Protease inhibitory activity of secretory leukocyte protease inhibitor ameliorates murine experimental colitis by protecting the intestinal epithelial barrier

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestine, and the dysfunction of intestinal epithelial barrier (IEB) may trigger the onset of IBD. Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that has been implicated in the tissue-protective effect in the skin and lung. We found that SLPI was induced in lipopolysaccharides-treated colon carcinoma cell line and in the colon of dextran sulfate sodium (DSS)-treated mice. SLPI-deficient mice were administered DSS to induce colitis and sustained severe inflammation compared with wild-type mice. The colonic mucosa of SLPI-deficient mice showed more severe inflammation with neutrophil infiltration and higher levels of proinflammatory cytokines compared with control mice. Moreover, neutrophil elastase (NE) activity in SLPI-deficient mice was increased and IEB function was severely impaired in the colon, accompanied with the increased number of apoptotic cells. Importantly, we demonstrated that DSS-induced colitis was ameliorated by administration of protease inhibitor SSR69071 and recombinant SLPI. These results suggest that the protease inhibitory activity of SLPI protects from colitis by preventing IEB dysfunction caused by excessive NE activity, which provides insight into the novel function of SLPI in the regulation of gut homeostasis and therapeutic approaches for IBD.     

Protease expression in dedifferentiated parosteal osteosarcoma

BACKGROUND: Parosteal osteosarcoma with dedifferentiation provides a useful model to study tumor progression from an indolent locally aggressive neoplasm to highly lethal metastasizing malignancy. Up-regulation of the proteolytic enzymes participating in stromal degradation is known to promote invasive growth and metastasis of several human and experimental tumors. METHODS: The expression patterns of urokinasase plasminogen activator (u-PA), its cell-surface receptor (u-PAR), and cathepsin B were analyzed by immunohistochemical techniques in 11 cases of parosteal osteosarcoma and in 4 cases of dedifferentiated parosteal osteosarcoma. RESULTS: Both enzymes and the receptor were coexpressed in most tumor cells of parosteal and dedifferentiated parosteal osteosarcoma. Their expression was strikingly enhanced in the dedifferentiated high-grade component of the tumors. Tumor cells involved in bone production (ie, those adjacent to tumor produced bone trabeculae) exhibited equally strong expression of u-PA, u-PAR, and cathepsin B, regardless of their histologic grade. Expression of u-PA, u-PAR, and cathepsin B was undetectable in the "normalized" cells embedded in the well-developed tumor bone trabeculae. CONCLUSION: These data indicate that u-PA and its interacting molecules, such as u-PAR and cathepsin B, may have some contributory effects on the metastatic potential of tumor cells in dedifferentiated parosteal osteosarcoma.     

Proglumide attenuates experimental colitis in rats.

Ulcerative colitis is associated with altered contractile activity and transit time of colon. On the other hand, cholecystokinin (CCK) has been shown to play an important role in regulation of gastrointestinal motor function including colonic contraction and transit. In the present study, an attempt was made to study the effect of proglumide, a CCK receptor antagonist on experimental colitis in rats. Experimental colitis was induced in male Sprague-Dawley rats by instilling 1 ml of 4% acetic acid followed by flushing with 0.5 ml air. The rats were kept in a head-down position for 30s. Finally, each rat received 1.5 ml colonic wash with 1.5 ml saline. Four groups of rats received proglumide orally (0, 250, 500 and 1000mg/kg). The first dose of proglumide was given 1 h before acetic acid challenge, whereas the second dose of proglumide was given 25 h after the first dose. Sham control rats received an equal volume of saline instead of acetic acid. Forty-eight hours after the acetic acid challenge, the colon was removed, weighed and split longitudinally and scored for injury. Part of the colon was used for histopathological study as well as analysis of myeloperoxidase (MPO) activity (as a marker of neutrophil activity). Acetic acid produced severe diarrhea and exfoliation of the colonic epithelium accompanied by extensive destruction of the mucosal interstitium. Proglumide dose dependently protected rats against acetic acid-induced increase in colon weight, diarrhea, MPO activity and colonic injury. Inhibition of CCK exerts a beneficial effect in experimental colitis. Further studies are warranted to determine the mechanism of protection and the therapeutic potential of CCK inhibitors.     

