD patients (n = 171). Ex vivo PAP secretion in intestinal tissue was measured in 56 IBD patients and 13 healthy controls. Cellular origin of PAP was determined by immunohistochemistry. The effects of exogenous PAP on nuclear factor κB (NFκB) activation, proinflammatory cytokine production, and endothelial adhesion molecule expression were also analysed ex vivo. Results: Patients with active IBD had increased serum PAP levels compared with controls, and these levels correlated with clinical and endoscopic disease severity. Ex vivo intestinal PAP synthesis was increased in active IBD and correlated with endoscopic and histological severity of inflammatory lesions. PAP localised to colonic Paneth cells. Incubation of mucosa from active Crohn’s disease with PAP dose dependently reduced proinflammatory cytokines secretion. PAP prevented TNF-α induced NFκB activation in monocytic, epithelial, and endothelial cells and reduced proinflammatory cytokine mRNA levels and adhesion molecule expression. Conclusions: PAP is synthesised by Paneth cells and is overexpressed in colonic tissue of active IBD. PAP inhibits NFκB activation and downregulates cytokine production and adhesion molecule expression in inflamed tissue. It may represent an anti-inflammatory mechanism and new therapeutic strategy in IBD.

Role of P-Selectin and ICAM-1 in Pancreatitis-Induced Lung Inflammation in Rats: Significance of Oxidative Stress

OBJECTIVE To investigate the role of P-selectin and intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of lung injury associated with pancreatitis, and the relation between xanthine oxidase-derived oxidants and expression of these adhesion molecules. SUMMARY BACKGROUND DATA In acute pancreatitis, acute respiratory distress syndrome occurs in the early stages of disease. This process is mediated by neutrophil infiltration. METHODS Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. ICAM-1 and P-selectin expression was measured using radiolabeled monoclonal antibodies. Neutrophil infiltration and plasma levels of xanthine oxidase were also evaluated. RESULTS Pancreatitis induces increases in P-selectin expression in lung, whereas ICAM-1 is unchanged from baseline levels. Immunoneutralization of either P-selectin or ICAM-1 prevents the infiltration of neutrophils into the lung. Xanthine and xanthine oxidase activity were increased after induction of pancreatitis. Xanthine oxidase inhibition prevents the upregulation of P-selectin in lung and neutrophil infiltration. CONCLUSIONS During acute pancreatitis, P-selectin is upregulated in the pulmonary endothelium and is a key determinant of leukocyte recruitment. Constitutive ICAM-1 is also involved in the process of cell infiltration into the lung. The increased expression of P-selectin appears to be triggered by a mechanism dependent on free radicals generated by xanthine oxidase released by the damaged pancreas.

Oral N-acetylcysteine attenuates the rat pulmonary inflammatory response to antigen

Oxidative stress is involved in the pathophysiology of inflammatory airway diseases including asthma; therefore, antioxidants might be of clinical benefit in asthma treatment. In the present study, the effects of N‐acetylcysteine on sensitised brown Norway rats were examined. N‐Acetylcysteine (3 mmol·kg body weight−1 administered orally) was given daily for 1 week before challenge and various antigen-induced pulmonary responses were studied. Antigen exposure increased lipid peroxidation in bronchoalveolar lavage fluid (BALF) and oxidised glutathione levels in lung tissue 2 h after challenge. Lung nuclear transcription factor‐κB‐binding activity was increased 2 h after challenge, and BALF tumour necrosis factor‐α and inducible nitric oxide synthase expression in lungs peaked 4 h after challenge. Expression of intercellular adhesion molecule‐1 and mucin MUC5AC was also increased 4 h after challenge. These changes in oxidant status, transcription factor activation, and inflammatory cytokine and gene expression were reduced by N‐acetylcysteine. This thiol did not affect the immediate bronchospasm reaction to antigen in anaesthetised rats but inhibited airways hyperresponsiveness to 5‐hydroxytryptamine and the augmented eosinophil numbers in BALF, which appear 24 h after exposure of conscious rats to antigen aerosol, and abolished antigen-induced extravasation of Evans blue into BALF. These results indicate that oral N‐acetylcysteine exerts an antioxidant protective effect and attenuates pulmonary inflammation in experimental asthma.

