Gerhard Rogler

Cytokines in Inflammatory Bowel Disease

Abstract. Cytokines play a central role in the modulation of the intestinal immune system. They are produced by lymphocytes (especially T cells of the Th1 and Th2 phenotypes), monocytes, intestinal macrophages, granulocytes, epithelial cells, endothelial cells, and fibroblasts. They have proinflammatory functions [interleukin-1 (IL-1), tumor necrosis factor (TNF), IL-6, IL-8, IL-12] or antiinflammatory functions [interleukin-1 receptor antagonist (IL-1ra), IL-4, IL-10, IL-11, transforming growth factor β (TGFβ)]. Mucosal and systemic concentrations of many pro- and antiinflammatory cytokines are elevated in inflammatory bowel disease (IBD). An imbalance between proinflammatory and antiinflammatory cytokines was found for the IL-1/IL-1ra ratio in the inflamed mucosa of patients with Crohn’s disease, ulcerative colitis, diverticulitis, and infectious colitis. Furthermore, the inhibition of proinflammatory cytokines and the supplementations with antiinflammatory cytokines reduced inflammation in animal models, such as the dextran sulfate colitis (DSS) model, the trinitrobenzene sulfonic acid (TNBS) model, or the genetically engineered model of IL-10 knockout mice. Based on these findings a rationale for cytokine treatment was defined. The first clinical trials using neutralizing monoclonal antibodies against TNFα (cA2) or the antiinflammatory cytokine IL-10 have shown promising results. However, many questions must be answered before cytokines can be considered standard therapy for IBD.

Isolation and phenotypic characterization of colonic macrophages

Macrophages play an important role in the intestinal mucosal immune system. However, they are a poorly defined cell population. We therefore determined their phenotype in normal colonic mucosa. Macrophages were isolated from colonic biopsies and surgical specimens by collagenase digestion. Colonic macrophages were positively sorted by anti‐CD33 magnetic beads. Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, CD44, CD11b, CD11c, CD64, HLA‐DR, CD80, CD86 and CD3/CD19 expression. CD33 was evaluated as a positive marker for intestinal macrophages. CD33+ cells isolated from normal colonic mucosa showed co‐expression of the established intracellular macrophage marker CD68 in FACS analysis. CD33+ cells were capable of phagocytosis. Isolation of this cell population by magnetic anti‐CD33 beads and culture resulted in a 4.2–40‐fold increase in IL‐1β and 4.5–44‐fold increase in tumour necrosis factor‐alpha (TNF‐α) secretion compared with unsorted lamina propria mononuclear cells (LPMC). Of the CD33+ cells, 90.9u2003±u20036.9% (meanu2003±u2003s.d.) were CD44+. However, macrophages from colonic mucosa showed only a low expression of CD14 (10.5u2003±u20033.8%), CD16 (10.1u2003±u20033.9%), HLA‐DR (27.3u2003±u20039.2%), CD11b (17.4u2003±u20036.8%), CD11c (17.8u2003±u200310.4%). Furthermore, expression of CD80 (9.2u2003±u20034.2%) and CD86 (15.1u2003±u20037.3%) was low, suggesting a low ability of normal intestinal macrophages to activate T cells and T cell‐mediated immune responses. We conclude that CD33 is useful for the isolation and flow cytometric characterization of colonic macrophages. These cells exhibit a single phenotype in normal mucosa (CD33++, CD44++, CD14−, CD16−, CD11b−, CD11c−, HLA‐DRlow, CD80−, CD86−) lacking lipopolysaccharide (LPS) receptor and costimulatory molecules.

