Donna-Marie McCafferty

Nitric oxide and intestinal inflammation

Inflammation of the intestinal tract remains a very serious concern in the clinical setting. Unfortunately, to date, the mechanisms underlying many inflammatory conditions such as sepsis or inflammatory bowel diseases are poorly understood and our therapeutic interventions are less than ideal. Over the past decade, an abundance of research has been directed toward the role of nitric oxide (NO) in intestinal inflammation. It has become apparent that NO might have a dichotomous role as both a beneficial and detrimental molecule. Nitric oxide is a weak radical produced from L-arginine via the enzyme nitric oxide synthase (NOS). NOS exists in three distinct isoforms; constitutively (cNOS) expressed neuronal NOS (NOS1 or nNOS) and endothelial NOS (NOS3 or eNOS) or an inducible isoform (NOS2 or iNOS) capable of high production output of NO during inflammation. Constitutively expressed NOS has been shown to be critical to normal physiology and inhibition of these enzymes (nNOS or eNOS) caused damage. It has been proposed that the high output production of NO from iNOS causes injury, perhaps through the generation of potent radicals such as peroxynitrite and hence may explain the apparent dichotomous role of NO. However, recent studies have challenged this simple paradigm providing evidence that iNOS may have some protective role in some inflammatory models. Moreover, the importance of peroxynitrite has been questioned. In this review we discuss the role of cNOS and iNOS in intestinal inflammation and provide an overview of peroxynitrite in intestinal inflammation, highlighting some of the controversy that exists.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis

Background: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohns disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD‐like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase‐1‐dependent processing of inflammatory mediators, such as IL‐1&bgr; and IL‐18. Methods: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3−/− and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6‐trinitrobenzenesulfonic acid models of experimental colitis. Results: Nlrp3−/− mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL‐1&bgr;, reduced antiinflammatory cytokine IL‐10, and reduced protective growth factor TGF‐&bgr;. Macrophages isolated from Nlrp3−/− mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3‐deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3−/− mice displayed altered colonic &bgr;‐defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. Conclusions: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2011)

Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice

BACKGROUND Studies using inhibitors of nitric oxide synthase (NOS) to date are inconclusive regarding the role of inducible NOS (iNOS) in intestinal inflammation. AIMS (1) To examine the role of iNOS in the development of chronic intestinal inflammation; (2) to identify the cellular source(s) of iNOS. METHODS Colitis was induced by an intrarectal instillation of trinitrobenzene sulphonic acid (TNBS, 60 mg/ml, 30% ethanol), in wild type (control) or iNOS deficient mice. Mice were studied over 14 days; the colons were scored for injury and granulocyte infiltration was quantified. Blood to lumen leakage of51Cr-EDTA was measured as a quantitative index of mucosal damage. RESULTS At 24 and 72 hours, iNOS deficient mice had significantly increased macroscopic inflammation compared with wild type mice. Granulocyte infiltration increased significantly at 24 hours and remained elevated in iNOS deficient mice at 72 hours, but significantly decreased in controls. However, by seven days post-TNBS macroscopic damage, microscopic histology, granulocyte infiltration, and mucosal permeability did not differ between wild type and iNOS deficient mice. A four- to fivefold increase in iNOS mRNA was observed in wild type mice at 72 hours and seven days post-TNBS and was absent in iNOS deficient mice. Immunohistochemistry techniques showed that iNOS expression was predominantly localised in neutrophils, with some staining also in macrophages. CONCLUSIONS These results suggest that leucocyte derived iNOS ameliorates the early phase, but does not impact on the chronic phase of TNBS induced colitis despite the presence of iNOS.

