Nobuo Tomiyasu

STAT3 activation via interleukin 6 trans-signalling contributes to ileitis in SAMP1/Yit mice

Background and aim: SAMP1/Yit mice spontaneously develops intestinal inflammation. Previously, we demonstrated that the signal transducer and activator of transcription (STAT)-3/suppressor of cytokine signalling (SOCS)-3 pathway is pivotal in human inflammatory bowel disease. In our studies in SAMP1/Yit mice, the aim was to investigate whether STAT3 activation contributes to ileitis and to examine the therapeutic effects of this signal blockade. Methods: Intestinal expression of phospho-STAT3 in SAMP1/Yit mice and control AKR/J mice was examined by western blotting and immunohistochemistry. SOCS3 and interleukin 6 (IL-6) mRNA were determined by northern blotting and reverse transcription-polymerase chain reaction, respectively. We also examined the effects of intravenously injected hyper-IL-6, an IL-6/soluble IL-6 receptor fusion protein, and of soluble gp130-Fc, a specific inhibitor of soluble IL-6 receptor signalling, on STAT3 phosphorylation and disease severity in SAMP1/Yit mice. Results: Phospho-STAT3 was expressed strongly during the disease course in SAMP1/Yit mice but only transiently in AKR/J mice. Phospho-STAT3 was localised to epithelial and mononuclear cells in the diseased intestine of SAMP1/Yit mice. SOCS3 as well as IL-6 mRNAs were expressed in affected intestine. Administration of hyper-IL-6 caused disease exacerbation and enhancement of STAT3 phosphorylation. In contrast, soluble gp130-Fc administration ameliorated the disease and suppressed STAT3 phosphorylation. Conclusion: STAT3 signalling is critical in the development of intestinal inflammation in SAMP1/Yit mice. Blockade of this signalling pathway by soluble gp130-Fc may have therapeutic effects in inflammatory bowel disease.

Interleukin-10 in the Pathophysiology of Inflammatory Bowel Disease: Increased Serum Concentrations During the Recovery Phase

Using a specific enzyme-linked immunosorbent assay, IL-10 concentrations were measured in serum from 62 patients with ulcerative colitis (UC), 43 with Crohns disease (CD), 25 with other colitides, and 44 normal control subjects. Serum IL-10 concentrations were increased in patients with active UC but not in those with active CD when compared with normal control subjects. A time course study showed that in patients with UC and CD, serum concentrations of IL-6 and C-reactive protein increased during the acute phase and returned to normal as patients go into remission. Notably, serum IL-10 concentrations increased during the phase of disease resolution and declined thereafter regardless of the treatment modality. Gel filtration analysis indicated that IL-10 circulated predominantly as a dimer. In conclusion, this study shows that serum IL-10 is increased during disease recovery in patients with inflammatory bowel disease, and may be a helpful marker in monitoring disease status.

Diminished cytokine signalling against bacterial components in mononuclear leucocytes from ulcerative colitis patients after leukocytapheresis

Infiltration by circulating inflammatory cells is a prominent local inflammatory feature of ulcerative colitis (UC). Several trials have suggested that leukocytapheresis by filtration can benefit patients with active UC. We investigated how this therapy might modulate the inflammatory response. Patients with active UC who were beginning repeated filtration leukocytapheresis were studied. Mononuclear cell preparations were obtained from blood before and after the first treatment, and expression of cytokine signalling components and the cell‐proliferative response were analysed in vitro. Leukocytapheresis reduced lipopolysaccharide‐induced production of proinflammatory cytokines (interleukin‐1, ‐6, ‐8 and tumour necrosis factor‐α, P < 0·05 for all) and activation of intracellular signalling components (nuclear factor‐κB, mitogen‐activated protein kinases, and signal transducer and activator of transcription‐3), as well as surface expression of toll‐like receptor‐4 (P < 0·05) in mononuclear cells. The therapy also reduced the cell‐proliferative response by mononuclear cells stimulated with sonicated bacterial preparations from autologous intestine (P < 0·05). These results indicate that activated mononuclear cells in the peripheral blood of patients with active UC are removed by leukocytapheresis and replaced by cells with a lower activation status. This replacement may partly explain the therapeutic benefit.