Abdominal MRI after enteroclysis or with oral contrast in patients with suspected or proven Crohn's disease

BACKGROUND & AIMSnDiagnostic results of magnetic resonance (MR) enteroclysis correlate highly with those from conventional enteroclysis; nevertheless, intubation of the patient and positioning of an intestinal tube is still necessary for the examination, which is often remembered as the most embarrassing part of the examination by the patient. A more comfortable and highly sensitive examination of the small bowel therefore would increase patient acceptance for recurring examinations, which are often necessary, for example, in patients with Crohns disease. This study evaluates the diagnostic efficacy of abdominal MR imaging (MRI) of the small bowel after drinking contrast agent only compared with conventional enteroclysis and abdominal MRI performed after enteroclysis in patients with suspected or proven Crohns disease.nnnMETHODSnTwenty-one patients with Crohns disease referred for conventional enteroclysis underwent abdominal MRI after enteroclysis. Additionally, 1 to 3 days before or after these examinations, abdominal MRI was performed using only orally administered contrast. All MRI examinations were performed using a 1.5T scanner.nnnRESULTSnAll pathological findings on conventional enteroclysis were shown correctly with MRI after enteroclysis and MRI after oral contrast only. Additional information by MRI was obtained in 6 of 21 patients. No statistically significant differences were found in assessing the diagnostic efficacy of the 3 examinations.nnnCONCLUSIONSnAbdominal MRI with oral contrast only can be used as a diagnostic tool for evaluation of the small bowel in patients with Crohns disease and has the potential to replace conventional enteroclysis as follow-up.

Comparison of capsule endoscopy and magnetic resonance (MR) enteroclysis in suspected small bowel disease

Background and aimsSmall bowel MR enteroclysis and wireless capsule endoscopy (WCE) are new diagnostic tools for the investigation of the small bowel. The aim of this study was to compare the diagnostic yield of WCE with MR enteroclysis in the detection of small bowel pathologies.MethodsA total of 36 patients were included in the study. Indications for imaging of the small bowel were proven or suspected small bowel Crohn’s disease (CD; n=18), obscure gastrointestinal (GI) bleeding (n=14) and tumour surveillance (n=4).ResultsIn patients with Crohn’s disease WCE detected significantly more inflammatory lesions in the first two segments of the small bowel compared with MR enteroclysis (12 patients vs. 1 patient, p=0.016). In 5 out of 14 (36%) patients with GI bleeding, angiodysplasia was detected as a possible bleeding source. Three of these patients had active bleeding sites detected by WCE. One patient had scattered inflammation of the mucosa. MR enteroclysis did not reveal any intestinal abnormalities in this patient group. MR enteroclysis provided extraintestinal pathologies in 10 out of 36 (28%) patients.ConclusionIn patients with Crohn’s disease WCE revealed significantly more inflammatory lesions in the proximal and middle part of the small bowel in comparison to MR enteroclysis, whereas in patients with obscure GI bleeding WCE was superior to MR enteroclysis.

HDL-Mediated Efflux of Intracellular Cholesterol Is Impaired in Fibroblasts From Tangier Disease Patients

To further elucidate the cellular mechanisms leading to HDL deficiency in Tangier disease, HDL-mediated cholesterol efflux was studied in cultured skin fibroblasts from Tangier patients. Both Tangier and control fibroblasts show specific saturable binding of HDL3 to the cell membrane (Bmax = 70 and 52 ng/mg protein, respectively; Kd = 8.8 and 10.6 micrograms/mL, respectively). There was no appreciable uptake of HDL3 by Tangier and control fibroblasts, indicating that cholesterol efflux from fibroblasts occurs at the cell membrane. When cellular cholesterol was labeled to equilibrium by [14C]cholesterol incubation for 48 hours at 37 degrees C, HDL3-mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnormality in HDL3-mediated cholesterol efflux more precisely, several additional experiments were performed. First, membrane desorption of cholesterol was determined after cell membranes were labeled with [14C]cholesterol for 3 hours at 15 degrees C. With this labeling protocol, there was no difference in HDL3-mediated cholesterol efflux between control and Tangier fibroblasts. Second, efflux of newly synthesized sterols was determined after incorporation of the precursor [14C]mevalonolactone. Under these conditions, specific HDL3-mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were labeled by incubation with reconstituted [3H]cholesteryl-linoleate-LDL. Efflux of LDL-derived cholesterol was only slightly reduced for the first 4 hours of incubation. After 12 hours, there was no difference between control and Tangier cells. The combined data indicate that the reduced efflux of cholesterol from Tangier fibroblasts observed after homogeneous labeling is due to severely reduced efflux of newly synthesized sterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of magnetic resonance imaging colonography with conventional colonoscopy for the assessment of intestinal inflammation in patients with inflammatory bowel disease: a feasibility study

