interleukin-8 and Inflammatory-Bowel-Diseases

Inflammation has a significant role in the onset and progression of inflammatory bowel disease (IBD). Increasing attention has been paid to the use of acupuncture in IBD patients; however, its regulatory effects on inflammatory factors in IBD still require validation. Here, we systematically evaluated the effects of acupuncture on inflammatory factors in IBD patients.. Eight electronic databases were searched for studies that met the inclusion criteria. After evaluating the quality of the studies selected by two reviewers, the meta-analysis was performed to assess the efficacy of acupuncture in IBD patients and the impact on inflammatory factors (TNF-α, IL-1, IL-8 and IL-10).. Four randomized controlled trials with a total of 228 patients satisfied the inclusion criteria. Acupuncture has a positive therapeutic impact on IBD (MD = 1.22, 95% CI [1.07, 1.39], P = 0.003). Moreover, it regulates the levels of TNF-α (MD =-60.58, 95% CI [-100.30, -20.89], P = 0.003), IL-8 (MD =-56.40, 95% CI [-60.02, -52.14], P < 0.00001) and IL-10 (MD =35.96, 95% CI [11.02, 60.91], P = 0.005) in IBD patients. However, the P value of meta-analysis in IL-1 great than 0.05.(MD =-27.90, 95% CI [-97.82, 42.02], P = 0.11).. Acupuncture has a positive therapeutic impact on IBD and can effectively regulate inflammatory factors in IBD patients. TNF-α, IL-8 and IL-10 are more appropriate inflammatory indicators for clinically evaluating the anti-inflammatory response in the blood of IBD patients by acupuncture.

Topics: Acupuncture Therapy; Anti-Inflammatory Agents; Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-10; Interleukin-8; Tumor Necrosis Factor-alpha

Whether interleukin (IL)-8, IL-10, and IL-18 polymorphisms influence predisposition of inflammatory bowel disease (IBD) remains uncertain.. The authors conducted a meta-analysis to explore relationships between IL-8, IL-10, or IL-18 polymorphisms and predisposition of IBD by merging the results of eligible literatures.. A thorough literature search in MEDLINE, Embase, Wanfang, VIP, and CNKI was conducted by the authors to identify eligible literatures, and 33 literatures were finally selected for merged analyses.. We found that genotypic frequencies of IL-8 rs4073, IL-10 rs1800871, IL-10 rs1800872, and IL-10 rs1800896 polymorphisms among cases with IBD and population-based controls differed significantly. Moreover, we found that genotypic frequencies of IL-8 rs4073, IL-10 rs1800871, and IL-18 rs1946518 polymorphisms among cases with IBD and population-based controls of Asian origin differed significantly, whereas genotypic frequency of IL-10 rs1800896 polymorphism among cases with IBD and population-based controls of Caucasian origin also differed significantly. Furthermore, genotypic frequency of IL-18 rs187238 polymorphism among cases with Crohn's disease (CD) and population-based controls also differed significantly.. The present meta-analysis shows that IL-8 rs4073, IL-10 rs1800871, IL-10 rs1800872, IL-10 rs1800896, and IL-18 rs1946518 polymorphisms may influence predisposition of IBD. Furthermore, IL-18 rs187238 polymorphism may influence predisposition of CD, but not predisposition of ulcerative colitis.

Topics: Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Inflammatory Bowel Diseases; Interleukin-10; Interleukin-18; Interleukin-8; Polymorphism, Single Nucleotide

The aim of this review was to analyze and synthesize recent data on the role of interleukin 8/CXCL8 in key mechanisms of the inflammatory process and carcinogenesis in patients with inflammatory bowel disease (IBD). Currently, the study of the relationship between the pathogenesis of cancer and chronic inflammation remains an urgent task for many researchers. It has been proven that the strongest correlation between the duration of inflammation and oncogenesis is observed in colorectal cancer (CRC). The discovery of interleukin 8 / CXCL8 as one of the powerful mediators of inflammation has expanded the general understanding of its contribution to the pathogenesis of IBD. This review emphasizes the role of CXCL8 in carcinogenesis in IBD, presents the results of studies on the molecular mechanisms of CXCL8 expression in IBD and CRC, and describes its properties as one of the main angiogenic and pro-inflammatory factors that regulate the cancer cells proliferation. Study of the effects of CXCL8 on signaling pathways activation may serve as a prerequisite for the search for and discovery of new therapeutic approaches, that will reduce the risk of CRC in patients with IBD.