Lack of TNFR2 expression by CD4+ T cells exacerbates experimental colitis

TNF plays fundamental roles in the induction and perpetuation of inflammation. The effects of TNF are mediated through TNF receptor (TNFR) 1 or 2. As these two receptors mediate different functions, selective targeting of one receptor may represent a more specific treatment for inflammatory disorders than the complete blocking of TNF. TNFR2 expression is up‐regulated in inflammatory bowel disease. Hence, we directly assessed the role of TNFR2 signaling in the CD4⁺ T‐cell transfer model of colitis using TNFR2^?/? or WT mice as donors of colitogenic CD4⁺CD45RB^hi T cells for transfer into syngeneic RAG2^?/? or RAG2^?/?TNFR2^?/? recipient mice. Although the absence of TNFR2 expression by non‐lymphoid cells of the recipient mice does not influence the course of colitis, transfer of TNFR2^?/? CD4⁺ T cells leads to an accelerated onset of disease and to more severe signs of inflammation. The enhanced colitogenic potential of TNFR2^?/? CD4⁺ T cells is associated with reduced activation‐induced cell death, resulting in an increased accumulation of TNFR2^?/? CD4⁺ T cells. Hence, TNFR2 signaling is crucial for the TNF‐dependent contraction of the disease‐inducing T cells. Therefore, a selective blocking of TNFR2 may lead to exacerbation rather than attenuation of T‐cell‐mediated inflammatory disorders.     

Transmucosal potential difference in experimental colitis in rats

Colon transmucosal potential difference (TPD), macro- and microscopic lesions, myeloperoxidase activity, and leukotriene levels were studied after the induction of experimental colitis in the rat. Forty-three male Wistar rats were subjected to the instillation of 200 mg/ml 2,4,6-trinitrobenzenesulfonic acid (TNB) solution through a rectal cannula. TPD measurements were made at different distances from the anus before and 24 h and one, two, three, and four weeks after lesion induction. Leukotriene B₄ levels were assayed by intracolonic dialysis 24 h and one, two, three and four weeks after lesion induction. Macro- and microscopic evaluations were made of the bowel lesions, and myeloperoxidase activity was assayed. The mean basal TPD was ?46.06 mV at 1 cm from the anus, and +10.86 mV in the proximal colon. Twenty-four hours after lesion induction the values proved markedly positive. This was correlated with an abrupt increase in LTB₄ levels and myeloperoxidase activity. After one week the TPD values exhibited a greater electronegativity, returning to basal values by the fourth week after lesion induction. This coincided with an improved macroscopic lesion index, LTB₄ levels, and myeloperoxidase activity. In conclusion, TPD is a useful indicator of acute colonic lesions and correlates well with LTB₄ and myeloperoxidase assays. Moreover, the parameter is able to delimit lesion evolution, reflecting possiblead integrum restoration of the bowel mucosa.     

Differential angiogenic regulation of experimental colitis

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract with unknown multifactorial etiology that, among other things, result in alteration and dysfunction of the intestinal microvasculature. Clinical observations of increased colon microvascular density during IBD have been made. However, there have been no reports investigating the physiological or pathological importance of angiogenic stimulation during the development of intestinal inflammation. Here we report that the dextran sodium sulfate and CD4+CD45RBhigh T-cell transfer models of colitis stimulate angiogenesis that results in increased blood vessel density concomitant with increased histopathology, suggesting that the neovasculature contributes to tissue damage during colitis. We also show that leukocyte infiltration is an obligatory requirement for the stimulation of angiogenesis. The angiogenic response during experimental colitis was differentially regulated in that the production of various angiogenic mediators was diverse between the two models with only a small group of molecules being similarly controlled. Importantly, treatment with the anti-angiogenic agent thalidomide or ATN-161 significantly reduced angiogenic activity and associated tissue histopathology during experimental colitis. Our findings identify a direct pathological link between angiogenesis and the development of experimental colitis, representing a novel therapeutic target for IBD.     