Aim: Magnetic resonance imaging (MRI) based colonography represents a new imaging tool which has mainly been investigated for polyp screening. To evaluate this approach for patients with inflammatory bowel disease (IBD), we compared MRI based colonography with conventional colonoscopy for assessing the presence and extent of colonic inflammation. Patients and methods: In 22 consecutive patients with suspected or known IBD, MRI colonography was performed immediately before conventional colonoscopy. After bowel cleansing, a T1 positive contrast agent was applied rectally. In addition to T2 weighted sequences, T1 weighted two dimensional and three dimensional Flash acquisitions as well as volume rendered virtual endoscopy were performed. All images were evaluated with regard to typical MRI features of inflammation. The results were compared with colonoscopy findings. Results: Distension and image quality was assessed as good to fair in 97.4% of all colonic segments. Only four of 154 segments were considered non-diagnostic. With colonoscopy serving as the gold standard, the sensitivity for correctly identifying inflammation on a per segment analysis of the colon was 31.6% for Crohn’s disease (CD) and 58.8% for ulcerative colitis (UC). In CD, in most cases mild inflammation was not diagnosed by MRI while in UC even severe inflammation was not always depicted by MRI. Virtual endoscopy did not add any relevant information. Conclusion: MRI based colonography is not suitable for adequately assessing the extent of colonic inflammation in patients with IBD. Only severe colonic inflammation in patients with CD can be sufficiently visualised.

Profiling adipocytokine secretion from creeping fat in Crohn's disease

Background: Adipose tissue is recognized as a compartment secreting highly active molecules. Creeping fat represents a characteristic feature of Crohns disease (CD). Proinflammatory or anti‐inflammatory adipose‐derived secretory products, now generally called adipocytokines, may play a role in the pathogenesis of CD. Materials and Methods: Adipose tissue specimens were obtained from creeping fat contiguous to the involved intestine of 10 patients with CD. Mesenteric adipose tissue specimens resected from 13 patients with colon cancer (CC) and from 7 patients with diverticulitis served as controls. Three fat tissue specimen per well and 6 to 8 wells per patient were incubated ex vivo for 24 h. The release of adiponectin, resistin, leptin, interleukin‐6, macrophage colony‐stimulating factor, monocyte chemotactic protein‐1, and migration inhibitory factor was measured by ELISA. The expression of AdipoR1 and AdipoR2 receptors was investigated by real‐time quantitative polymerase chain reaction in a subset of adipose tissues. Results: The secretion of adiponectin and macrophage colony‐stimulating factor, as well as leptin and migration inhibitory factor, was significantly upregulated in CD compared with CC and diverticulitis or CC only, respectively. Resistin, interleukin‐6, and monocyte chemotactic protein‐1 were not specifically induced in CD but were associated with unspecific inflammation. Adiponectin receptor expression was not different in CC, CD, or diverticulitis. Steroid treatment in CD patients had differential effects on the ex vivo secretion of adipocytokines. Conclusions: A specific secretion pattern of proinflammatory and anti‐inflammatory adipocytokines indicates the significance of adipose tissue in intestinal inflammation in CD. Future investigations of this intestinal compartment may provide novelinsights into the pathophysiological role of creeping fat and CD.

In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis.

Recently we demonstrated that in inflammatory bowel disease (IBD) macrophage‐oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti‐oxidative potential. Using the dextran sulphate sodium (DSS)‐induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600u2003µg/mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti‐CD3 antibody in the presence of interleukin (IL)‐2 (final concentration 10u2003U/ml). After incubation for 24 h, IL‐1β, IL‐6, IL‐12 tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ levels in supernatants were analysed by the beadlyte® cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3·2 with acteoside versus 5·2 with phosphate‐buffered saline (PBS) (Pu2003<u20030·02). In chronic colitis both 120u2003µg (3·3 versus 5·2) or 600u2003µg acteoside (3·0 versus 5·2) significantly ameliorated colitis (both Pu2003<u20030·02). Stimulated MLN from mice with chronic DSS‐induced colitis treated with acteoside showed a significant down‐regulation of IFN‐γ secretion (195u2003pg/ml with 600u2003µg acteoside versus 612u2003pg/ml with PBS, Pu2003<u20030·02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.