Increased Circulating Concentrations of Growth-Related Oncogene (GRO)-α in Patients with Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory state associated with increased risk of intestinal cancers. The aim of this study is to examine serum concentrations of growth-related oncogene (GRO)-α, a cytokine with inflammatory and growth-regulatory properties, in patients with IBD. We measured serum concentrations of GRO-α in 60 patients with ulcerative colitis, 42 patients with Crohns disease, 16 patients with other colitides, 12 patients with colorectal cancer, and 40 normal subjects using an enzyme-linked immunosorbent assay. We then analyzed how the cytokine was related to clinical and laboratory variables. Serum GRO-α concentrations in patients with active IBD were significantly higher than those in patients with quiescent disease, which in turn were higher than those in normal controls. Concentrations in patients with active ulcerative colitis were higher than in patients with active Crohns disease. Analysis of paired serum samples showed a decrease in GRO-α after initiation of therapy. Furthermore, serum GRO-α correlated well with laboratory markers of IBD activity. We conclude that GRO-α may have an important role in development of IBD, and might itself be used as a marker of activity. Manipulation of GRO-α function might prove therapeutically useful.

A form of circulating interleukin-6 receptor component soluble gp130 as a potential interleukin-6 inhibitor in inflammatory bowel disease

The presence and the role of soluble gp130, the soluble form of a component of the interleukin (IL)‐6 receptor complex, were investigated in inflammatory bowel disease. The serum concentrations of soluble gp130 were increased in ulcerative colitis (active disease, median, 93·5 ng/ml; interquartile range, 26–125 ng/ml; inactive disease, 81 ng/ml, 24·8–137·3 ng/ml) and to a lesser extent in Crohns disease (active disease, 66 ng/ml, 44·4–87·6 ng/ml; inactive disease, 63 ng/ml, 43·5–82·5 ng/ml) compared to normal controls (43 ng/ml, 27–59 ng/ml). Paired analysis of serum samples showed a decrease of IL‐6 and soluble IL‐6 receptor concentrations in both diseases and an increase of soluble gp130 concentrations, especially in ulcerative colitis, just after the resolution of disease exacerbation. Size fractionation of the serum revealed that a part of the IL‐6 co‐eluted with soluble gp130 and soluble IL‐6 receptor. The IL‐6‐induced proliferation of murine B9 hybridoma was enhanced by recombinant soluble IL‐6 receptor, whereas the proliferation was inhibited by recombinant soluble gp130. These results indicate that soluble gp130 may function as a natural inhibitor of the IL‐6 actions in inflammatory bowel disease.

Activation of c-Jun N-terminal kinase (JNK) signalling in experimentally induced gastric lesions in rats

The c‐Jun N‐terminal kinase (JNK) participates in intracellular signalling cascades that mediate inflammatory responses. Therefore, the JNK signalling may be involved in gastric injury and inhibition of this pathway may form the basis of a new strategy for the treatment of gastric injury. The aim of this study was to determine whether JNK participates in the formation of gastric lesions in an experimental model. Acute gastric injury was induced in Sprague‐Dawley rats by intragastric administration of 100% ethanol. The amount of phospho‐JNK in the rat stomach was determined using immunohistochemistry and Western analysis. Animals received subcutaneous injections of a specific JNK inhibitor SP600125 or vehicle and the extent of mucosal damage in the stomach was determined. Western analysis revealed early phosphorylation of JNK and, to a lesser extent, p38 as well as late phosphorylation of the p42/44 extracellular signal‐related kinases during the development of gastric lesions. JNK was phosphorylated in epithelial cells and in occasional mononuclear cells present at lesion sites. These cells were rarely found in samples from control specimens. Treatment with SP600125 significantly reduced the extent of gastric lesions. These findings indicate that experimental gastric injury is associated with activation of the JNK signalling pathway, and also suggest that JNK inhibitors may play a role in the treatment of gastric injury in humans.

Efficacy of Vascular Endothelial Growth Factor in the Treatment of Experimental Gastric Injury