Role of p38 Mitogen-Activated Protein Kinase in Chemokine-Induced Emigration and Chemotaxis In Vivo

It has been proposed that L-selectin engagement with ligand activates p38 mitogen-activated protein kinase (MAPK) and can impact on downstream events of leukocyte rolling, including adhesion, and emigration. Using a novel chemotactic assay in vivo, we visualized slow release of chemokine from an agarose gel positioned 350 μm from a postcapillary venule, which induced directed migration (chemotaxis) of neutrophils. In this system, keratinocyte-derived cytokine induced phosphorylation of p38 MAPK, which phosphorylated a downstream protein (ATF-2). This latter event was blocked by the concentration of p38 inhibitors used in this study. Mice were treated with two different p38 inhibitors: SKF86002 and SB203580. Neither inhibitor affected rolling or adhesion in microvessels. Intravenous treatment with SFK86002 (5, 10, and 20 mg/kg) 30 min before the inflammatory stimulus inhibited the total number of emigrated cells at a dose of 20 mg/kg (62%, p < 0.05), despite the presence of many adherent cells within the vessels. A similar inhibition was observed with 20 mg/kg of a second p38 inhibitor SB203580 (67%, p < 0.05). In addition to emigration, both p38 inhibitors impaired the ability of emigrated cells to migrate through the tissue toward the chemotactic stimulus. In fact, the majority of emigrated leukocytes in p38 inhibitor-treated animals remained within 50 μm of the venule. Superfusion of the tissue with SKF86002 (0.7 mM) to impact only on emigrated and not vascular leukocytes resulted in no impairment in emigration, but in a significant reduction in chemotaxis away from the vessel wall. Again, the majority of emigrated leukocytes remained within 50 μm of the blood vessel. Our results suggest that p38 does not affect rolling or adhesion, but that it is involved in leukocyte emigration and chemotaxis through interstitium in response to keratinocyte-derived cytokine in vivo.

Indomethacin-induced gastric injury and leukocyte adherence in arthritic versus healthy rats

BACKGROUND & AIMS Arthritic patients are at greater risk of developing nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers than other NSAID users. Leukocyte adherence to the vascular endothelium has been suggested to play an important role in the pathogenesis of experimental NSAID-associated gastropathy. The aim of this study was to examine the role of leukocyte adherence in NSAID-induced gastric injury in healthy and adjuvant-induced arthritic rats. METHODS Leukocyte adherence was examined using intravital microscopy before and after indomethacin administration. The role of CD18 and intercellular adhesion molecule 1 (ICAM-1) in indomethacin-induced gastric injury and leukocyte adherence was examined using specific monoclonal antibodies against these molecules. RESULTS Indomethacin (5-10 mg/kg) caused significantly more gastric damage in arthritic than normal rats, and the former group had significantly greater levels of leukocyte adherence to mesenteric postcapillary venules under basal conditions. Dexamethasone markedly reduced basal and indomethacin-induced leukocyte adherence and the extent of gastric damage. In arthritic rats, pretreatment with a monoclonal antibody directed against ICAM-1 significantly reduced gastric damage and leukocyte adherence to the levels observed in healthy rats, whereas an antibody directed against CD18 had no effect. CONCLUSIONS Indomethacin-induced damage in healthy and arthritic rats is largely dependent on ICAM-1 expression. The increased susceptibility of arthritic rats to indomethacin-induced gastric injury may be partly related to elevated levels of ICAM-1-dependent leukocyte adherence.

Tissue-Selective Inhibition of Prostaglandin Synthesis in Rat by Tepoxalin: Anti-inflammatory Without Gastropathy?