Soluble CD83 ameliorates experimental colitis in mice

The physiological balance between pro- and anti-inflammatory processes is dysregulated in inflammatory bowel diseases (IBD) as in Crohn's disease and ulcerative colitis. Conventional therapy uses anti-inflammatory and immunosuppressive corticosteroids to treat acute-phase symptoms. However, low remission rate and strong side effects of these therapies are not satisfying. Thus, there is a high medical need for new therapeutic strategies. Soluble CD83, the extracellular domain of the transmembrane CD83 molecule, has been reported to have interesting therapeutic and immunosuppressive properties by suppressing dendritic cell (DC)-mediated T-cell activation and inducing tolerogenic DCs. However, the expression and function of CD83 in IBD is still unknown. Here, we show that CD83 expression is upregulated by different leukocyte populations in a chemical-induced murine colitis model. Furthermore, in this study the potential of sCD83 to modulate colitis using an experimental murine colitis model was investigated. Strikingly, sCD83 ameliorated the clinical disease symptoms, drastically reduced mortality, and strongly decreased inflammatory cytokine expression in mesenteric lymph nodes and colon. The infiltration of macrophages and granulocytes into colonic tissues was vigorously inhibited. Mechanistically, we could show that sCD83-induced expression of indolamine 2,3-dioxygenase is essential for its protective effects.     

Intestinal nematode infection ameliorates experimental colitis in mice

Epidemiological studies suggest that inflammatory bowel disease (IBD) is common in developed countries and rare in countries where intestinal nematode infections are common. T cells are critical in many immune responses, including those associated with IBD and nematode infection. Among the distinct T helper (Th) cell subsets, Th1-type immune response is predominantly associated with Crohn's disease, while many nematode infections generate a strong Th2 response. The reciprocal cross regulation between Th1 and Th2 cells suggests that generation of a Th2 response by nematodes could prevent or reduce the effects of Th1-mediated diseases. In the present study, we investigated the effect of polarizing the immune response toward the Th2 type, using intestinal nematode infection, on subsequent experimental colitis. Mice were infected with the intestinal nematode Trichinella spiralis and allowed to recover before colitis was induced with dinitrobenzene sulfonic acid. The mice were sacrificed postcolitis to assess colonic damage macroscopically, histologically, and by myeloperoxidase (MPO) activity and Th cytokines. Prior nematode infection reduced the severity of colitis both macroscopically and histologically together with a decreased mortality and was correlated with a down-regulation of MPO activity, Th1-type cytokine expression in colonic tissue, and emergence of a Th2-type immune response. These results indicate a protective role of nematode infection in Th1 cell-driven inflammation and prompt consideration of a novel therapeutic strategy in IBD based on immunological distraction.     

Compromised neuroimmune status in rats with experimental colitis

In colitis, chronic and recurrent inflammation is associated with a breakdown in host defence mechanisms that leads to local and systemic infection. Whether this is due to a compromised neuroimmune response has not been studied. Our aim was to determine if colitis altered the host neuroimmune response as reflected in either body temperature rhythm or the febrile responses to lipopolysaccharide (LPS). Body temperature was monitored by telemetry from conscious, unrestrained male rats treated with trinitrobenzene sulphonic acid or saline. Twenty-six days after initial induction, colitis was reactivated. Animals were given LPS (50 μg kg⁻¹ Escherichia coli LPS) during colitis and after reactivation. At the peak of colitis, treated rats showed a disruption of circadian body temperature rhythm, manifested as day-time fever followed by night-time hypothermia. In response to LPS, controls displayed a characteristic fever, whereas treated animals had a significantly reduced fever and low plasma levels of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). During reactivation of colitis, treated animals did not mount a fever or exhibit increased plasma levels of IL-6 and TNF-α after LPS. We conclude that experimental colitis is associated with a compromised neuroimmune status.     

Protease inhibitor SERPINA1 expression in epithelial ovarian cancer

Epithelial ovarian cancer is the most lethal gynecologic cancer with a 5 years survival rate of 30–40% in patients diagnosed with high-grade invasive disease (TOV). This is in stark contrast to the 95% 5 years survival rate in ovarian cancer patients diagnosed with low malignant potential (LMP) disease. The progression from localized tumor to invasive metastasis involves matrix proteolysis. Protease inhibitors are thought to play a key role by limiting this process. Using the Affymetrix HG-U133A GeneChip array, we have studied all serine protease inhibitors and found several serpin family members that are differentially expressed between LMP and TOV serous tumors. SERPINA1 was selected for further study due to its high expression in the majority of LMP tumors and its low expression in TOV tumors; observations that were also validated by quantitative-PCR (Q-PCR). To study the effects of its over expression on different tumorigenic parameters, SERPINA1 was cloned in the pcDNA3.1+ plasmid which was subsequently used to derive stable clones from two invasive ovarian cancer cell lines, TOV-112D and TOV-1946. We found no effect of SERPINA1 over expression on tumor growth in SCID mice although cell migration and invasion were affected in in vitro assays. There was also no association between patient survival and SERPINA1 immunostaining, however, SERPINA1 localization was different in LMP (nuclear) and TOV (cytoplasmic) tumors. SERPINA1 remains an interesting candidate since protein homeostasis, regulated by proteases and their inhibitors, should be studied holistically in order to assess their full impact in tumor progression.     