Topics: Carcinogenesis; Colorectal Neoplasms; Humans; Inflammatory Bowel Diseases; Interleukin-8; Signal Transduction

The production of interleukin (IL)-26 was initially attributed to T cells, and in particular to Th17 cells. However, more recent findings indicate IL-26 production in natural killer (NK) cells, macrophages and fibroblast-like cells as well. It is known that IL-26 binds to the IL-20R1/IL-10R2 receptor complex on certain target cells, where it causes specific intracellular signaling and the secretion of IL-1β, IL-8 and TNF-α. In line with this type of proinflammatory role, IL-26 also increases chemotaxis of human neutrophils. Interestingly, high levels of IL-26 are present even in normal human airways, and endotoxin exposure further enhances these levels; this indicates involvement in antibacterial host defense. Studies on acute inflammatory disorders are few but there are studies showing the involvement of IL-26 in rheumatoid arthritis and inflammatory bowel disease. In conclusion, IL-26 is emerging as a potentially important player in host defense and may also be a pathogenic factor in the chronic inflammatory disorders of humans.

Topics: Arthritis, Rheumatoid; Chemotaxis; Chronic Disease; Humans; Immunity, Innate; Inflammation; Inflammatory Bowel Diseases; Interleukin-10 Receptor beta Subunit; Interleukin-1beta; Interleukin-8; Interleukins; Killer Cells, Natural; Macrophages; Receptors, Interleukin; Signal Transduction; Th17 Cells; Tumor Necrosis Factor-alpha

Many studies demonstrate that intestinal inflammation is either initiated or exaggerated by a component of the normal microbiota, most likely commensal bacteria or products derived from these organisms. We review the nature of human inflammatory bowel disease, the evidence for the involvement of the normal bacterial flora in these disorders and the relevance of maintaining the integrity of the epithelial barrier. Moreover, we, and others, have shown abnormal mitochondria structure in tissue resections from patients with inflammatory bowel disease and tissues from rodents that demonstrated psychological stress-induced increases in epithelial permeability. Thus, we also consider the possibility that a defect in epithelial mitochondrial function would predispose an individual to respond to their commensal bacteria flora--no longer considering them as a beneficial passive inhabitant, but rather perceiving them as a threatening and pro-inflammatory stimulus. In support of this postulate, we discuss our recent findings from an in vitro model showing that the human colon-derived T84 cell line exposed to the metabolic stressor, dinitrophenol, and the non-pathogenic, non-invasive, Escherichia coli (strain HB101) display a loss of barrier function, increased signal transduction and increased production of the chemokine, interleukin 8.

Topics: Animals; Epithelial Cells; Humans; Immunity, Mucosal; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Permeability

An imbalance of T helper cell type 1 (Th1) versus type 2 (Th2) polarization in favor of Th1 cell subsets appears to be a key pathogenic mechanism in chronic inflammatory bowel disease (IBD), in particular in Crohn's disease. The interferon gamma-inducing factor interleukin (IL)-18 acts in strong synergism with the Th1 polarizing cytokine IL-12. Recent studies provide evidence for the participation of IL-18 in the pathogenesis of IBD: IL-18 expression is increased in inflamed lesions of Crohn's disease patients and neutralization of IL-18 in different models of experimental colitis resulted in a dramatic amelioration of disease severity. IL-18 and IL-1beta are cleaved and thereby activated by the interleukin-1beta converting enzyme (ICE). Activation of ICE also occurs during different types of infectious colitis, and ICE expression and subsequent release of IL-1beta and IL-18 significantly contribute to intestinal inflammation. ICE knockout mice as well as mice treated with the ICE inhibitor pralnacasan are protected against experimental mucosal inflammation. Thus, inhibition of ICE represents an intriguing new target that requires further investigation in animal models.

Topics: Animals; Caspase 1; Caspase Inhibitors; Enzyme Inhibitors; Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-18; Interleukin-8