BACKGROUND Inhibition of prostaglandin synthesis is likely a primary mechanism for both the anti-inflammatory and ulcerogenic effects of nonsteroidal anti-inflammatory drugs (NSAIDs). The present study examined the mechanism underlying the ability of a novel anti-inflammatory drug, tepoxalin, to suppress prostaglandin synthesis without inducing gastric mucosal injury. METHODS The effects on prostaglandin synthesis by various tissues of tepoxalin, diclofenac, and indomethacin were examined in vivo and in vitro. These compounds were also studied in two inflammation models. The capacity of indomethacin and tepoxalin to induced antral ulceration in the rabbit was compared. RESULTS In most tissues, tepoxalin was a weaker inhibitor of prostaglandin synthesis than the two NSAIDs. However, at a site of peripheral inflammation, tepoxalin was comparable with the NSAIDs in suppressing prostaglandin synthesis and in exerting anti-inflammatory effects. Indomethacin induced penetrating antral ulcers in rabbits whereas tepoxalin produced no detectable mucosal injury. CONCLUSIONS The ability of tepoxalin to suppress inflammation without causing gastric mucosal injury appears to be related to its differential suppression of prostaglandin synthesis in various tissues. Compounds that selectively inhibit prostaglandin synthesis at sites of inflammation may represent a class of anti-inflammatory drugs without detrimental effects on the stomach.

The atypical cannabinoid O-1602 protects against experimental colitis and inhibits neutrophil recruitment.

Background: Cannabinoids are known to reduce intestinal inflammation. Atypical cannabinoids produce pharmacological effects via unidentified targets. We were interested in whether the atypical cannabinoid O‐1602, reportedly an agonist of the putative cannabinoid receptor GPR55, reduces disease severity of dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS)‐induced colitis in C57BL/6N and CD1 mice. Methods: DSS (2.5% and 4%) was supplied in drinking water for 1 week while TNBS (4 mg) was applied as a single intrarectal bolus. Results: Both treatments caused severe colitis. Injection of O‐1602 (5 mg/kg intraperitoneally) significantly reduced macroscopic and histological colitis scores, and myeloperoxidase activity. The protective effect was still present in cannabinoid receptor 1 (CB1) and 2 (CB2) double knockout mice and mice lacking the GPR55 gene. To investigate a potential mechanism underlying the protection by O‐1602 we performed neutrophil chemotactic assays. O‐1602 concentration‐dependently inhibited migration of murine neutrophils to keratinocyte‐derived chemokine (KC), N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), and the N‐formyl‐peptide receptor ligand WKYMVm. The inhibitory effect of O‐1602 was preserved in neutrophils from CB1/CB2 double knockout and GPR55 knockout mice. No differences were seen in locomotor activity between O‐1602‐treated and control mice, indicating lack of central sedation by this compound. Conclusions: Our data demonstrate that O‐1602 is protective against experimentally induced colitis and inhibits neutrophil recruitment independently of CB1, CB2, and GPR55 receptors. Thus, atypical cannabinoids represent a novel class of therapeutics that may be useful for the treatment of inflammatory bowel diseases. (Inflamm Bowel Dis 2010;)

Interleukin-10-independent anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is an important intestinal growth factor with anti-inflammatory activity. We hypothesized that GLP-2 decreases mucosal inflammation and the associated increased epithelial proliferation by downregulation of Th1 cytokines attributable to reprogramming of lamina propria immune regulatory cells via an interleukin-10 (IL-10)-independent pathway. The effects of GLP-2 treatment were studied using the IL-10-deficient (IL-10(-/-)) mouse model of colitis. Wild-type and IL-10(-/-) mice received saline or GLP-2 (50 microg/kg sc) treatment for 5 days. GLP-2 treatment resulted in significant amelioration of animal weight loss and reduced intestinal inflammation as assessed by histopathology and myeloperoxidase levels compared with saline-treated animals. In colitis animals, GLP-2 treatment also reduced crypt cell proliferation and crypt cell apoptosis. Proinflammatory (IL-1beta, TNF-alpha, IFN-gamma,) cytokine protein levels were significantly reduced after GLP-2 treatment, whereas IL-4 was significantly increased and IL-6 production was unchanged. Fluorescence-activated cell sorting analysis of lamina propria cells demonstrated a decrease in the CD4(+) T cell population following GLP-2 treatment in colitic mice and an increase in CD11b(+)/F4/80(+) macrophages but no change in CD25(+)FoxP3 T cells or CD11c(+) dendritic cells. In colitis animals, intracellular cytokine analysis demonstrated that GLP-2 decreased lamina propria macrophage TNF-alpha production but increased IGF-1 production, whereas transforming growth factor-beta was unchanged. GLP-2-mediated reduction of crypt cell proliferation was associated with an increase in intestinal epithelial cell suppressor of cytokine signaling (SOCS)-3 expression and reduced STAT-3 signaling. This study shows that the anti-inflammatory effects of GLP-2 are IL-10 independent and that GLP-2 alters the mucosal response of inflamed intestinal epithelial cells and macrophages. In addition, the suggested mechanism of the reduction in inflammation-induced proliferation is attributable to GLP-2 activation of the SOCS-3 pathway, which antagonizes the IL-6-mediated increase in STAT-3 signaling.