Spontaneous chronic colitis in TCR alpha-mutant mice; an experimental model of human ulcerative colitis

Mice with targeted disruption of the T cell receptor alpha gene (TCR alpha-/-) spontaneously develop chronic colitis. Colonic inflammation begins at 6-8 weeks of age and chronic colitis is established in about 60% of mice by 16-20 weeks of age. The disease is also associated with autoantibodies (anti-tropomyosin antibodies, anti-neutrophil cytoplasmic antibodies) and an oligoclonal immune response to luminal bacterial antigens. Although T cells, but not B cells or autoantibodies, are essential for the development of colitis, B cells and/or autoantibodies may have a regulatory role in the pathogenesis of this colitis because the colitis is more severe in B cell deficient TCR alpha-/- mice. Cytokines, specifically IL-4 and IL-1, also play an important role in the development of colitis in TCR alpha-/- mice. Enteric bacteria located in the large intestine are an important factor in the pathogenesis of this colitis because germ-free TCR alpha-/- mice do not develop colitis and appendectomy at an early age delays the onset of this colitis. The colitis in TCR alpha-/- mice resembles human ulcerative colitis and provides a useful model to study the pathogenesis of human inflammatory bowel disease.     

Pathophysiological role of nitric oxide in rat experimental colitis

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of ulcerative colitis. A 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS) colitis model was established to examine the effect of selective iNOS inhibition, by S-(2-aminoethyl) isothiouronium bromide (ITU), on colonic mucosal cell damage and inflammation. Rats, killed 7 days after TNBS, had increased colonic mucosal levels of iNOS and interleukin-8 (IL-8), in addition to severe colonic inflammation which was characterized by significantly increased colon weight, damage score and colonic myeloperoxidase activity (MPO) (a marker of neutrophil influx). TNBS-treated rats had markedly decreased body weight and thymus weight. Administration of colitic rats with ITU significantly inhibited iNOS activity/expression and tended to reduce mucosal levels of IL-8, but no effect on MPO activity was observed. Following ITU therapy, colitic rats had reduced colonic damage and losses in body weight and thymus weight were reversed. Improvement of TNBS colitis by ITU suggested that excess NO, produced by iNOS, may have contributed to the initiation/ amplification of colonic disease, by mechanisms including enhancement of IL-8 releaseNO-mediated enhancement of pro-inflammatory cytokine release was further investigated in vitro. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated release of nitrite, lactate dehydrogenase (LDH), TNFα, IL-1β and IL-8 from rat peritoneal macrophages, all of which were significantly reduced by ITU. This suggests that NO-mediated cell damage enhances pro-inflammatory mediator release from macrophages. In addition, enhancement of IL-8 and TNFα release was also partially NO-dependent in activated peritoneal neutrophils. Therefore, the amelioration of TNBS colitis by ITU could include inhibition of NO-mediated pro-inflammatory cytokine release.     

The role of histamine in experimental ulcerative colitis in rats

Inflammation Research - .     

共刺激分子及黏附分子在大鼠实验性结肠炎中的表达研究

目的：探讨共刺激分子及黏附分子在大鼠实验性结肠炎发病中的作用。方法：建立三硝基苯磺酸诱导的实验性结肠炎大鼠模型，并予以泼尼松或柳氮磺胺吡啶治疗，设立正常对照及未治疗组对照，进行组织学评分；流式细胞仪检测各组外周血、脾脏和结肠上皮内单个核细胞CD28、CTLA-4及CD11b的表达。结果：实验性结肠炎大鼠在外周血、脾脏和结肠表达CD11b、CD28及CTLA-4均较正常对照组升高，有统计学意义，而经泼尼松或柳氮磺胺吡啶治疗后在结肠的CD11b和CD28的表达有所回落，而CTLA-4升高更加明显，均有统计学意义。结论：黏附分子可能参与了大鼠实验性结肠炎的发生，CD28与CTLA-4表达失调可能是发病机制之一，泼尼松和柳氮磺胺吡啶的治疗机制可能与共刺激分子及黏附分子有关。     

