Laura Gray

Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease

The inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.The inflammatory bowel diseases (IBD; Crohns disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.

Role of Nitric Oxide in the Regulation of Acute and Chronic Inflammation

Recent studies by a number of different laboratories have implicated nitric oxide (NO) as an important modulator of a variety of acute and chronic inflammatory disorders. A hallmark of inflammation is the adhesion of leukocytes to post-capillary venular endothelium and the infiltration of leukocytes into the tissue interstitium. Leukocyte adhesion and infiltration is known to be dependent on interaction of the leukocytes with the endothelial cell surface via a class of glycoproteins collectively known as endothelial cell adhesion molecules (ECAMs). Several recent studies suggest that NO may modulate cytokine-induced ECAM expression in cultured endothelial cells in vitro by regulating the activation of nuclear transcription factor kappa B (NF-kappaB). This discussion reviews some of the more recent studies that assess the role of the different NOS isoforms on the inflammatory response in vivo.

Mouse model of liver ischemia and reperfusion injury : method for studying reactive oxygen and nitrogen metabolites in vivo

The mouse model of liver ischemia and reperfusion injury has proven to be valuable for our understanding of the role that reactive oxygen and nitrogen metabolites play in postischemic tissue injury. This methods paper provides a detailed protocol for inducing partial liver ischemia followed by reperfusion. Liver ischemia is induced in anesthetized mice by cross-clamping the hepatic artery and portal vein for varying lengths of time, resulting in deprivation of blood flow to approximately 70% of the liver. Restoration of blood flow to the ischemic lobes enhances superoxide production concomitant with a rapid and marked decrease in the bioavailability of nitric oxide, resulting in alterations in the redox state of the liver in favor of a more oxidative environment. This hepatocellular oxidative stress induces the activation of oxidant-sensitive transcription factors followed by the upregulation of proinflammatory cytokines and mediators that ultimately lead to liver injury. This model can be induced in any strain or sex of mouse and requires 1-2 months of practice to become proficient in the surgery and animal manipulation. The roles of various reactive metabolites of oxygen and nitrogen may be evaluated using genetically engineered mice as well as selective molecular, cellular, and/or pharmacological agents.

Role of inducible nitric oxide synthase in the regulation of VCAM-1 expression in gut inflammation

The objectives of this study were to assess the role of the inducible isoform of nitric oxide synthase (iNOS) on vascular cell adhesion molecule 1 (VCAM-1) expression in vivo in an acute model of inflammation induced in iNOS-deficient (iNOS-/-) mice and compare these data to those obtained by pharmacological inhibition of iNOS in a CD4+ T lymphocyte-dependent model of chronic colitis. VCAM-1 expression was quantified in vivo using the dual radiolabel monoclonal antibody technique. We found that intraperitoneal injection of 10 μg/kg tumor necrosis factor-α (TNF-α) enhanced VCAM-1 expression by approximately twofold in the colon, cecum, and stomach but not small intestine in iNOS-/-mice compared with TNF-α-injected wild-type mice. Injection of wild-type mice with 25 μg/kg TNF-α further enhanced VCAM-1 expression by approximately twofold compared with wild-type mice injected with 10 μg/kg TNF-α; however, VCAM-1 expression was not further enhanced in any gastrointestinal organ system in iNOS-/- mice. In a second series of experiments, we found that continuous inhibition of iNOS using oral administration of N G-iminoethyl-l-lysine did not alter the enhanced levels of VCAM-1 expression in the colon nor did it alter the severity of colonic inflammation in SCID mice reconstituted with CD4+, CD45RBhigh T cells. We conclude that iNOS may regulate VCAM-1 expression in acute inflammation; however, this effect is modest and tissue specific and occurs only when VCAM-1 expression is submaximal. iNOS does not appear to modulate VCAM-1 expression in an immune model of chronic colitis.The objectives of this study were to assess the role of the inducible isoform of nitric oxide synthase (iNOS) on vascular cell adhesion molecule 1 (VCAM-1) expression in vivo in an acute model of inflammation induced in iNOS-deficient (iNOS-/-) mice and compare these data to those obtained by pharmacological inhibition of iNOS in a CD4+ T lymphocyte-dependent model of chronic colitis. VCAM-1 expression was quantified in vivo using the dual radiolabel monoclonal antibody technique. We found that intraperitoneal injection of 10 microg/kg tumor necrosis factor-alpha (TNF-alpha) enhanced VCAM-1 expression by approximately twofold in the colon, cecum, and stomach but not small intestine in iNOS-/- mice compared with TNF-alpha-injected wild-type mice. Injection of wild-type mice with 25 microg/kg TNF-alpha further enhanced VCAM-1 expression by approximately twofold compared with wild-type mice injected with 10 microg/kg TNF-alpha; however, VCAM-1 expression was not further enhanced in any gastrointestinal organ system in iNOS-/- mice. In a second series of experiments, we found that continuous inhibition of iNOS using oral administration of NG-iminoethyl-L-lysine did not alter the enhanced levels of VCAM-1 expression in the colon nor did it alter the severity of colonic inflammation in SCID mice reconstituted with CD4+, CD45RB(high) T cells. We conclude that iNOS may regulate VCAM-1 expression in acute inflammation; however, this effect is modest and tissue specific and occurs only when VCAM-1 expression is submaximal. iNOS does not appear to modulate VCAM-1 expression in an immune model of chronic colitis.

Cytokine and adhesion molecule expression in SCID mice reconstituted with CD4+ T cells.