Epithelial neutrophil-activating protein 78 (ENA-78) is a member of the CXC chemokines and acts as a potent chemoattractant and activator of neutrophil function. On stimulation in vitro, ENA-78 is highly expressed in many cell types. ENA-78 protein levels are strongly elevated in synovial fluid and blood of patients with rheumatoid arthritis. By in situ hybridization and immunofluorescence staining, ENA-78 has been recognized as a major CXC chemokine expressed in epithelial cells of the intestinal mucosa of patients with Crohn's disease, ulcerative colitis, and acute appendicitis. A high expression of ENA-78 and interleukin-8 (IL-8) was also observed in the exocrine tissue of patients with chronic pancreatitis (CP). It is interesting to note that expression of IP-10, MIP-1alpha, and MCP-1 is high in healthy pancreatic tissue but low in tissue of patients with CP, suggesting a mutually exclusive expression of the ELR-CXC vs. non-ELR-CXC/CC chemokines. High-resolution studies of intracellular chemokines has revealed specific immunoreactivity for ENA-78 associated with the endoplasmic reticulum of many cell types. In contrast, GROalpha immunoreactivity was exclusively localized in the nucleus. Despite their common effects on neutrophil functions, the differential intracellular localization of ENA-78 and GROalpha suggests additional roles for these two chemokines in normal cell biology.

Topics: Arthritis, Rheumatoid; Chemokine CXCL5; Chemokines, CXC; Chronic Disease; Humans; Inflammatory Bowel Diseases; Interleukin-8; Monocytes; Pancreatitis; Respiratory Distress Syndrome

Topics: Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-6; Interleukin-8; Interleukins; Tumor Necrosis Factor-alpha

Cytokines are important immunoregulatory mediators. Their contribution to the pathogenesis of acute and chronic gastroenterological disorders is obvious. Increased expression of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor (TNF) can be detected in inflammatory bowel disease. During the last few years it has also been recognized that activated leukocytes have an important role in the multisystem involvement of acute pancreatitis. Activation of leukocytes is an early event during severe acute pancreatitis, and it may be a pathogenetic factor in the severity of the disease. The review summarizes the recent findings in the field of inflammatory cytokines with particular attention of TNF, IL-1, IL-6, and IL-8 during severe acute pancreatitis and underscores the role of the activated leukocytes in the pathogenesis of complicated acute pancreatitis.

Topics: Acute Disease; Adjuvants, Immunologic; Cytokines; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-6; Interleukin-8; Pancreatitis; Severity of Illness Index; Tumor Necrosis Factor-alpha

Topics: Cytokines; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Neutrophils; Research Design

Single-incision laparoscopic (SIL) surgery is expanding, but its benefits, efficacy and safety compared with conventional laparoscopic (CL) surgery remain unclear. This pilot study examined clinical outcomes and biochemical markers of inflammation for colorectal resections by SIL and CL in a randomized controlled pilot trial.. Fifty patients undergoing elective colorectal resection were randomized to either SIL or CL. Primary outcomes were operating time and length of stay (LoS); secondary outcomes included combined length of scars, pain scores, complications, Quality of Life EQ5D-VAS and the inflammatory markers interleukin-6 (IL-6), IL-8 and C-reactive protein (CRP) at baseline, 2, 6, 24 and 72 h.. There was no difference in age, gender, body mass index, indications and site of surgery, American Society of Anesthesiologists grade or incidence of previous surgery between the groups. Except for one conversion from SIL to open surgery, surgery was completed as intended. No difference between SIL and CL was found for operating time [median 130 (72-220) vs 130 (90-317) min, respectively, P = 0.528], LoS [median 4 (3-8) vs 4 (2-19)days, P = 0.888] and time to first flatus [2 (1-4) vs 2 (1-5) days, P = 0.374]. The combined length of scars was significantly shorter for SIL [4 (2-18) vs 7 (5-8) cm, P < 0.001]; in each group, four postoperative complications occurred (16%). Postoperative pain scores were similar [mean 7.67 (interquartile range 4) vs 7.25 (interquartile range 3.75), P = 0.835] to day 3. EQ5D-VAS was no different for both groups at discharge [72.5 (40-90) vs 70 (30-100), P = 0.673] but slightly higher for CL at 3 months [79 (45-100) vs 90 (50-100), P = 0.033].The IL-6, IL-8 and CRP levels between both groups showed similar peaks and no significant differences.. SIL colorectal surgery by experienced laparoscopic surgeons appears to be safe and equivalent to CL, with no discernible difference in its effect on the physiological response to surgical trauma.