Pancreatitis-associated protein I suppresses NF-κB activation through a JAK/STAT-mediated mechanism in epithelial cells

Pancreatitis-associated protein I (PAP I), also known as HIP, p23, or Reg2 protein, has recently been implicated in the endogenous regulation of inflammation. Although it was initially characterized as a protein that is overexpressed in acute pancreatitis, PAP I has also been associated with a number of inflammatory diseases, such as Crohn’s disease. Knowing that PAP I and IL-10 responses share several features, we have used a pancreatic acinar cell line (AR42J) to assess the extent to which their expression is reciprocally regulated, and whether the JAK/STAT and NF-κB signaling pathways are involved in the suppression of inflammation mediated by PAP I. We observed that PAP I is induced in epithelial cells by IL-10 and by PAP I itself. In contrast, we found phosphorylation and nuclear translocation of STAT3 and induction of suppressor of cytokine signaling 3 in response to PAP I exposure. Finally, a JAK-specific inhibitor, tyrphostin AG490, markedly prevented PAP I-induced NF-κB inhibition, pointing to a cross-talk between JAK/STAT3 and NF-κB signaling pathways. Together, these findings indicate that PAP I inhibits the inflammatory response by blocking NF-κB activation through a STAT3-dependent mechanism. Important functional similarities to the anti-inflammatory cytokine IL-10 suggest that PAP I could play a role similar to that of IL-10 in epithelial cells.

Experimental acute pancreatitis in PAP/HIP knock-out mice

Background and aims: PAP/HIP was first reported as an additional pancreatic secretory protein expressed during the acute phase of pancreatitis. It was shown in vitro to be anti-apoptotic and anti-inflammatory. This study aims to look at whether PAP/HIP plays the same role in vivo. Methods: A model of caerulein-induced pancreatitis was used to compare the outcome of pancreatitis in PAP/HIP−/− and wild-type mice. Results: PAP/HIP−/− mice showed the normal phenotype at birth and normal postnatal development. Caerulein-induced pancreatic necrosis was, however, less severe in PAP/HIP−/− mice than in wild-type mice, as judged by lower amylasemia and lipasemia levels and smaller areas of necrosis. On the contrary, pancreas from PAP/HIP−/− mice was more sensitive to apoptosis, in agreement with the anti-apoptotic effect of PAP/HIP in vitro. Surprisingly, pancreatic inflammation was more extensive in PAP/HIP−/− mice, as judged from histological parameters, increased myeloperoxidase activity and increased pro-inflammatory cytokine expression. This result, in apparent contradiction with the limited necrosis observed in these mice, is, however, in agreement with the anti-inflammatory function previously reported in vitro for PAP/HIP. This is supported by the observation that activation of the STAT3/SOCS3 pathway was strongly decreased in the pancreas of PAP/HIP−/− mice and by the reversion of the apoptotic and inflammatory phenotypes upon administration of recombinant PAP/HIP to PAP/HIP−/− mice. Conclusion: The anti-apoptotic and anti-inflammatory functions described in vitro for PAP/HIP have physiological relevance in the pancreas in vivo during caerulein-induced pancreatitis.

Free radicals generated by xanthine oxidase mediate pancreatitis-associated organ failure.

In the present study we evaluate the possibilitythat xanthine oxidase released by damaged pancreas couldact as a source of oxidative damage in systemic tissuesduring the early stages of acute pancreatitis. This was accomplished by evaluating the effectsof xanthine oxidase inhibition with oxypurinol infusedinto the portal vein. Under these conditions, weinhibited the enzyme before it reached the liver and other distant organs, without inducing changesin the severity of pancreatic damage. Results indicatethat pancreatitis parallels increases in xanthineoxidase activity in plasma. Superoxide radicalsgenerated by this enzyme appears to be involved in thedecrease of reduced glutathione levels in the plasma andliver. In addition, xanthine oxidase inhibition preventsthe infiltration of neutrophils into the lungs. We conclude that oxygen free radicals generatedby xanthine and xanthine oxidase released to thebloodstream are involved in the systemic organ failureassociated with acute pancreatitis.