Improvement of arthritis and arthralgia after treatment with infliximab (Remicade) in a German prospective, open-label, multicenter trial in refractory Crohn's disease.

was normal (138 10/ l). Two years later he was still positive for hepatitis B surface antigen, HBeAg, and HBV DNA measured by polymerase chain reaction; the ALT activity was elevated to 54 IU/L (normal 40). Hepatitis C virus infection and autoaggressive disorders were excluded. Physical examination revealed no deviation from normal state; there were no hematological disturbances (platelet count 149 10/ l, red blood cell [RBC] count 4.42 10/ l, white blood cell [WBC] count 4.9 10/ l). Liver ultrasound and Doppler sonography revealed no abnormalities. We decided to treat him with lamivudine at a dose of 100 mg/day (3 mg/kg/day). After a month of treatment, decline in platelets to 35 10/ l was observed. WBC and RBC counts were within normal ranges; ALT activity was 34 IU/L. Lamivudine therapy was discontinued; he received immunoglobulin for 5 days and the platelet count returned to normal (158 10/ l). During 3 months of observation after lamivudine therapy discontinuation the platelet count was in the normal range. The boy still replicated HBV (HBeAg and HBV DNA positive) and ALT activity increased to 68 IU/L. We therefore decided to administer lamivudine again at a dose of 50 mg/day. After 3 days of therapy the platelet count decreased to 55 10/ l (WBC and RBC counts were within normal ranges). Antiplatelet and antinuclear antibodies were not detected. Marrow specimens showed normal cellularity and increased megakaryocytes. The boy had recovery of platelets a week after lamivudine discontinuation without any concomitant treatment; a month later the platelet count was 137 10/ l. At this moment, to our knowledge, thrombocytopenia during lamivudine therapy has not been reported. Possible causes of cytopenia during antiviral therapy (e.g., interferon) in patients with chronic hepatitis B are mechanisms such as bone marrow suppression (7), immune-mediated toxicity of the drug (7, 8), or increased sequestration of platelets in the liver and spleen (9). We speculate that the observed thrombocytopenia in our patient treated with lamivudine could be the consequence of immune peripheral destruction of platelets, mainly in the spleen, rather than myelosuppression. The results of this study indicate that, despite confirmed lamivudine therapy safety in adults, serious adverse effects can occur, and this drug should be prescribed for children only by physicians experienced in its use, who should be cautious about the hematologically adverse effects of this drug.

Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis.

Haem‐oxygenase‐1 (HO‐1) has been shown to exert anti‐inflammatory, anti‐apoptotic and anti‐proliferative effects. We investigated HO‐1 expression in patients with inflammatory bowel disease (IBD) and could demonstrate a scattered expression of HO‐1 in the intestinal epithelium of severely inflamed colonic mucosa of patients with IBD compared to control specimens such as diverticulitis, suggesting dysregulated expression in IBD. To further analyse potential mechanisms of HO‐1 induction in the intestine we employed an in vitro epithelial cell apoptosis model and an experimental colitis model. In vitro induction of HO‐1 by the HO‐1 inducer cobalt protoporphyrin (CoPP) resulted in a dose‐dependent down‐regulation of caspase‐3 activation in HT‐29 cells, indicating an anti‐apoptotic function of HO‐1 in the intestine. In vivo, preventive HO‐1 induction by CoPP in acute dextran sodium sulphate (DSS)‐induced colitis led to a significant down‐regulation of colonic inflammation (Pu2003<u20030·01) with a concomitant reduction in interferon (IFN)‐γu2003− but unaffected interleukin (IL)‐10‐secretion by isolated mesenteric lymph nodes (Pu2003<u20030·01). Additionally, TUNEL staining of colonic sections demonstrated fewer apoptotic epithelial cells in the colon of CoPP treated animals. No beneficial effects were observed if HO‐1 was induced by CoPP after the onset of acute colitis or in chronic DSS‐induced colitis. In conclusion, the data suggest a protective role of HO‐1 if it is induced before the onset of inflammation. However, as shown by the lack of effects in established acute or in chronic colitis, the induction of HO‐1 may not be a promising approach for the treatment of IBD.