Granulocyte-macrophage colony-stimulating factor (GM-CSF): a chemoattractive agent for murine leukocytes in vivo.

GM‐CSF is well recognized as a proliferative agent for hematopoietic cells and exerts a priming function on neutrophils. The aim of this study was to determine if GM‐CSF has a role as a neutrophil chemoattractant in vivo and if it can contribute to recruitment during intestinal inflammation. Initial studies in vitro, using the under‐agarose gel assay, determined that GM‐CSF can induce neutrophil migration at a much lower molar concentration than the fMLP‐like peptide WKYMVm (33.5–134 nM vs. 1–10 μM). GM‐CSF‐induced neutrophil migration was ablated (<95%) using neutrophils derived from GMCSFRβ−/− mice and significantly attenuated by 42% in PI3Kγ−/−neutrophils. In vivo, a significant increase in leukocyte recruitment was observed using intravital microscopy 4 h post‐GM‐CSF (10 μg/kg) injection, which was comparable with leukocyte recruitment induced by KC (40 μg/kg). GM‐CSF‐induced recruitment was abolished, and KC‐induced recruitment was maintained in GMCSFRβ−/− mice. Furthermore, in vivo migration of extravascular leukocytes was observed toward a gel containing GM‐CSF in WT but not GMCSFRβ−/− mice. Finally, in a model of intestinal inflammation (TNBS‐induced colitis), colonic neutrophil recruitment, assessed using the MPO assay, was attenuated significantly in anti‐GM‐CSF‐treated mice or GMCSFRβ−/− mice. These data demonstrate that GM‐CSF is a potent chemoattractant in vitro and can recruit neutrophils from the microvasculature and induce extravascular migration in vivo in a β subunit‐dependent manner. This property of GM‐CSF may contribute significantly to recruitment during intestinal inflammation.

Neuronal nitric oxide synthase (NOS) regulates leukocyte-endothelial cell interactions in endothelial NOS deficient mice

The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte – endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS−/− mice. Baseline leukocyte rolling and adhesion revealed no differences between wild‐type and eNOS−/− mice in either the cremasteric or intestinal microcirculations. Superfusion with L‐NAME (100 μM) caused a progressive and significant increase in leukocyte adhesion in both wild‐type and eNOS−/− mice, without detecting differences between the two strains of mice. Superfusion with 7‐nitroindazole (100 μM), a selective inhibitor of nNOS, had no effect on leukocyte adhesion in wild‐type animals. However, it increased leukocyte adhesion significantly in eNOS−/− mice, which was reversed by systemic L‐arginine pre‐administration. Stimulation of the microvasculature with H2O2 (100 μM) induced a transient elevation in leukocyte rolling in wild‐type mice. Conversely, the effect persisted during the entire 60 min of experimental protocol in eNOS−/− mice either with or without 7‐nitroindazole. Semi‐quantitative analysis by RT – PCR of the mRNA for nNOS levels in eNOS−/− and wild‐type animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS−/− mice. In conclusion, we have demonstrated that leukocyte‐endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS−/− mice. Interestingly, unlike eNOS there was insufficient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.