Sodium alginate as a novel therapeutic option in experimental colitis

The potential therapeutic effect of low-viscosity sodium alginate (LVA) was studied in a rat model of acute colitis induced by intracolonic administration of acetic acid. This experimental model produced a significant ulcerative colitis. Induction of colitis also significantly enhanced the serum and colonic mucosal cytokine (IL-6 and TNF-alpha) and eicosanoid (LTB4 and PGE2) levels, which paralleled with the severity of colitis. LVA solution was administered orally as drinking water at concentration of 0.5% (W/V) for 1 week. The tolerability and inhibitory effect of LVA on matrix metalloproteinase-2 (MMP-2) were tested using WEHI-164 cell line and zymography method. The results showed that LVA therapy is able to significantly reduce colonic damage score, histological lesion, serum and colonic mucosal IL-6, TNF-alpha, LTB4 and PGE2 levels in treated group compared with nontreated controls. Moreover, in vitro examinations revealed that treatment with LVA could diminish MMP-2 activity. It is concluded that LVA is able to suppress acetic acid-induced colitis in rats. Some of the action of LVA may be associated with its inhibitory effects on cytokine and eicosanoid production and MMP-2 activity. Our data suggest that LVA could potentially be a novel therapeutic option for inflammatory bowel disease.     

Subepithelial myofibroblasts and tenascin expression in microscopic colitis

AIMS: To assess differences in the pattern of subepithelial myofibroblasts and the expression of tenascin as a marker of extracellular matrix production in collagenous and lymphocytic colitis. METHODS AND RESULTS: Colorectal biopsies were studied from 122 patients with chronic diarrhoea and normal colonoscopy. The pathological diagnoses were collagenous colitis (n = 35), lymphocytic colitis (n = 37), mild non-specific chronic inflammation (n = 28) and normal mucosa (n = 18). Four cases showed features of collagenous colitis but with collagen bands <10 micro m thick. Normal mucosa from 14 patients without diarrhoea served as healthy control tissue. Immunohistochemical expression of alpha-smooth muscle actin (myofibroblast marker) and tenascin was evaluated in well-orientated sections. The expression of alpha-smooth muscle actin was significantly increased in collagenous colitis compared with all the other groups. Strong tenascin subepithelial expression was seen in all cases of collagenous colitis, including the four without full-blown features. The mean thickness of tenascin bands was greater than that obtained by conventional stains. CONCLUSIONS: There are clear differences, with respect to extracellular matrix remodelling, between collagenous and lymphocytic colitis. These results support the theory of matrix overproduction in the genesis of collagenous colitis.     

Suppression of experimental colitis by intestinal mononuclear phagocytes

The contribution of innate immunity to inflammatory bowel disease (IBD) remains an area of intense interest. Macrophages (M?) and dendritic cells (DC) are considered important factors in regulating the onset of IBD. The goal of this study was to determine if intestinal mononuclear phagocytes (iMNP) serve a pathological or protective role in dextran sulfate sodium (DSS)-induced colitis in mice. Using a conditional M?/DC depletion transgenic mouse line--M? Fas-induced apoptosis--to systemically deplete iMNP, DSS colitis histopathology was shown to be more severe in M?/DC-depleted compared with M?/DC-intact mice. Similarly, localized iMNP depletion by clodronate-encapsulated liposomes into C57BL/6, BALB/c, and CB.17/SCID mice also increased DSS colitis severity, as indicated by increased histopathology, weight loss, rectal bleeding, decreased stool consistency, and colon length compared with M?/DC-intact, DSS-treated mice. Histology revealed that iMNP depletion during DSS treatment led to increased neutrophilic inflammation, increased epithelial injury, and enhanced mucin depletion from Goblet cells. iMNP depletion did not further elevate DSS-induced expression of TNF-alpha and IFN-gamma mRNA but significantly increased expression of CXCL1 chemokine mRNA. Myeloperoxidase activity was increased in colons of M?/DC-depleted, DSS-treated mice, compared with DSS alone, coincident with increased neutrophil infiltration in diseased colons. Neutrophil depletion combined with M?/DC depletion prevented the increase in DSS colitis severity compared with M?/DC depletion alone. This study demonstrates that iMNP can serve a protective role during development of acute colitis and that protection is associated with M?/DC-mediated down-regulation of neutrophil infiltration.