Topics: Adult; Aged; Aged, 80 and over; C-Reactive Protein; Colectomy; Colorectal Neoplasms; Diverticular Diseases; Female; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Laparoscopy; Length of Stay; Male; Middle Aged; Operative Time; Pain, Postoperative; Pilot Projects; Postoperative Complications; Proctectomy; Quality of Life; Single-Blind Method; Young Adult

Around 30% of patients with inflammatory bowel disease (IBD) are refractory to current IBD drugs or relapse over time. Novel treatments are called for, and low dose Naltrexone (LDN) may provide a safe, easily accessible alternative treatment option for these patients. We investigated the potential of LDN to induce clinical response in therapy refractory IBD patients, and investigated its direct effects on epithelial barrier function.. Patients not in remission and not responding to conventional therapy were offered to initiate LDN as a concomitant treatment. In total 47 IBD patients prescribed LDN were followed prospectively for 12 weeks. Where available, endoscopic remission data, serum and biopsies were collected. Further the effect of Naltrexone on wound healing (scratch assay), cytokine production and endoplasmic reticulum (ER) stress (GRP78 and CHOP western blot analysis, immunohistochemistry) were investigated in HCT116 and CACO2 intestinal epithelial cells, human IBD intestinal organoids and patient samples.. Low dose Naltrexone induced clinical improvement in 74.5%, and remission in 25.5% of patients. Naltrexone improved wound healing and reduced ER stress induced by Tunicamycin, lipopolysaccharide or bacteria in epithelial barriers. Inflamed mucosa from IBD patients showed high ER stress levels, which was reduced in patients treated with LDN. Cytokine levels in neither epithelial cells nor serum from IBD patients were affected.. Naltrexone directly improves epithelial barrier function by improving wound healing and reducing mucosal ER stress levels. Low dose Naltrexone treatment is effective and safe, and could be considered for the treatment of therapy refractory IBD patients.

Topics: Adult; Cell Line, Tumor; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoscopy; Epithelial Cells; Female; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Male; Middle Aged; Naltrexone; Remission Induction; Wound Healing

Inflammatory Bowel Disease (IBD) patients are at an increased risk of developing herpes zoster (HZ), especially when immunosuppressed. HZ may be preventable with the herpes zoster vaccine (HZV), but many patients are not offered vaccination over concern regarding efficacy and fear of adverse events. Although the Center for Disease Control and Prevention recommends that low-dose immunosuppression is not a contraindication, few IBD patients on these medications are receiving HZV.. This study was a prospective clinical trial to assess the safety and immunogenicity of HZV among 2 groups of IBD patients. Group A consisted of 14 patients on low-dose immunomodulators and group B consisted of 25 patients either on 5-aminosalicylic acid or no IBD therapy. Blood samples were obtained to measure immune responses.. HZ specific immunoglobulin G rose significantly in both groups but the response was lower in the immunosuppressed group (P = 0.0002). Peripheral blood mononuclear cell secretion of Tumor necrosis factor-α in response to HZ antigen increased after HZV in group B, but not in group A. Interleukin-8 secretion increased in both groups, but the response was much higher in group B. There were no significant differences in adverse events between groups. No patients developed a HZ-like rash within 1 year after vaccination.. IBD patients on low-dose immunosuppressive therapy have a blunted immune response to HZV as compared with nonimmunosuppressed subjects. Despite this, immunosuppressed IBD patients are able to mount a statistically significant immune response. There were no serious adverse events to HZV.

Topics: Aged; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Viral; Cells, Cultured; Female; Herpes Zoster Vaccine; Humans; Immunoglobulin G; Immunosuppressive Agents; Inflammatory Bowel Diseases; Interleukin-8; Leukocytes, Mononuclear; Male; Mesalamine; Middle Aged; Prospective Studies; Tumor Necrosis Factor-alpha

The risk of inflammatory bowel disease (IBD) is substantially heightened if patients' first-degree relatives have it. The genetic and immune factors related to the disease have attracted great attention, including patients innate genetic polymorphisms. Interleukin-8 (IL-8) plays a vital role in digestive-system diseases, especially in gastrointestinal diseases.. The study intended to explore the expression of interleukin-8 (IL-8) in the colon tissues of patients with Crohn's disease and the correlation between its polymorphisms and the disease's occurrence.. The research team performed a prospective study.. The study took place in the Department of Gastroenterology at Zhuji People's Hospital of Zhejiang Province in Zhuji, China.. Participants were 100 patients with Crohn's disease at the hospital between November 2016 and June 2018 and 100 healthy individuals. The research team assigned participants with Crohn's disease to the Crohn's disease group and the healthy participants to the control group.. The research team: (1) determined differences in the protein expression of the IL-8 between the groups; (2) examined the conformity of the data to that of the Hardy-Weinberg equilibrium; (3) analyzed the differences in the genotypes and alleles for the IL-8 single nucleotide polymorphisms (SNPs) rs102039, rs103284 and rs105432 between the groups; and (4) for the Crohn's disease group, examined the differences in the disease's location and behavior for the participants with different genotypes.. The protein expression level of IL-8 in the colon tissues in Crohn's disease group was significantly higher than that in control group (P < .05). The genetic association analysis showed significant correlations between the polymorphisms rs103284 and rs105432 and alleles of the IL-8 gene and the occurrence of Crohn's disease (P < .05), but no associations existed between the gene polymorphism rs102039 and alleles and Crohn's disease (P > .05). Significant correlations existed between the IL-8 gene polymorphisms rs103284 and rs105432 and the disease's location and behavior (P < .05).. IL-8 had a significantly increased expression in the colon tissues of the participants with Crohn's disease, and some genotypes and alleles for the gene polymorphisms rs103284 and rs105432 were significantly higher in the Crohn's disease group than in the control group. In addition, the disease's location and behavior were significantly different for participants in the Crohn's disease group with different genotypes.