Summary: The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate‐buffered saline (PBS) developed clinical evidence of colitis at 6‐8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Thl and macrophage‐derived cytokines including interferon &ggr;, tumor necrosis factor‐&agr;, interleukin (IL)‐1&bgr;, IL‐6, IL‐12, and IL‐18 lymphotoxin‐&bgr;. In addition, message levels and vascular surface expression of ICAM‐1, VCAM‐1, and MAdCAM‐1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

Therapeutic evaluation of ex vivo-generated versus natural regulatory T-cells in a mouse model of chronic gut inflammation.

Abstract:The objectives of this study were to (a) evaluate and compare the ability of ex vivo-generated induced regulatory T cells (iTregs) and freshly isolated natural Tregs (nTregs) to reverse/attenuate preexisting intestinal inflammation in a mouse model of chronic colitis and (b) quantify the Treg-targeted gene expression profiles of these two Treg populations. We found that ex vivo-generated iTregs were significantly more potent than nTregs at attenuating preexisting colitis. This superior therapeutic activity was associated with increased accumulation of iTregs within the mesenteric lymph nodes and large and significant reductions in interleukin (IL)-6 and IL-17A expression in the colons of iTreg- versus nTreg-treated mice. The enhanced immunosuppressive activity of iTregs was not because of increased expression or stability of Foxp3 as iTregs and nTregs obtained from the mesenteric lymph nodes, and colons of reconstituted mice expressed similar levels of this important transcription factor. In addition, we observed a total of 27 genes that were either upregulated or downregulated in iTregs when compared with nTregs. Although iTregs were found to be superior at reversing established disease, their message levels of IL-10 and IL-35 and surface expression of the gut-homing molecules CCR9 and &agr;4&bgr;7 were significantly reduced when compared with nTregs. Taken together, our data demonstrate that ex vivo-generated iTregs are significantly more potent than nTregs at attenuating preexisting gut inflammation despite reduced expression of classical regulatory cytokines and gut-homing molecules. Our data suggest that the immunosuppressive activity of iTregs may be because of their ability to directly or indirectly decrease expression of IL-6 and IL-17A within the inflamed bowel.

Role of the Gut-associated and Secondary Lymphoid Tissue in the Induction of Chronic Colitis

Background: It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut‐associated lymphoid tissue (GALT; Peyers patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice. Methods: Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene‐1‐deficient mice (RAG−/−) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxin&bgr;‐deficient (LT&bgr;−/−) mice with RAG−/− animals to generate LT&bgr;−/− x RAG−/− offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4+CD45RBhigh) T‐cells were adoptively transferred into the different groups of RAG−/− recipients to induce chronic colitis. Results: We found that at 3‐5 wks following T‐cell transfer, all four of the surgically‐manipulated RAG−/− groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut‐homing markers nor were there any differences noted in IFN‐&ggr; and IL‐17 generation by mononuclear cells isolated among these surgically‐manipulated mice. Although we anticipated that LT&bgr;−/− x RAG−/− mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LT&bgr;−/− x RAG−/−mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RBhigh T‐cells into LT&bgr;−/− x RAG−/− mice or their littermate controls (LT&bgr;+/+ x RAG−/−) induced rapid and severe colitis in both groups. Conclusions: Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T‐and/or B‐cells may be required for the development of MLNs in LT&bgr;−/− mice. (Inflamm Bowel Dis 2011;)

Role of LFA-1 in the activation and trafficking of T cells: implications in the induction of chronic colitis.

Introduction: We have previously demonstrated that adoptive transfer of naïve CD4+ T cells devoid of lymphocyte function‐associated antigen‐1‐deficient (LFA‐1; CD11a/CD18) into recombination activating gene‐1 (RAG‐1) deficient (RAG−/−) mice fails to induce chronic colitis whereas transfer of wild type (WT) T‐cells induces unrelenting and chronic disease. Methods: The objectives of this study were to assess the role of lymphocyte function‐associated antigen‐1 (LFA‐1) in enteric antigen (EAg)‐induced activation of T cells in vitro and in vivo and to define the importance of this integrin in promoting trafficking of T cells to the mesenteric lymph nodes (MLNs) and colon. Results: We found that EAg‐pulsed dendritic cells (DCs) induced proliferation of LFA‐1‐deficient (CD11a−/−) CD4+ T cells that was very similar to that induced using WT T cells, suggesting that LFA‐1 is not required for activation/proliferation of T cells in vitro. Coculture of WT or CD11a−/− T cells with EAg‐pulsed DCs induced the generation of similar amounts of interferon‐gamma, interleukin (IL)‐4, and IL‐10, whereas IL‐17A production was reduced ≈2‐fold in cocultures with CD11a−/− T cells. Short‐term (20–22 hours) trafficking studies demonstrated that while both WT and CD11a−/− T cells migrated equally well into the spleen, liver, lungs, small intestine, cecum, and colon, trafficking of CD11a−/− T cells to the MLNs was reduced by 50% when compared to WT T cells. When the observation period was extended to 3–7 days posttransfer, we observed ≈2–3‐fold more WT T cells within the MLNs and colon than CD11a−/− T cells, whereas T‐cell proliferation (as measured by CFSE dilution) was comparable in both populations. Conclusions: Taken together, our data suggest that LFA‐1 is not required for EAg‐induced activation of CD4+ T cells in vitro or in vivo but is required for trafficking of T cells to the MLNs and homing of colitogenic effector cells to the colon where they initiate chronic gut inflammation. (Inflamm Bowel Dis 2012;)