Background/Aims: Studies indicate that angiogenesis is important in tissue healing. However, the role of vascular endothelial growth factor (VEGF) in tissue healing has not been established. The aim of the study is to determine whether VEGF has a gastroprotective role in experimental gastric injury. Methods: Acute gastric injury was induced in Sprague-Dawley rats by intragastric administration of 100% ethanol. Expression of gastric VEGF was determined in tissue homogenates by enzyme-linked immunosorbent assay and in paraffin-embedded sections by immunohistochemistry. The effect of systemic administration of anti-VEGF antibodies and recombinant VEGF on injury severity was assessed macroscopically and microscopically. Results: Gastric VEGF concentrations peaked at 6 h and again 3 days after acute injury. The presence of VEGF was demonstrated in epithelial cells and in mononuclear cells. Blocking endogenous VEGF effects with anti-VEGF antibodies exacerbated mucosal injury. Administration of recombinant VEGF after the onset of injury reduced the severity of mucosal injury, irrespective of the timing of initial treatment with VEGF. Immunohistochemical detection of vascular endothelial cells revealed that the VEGF-induced mucosal repair is closely related to the degree of angiogenesis. Conclusion: The results provide strong evidence for the role of VEGF in the repair of tissue damage induced by ethanol. The results also show how VEGF may be used in a clinical setting to treat some acute gastric lesions.

Transcription Factor-Targeted Therapies in Inflammatory Bowel Disease

Although the causes of inflammatory bowel disease currently are not fully understood, increasing evidence implicates cytokines as key factors in the development of this disorder. The rationale for cytokine-targeted therapy for inflammatory bowel disease has been refined significantly, and clinical studies have been initiated. Recent investigations have focused on transcription factors that regulate production and activation of cytokines, including the nuclear factor-ĸB, the p38 mitogen-activated protein kinase, the peroxisome proliferator-activated receptor-γ, and the Janus kinases/signal transducers and activator of transcription pathways. Although their exact role in inflammatory bowel disease is still unknown, further studies may lead to identification of additional possible targets for therapeutic intervention that could improve management of the disease.

High Affinity of Ecabet Sodium for Inflamed Colonic Mucosa in Ulcerative Colitis

To the Editor: The pathogenesis of ulcerative colitis (UC) is unknown, but may be associated with inability of the colonic mucosa to protect itself from luminal challenges and/or with inappropriate effective repair following colonic injury. Therapeutic approaches aiming to promote the repair of the injured mucosa have included the use of mucosal protectants. Ecabet sodium, purified from pine resin, has been used widely in Japan as a nonabsorbable protectant in treating gastric ulcer [1]. Recently, benefit from ecabet sodium given by enema has been reported in treatment of UC [2] as well as in experimental colitis [3]. In the stomach, ecabet sodium has been found to adhere selectively to ulcerated gastric mucosa, showing a high affinity for injured gastric epithelium. In gastric ulcer, this agent promotes mucosal integrity, possibly by enhancing epithelial restitution, facilitating mucus synthesis and secretion, inducing prostaglandin production, and increasing mucosal blood flow [4, 5]. In intestinal epithelial cells in vitro, ecabet sodium activated extracellular signal-regulated kinases 1 and 2, and also induced cyclooxygenase-2 [6]. However, whether ecabet sodium adheres preferentially to inflamed colon as it does to ulcerated stomach remains unclear. We, therefore, evaluated the affinity of ecabet sodium for colonic mucosa in patients with UC. Seven patients with active UC were studied (4 male, 3 female; mean age, 36 years; mean disease duration, 5.4 years). Patients had either left-sided colitis (n = 3) or proctitis (n = 4), and represented a mild (n = 5) or mod-

Mechanisms Underlying the Effects of Leukocyte Apheresis with a Fiber Filter in a Rat Model of Dextran Sulfate Sodium-Induced Colitis

While several clinical trials have suggested that leukocytapheresis (LCAP) by filtration can benefit patients with active ulcerative colitis, the mechanisms underlying these benefits are largely unknown. The aim of this study was to address the mechanisms that may underlie the therapeutic effects of LCAP using a dextran sulfate sodium-induced colitis model in rats. Treatment with the active column, but not the sham column, improved disease severity by down-regulating pro-inflammatory events, including the cell-proliferative responses and inflammatory cytokine and reactive oxygen production, as well as by up-regulating protective events, including hepatocyte growth factor production, bone marrow-derived endothelial progenitor cell induction, and colonic blood flow levels, which were mediated predominantly by calcitonin gene-related peptide. The improvement was also associated with the increase of Ki-67 labeling in the colonic epithelium. In conclusion, the LCAP procedure was used in a dextran sulfate sodium-induced colitis model in rats under extracorporeal circulation conditions. This approach down-regulated pro-inflammatory events and up-regulated protective events in association with disease improvement. These data suggest that LCAP is feasible in animals and should shed light on the mechanisms of LCAP in clinical settings.