Topics: Crohn Disease; Humans; Inflammatory Bowel Diseases; Interleukin-8; Polymorphism, Single Nucleotide; Prospective Studies

Endoplasmic reticulum (ER) stress contribute to inflammatory bowel disease (IBD). However, the mechanistic link between toll-like receptor 4 (TLR4) and ER stress in IBD remains elusive. This study aimed to investigate the mechanism by which ER stress and TLR4 promote inflammation in IBD. IBD mouse model was established by the induction of TNBS, and Grp78 and TLR4 in intestine tissues were detected by immunohistochemistry. THP-1 cells were treated with lipopolysaccharides (LPS), ER stress inducer or inhibitor tauroursodeoxycholic acid (TUDCA), or p38 MAPK inhibitor. The activation of MAPK signaling was detected by Western blot, and the production and secretion of inflammatory factors were detected by PCR and ELISA. We found that the expression levels of TLR4 and GRP78 were significantly higher in the intestine of IBD model mice compared with control mice but were significantly lower in the intestine of IBD model mice treated with ER stress inhibitor TUDCA. ER stress inducer significantly increased while ER stress inhibitor TUDCA significantly decreased the expression and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated by LPS. Only p38 MAPK signaling was activated in THP-1 cells treated by ER stress inducer. Furthermore, p38 inhibitor SB203580 inhibited the production and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated with LPS. In conclusion, TLR4 promotes ER stress induced inflammation in IBD, and the effects may be mediated by p38 MAPK signaling. TLR4 and p38 MAPK signaling are novel therapeutic targets for IBD.

Topics: Animals; Endoplasmic Reticulum Stress; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Lipopolysaccharides; Mice; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Present studies have shown that Flos Chrysanthemi has anti-inflammatory and other effects and regulates intestinal function, while the chrysanthemum stem and leaf as non-medicinal parts of chrysanthemum have similar chemical components with chrysanthemum, but the activity and mechanisms are rarely elucidated. Therefore, this study used a DSS-induced zebrafish inflammatory bowel disease model to study the anti-inflammatory and antioxidant effects of chrysanthemum stem and leaf extracts. The results indicate that DSS induction leads to increased secretion of acidic mucin in the intestines of juvenile fish, enlargement of the intestinal lumen and the emergence of intestinal inflammation. Compared with the model group, each administration group differentially inhibited the expression of IL-1β, IL-8 and MMP9 in DSS-induced zebrafish, while upregulating the activity of superoxide dismutase. The quantitative analysis results showed that the flavonoids (including Linarin, Diosmetin-7-glucoside, Tilianin, etc.) and phenolic acids (including Isochlorogenic acid C, Isochlorogenic acid A, 1,3-Dicaffeoylquinic acid, etc.) in the alcohol extract were closely related with both anti-inflammatory and antioxidant activity, while the polysaccharides were also shown a certain anti-inflammatory and antioxidant activity. In conclusion, this study suggests that the flavonoids, phenolic acids and polysaccharides from chrysanthemum stem and leaf extracts can improve inflammatory bowel disease of zebrafish by regulating the expressions of IL-1β, IL-8 and MMP9.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Chrysanthemum; Drugs, Chinese Herbal; Flavonoids; Inflammatory Bowel Diseases; Interleukin-8; Matrix Metalloproteinase 9; Plant Extracts; Zebrafish

Celiac disease (CeD) and inflammatory bowel disease (IBD) are accompanied by impaired immune responses. To study the immune regulation of these diseases, we evaluated the expression levels of pro-inflammatory (IL-8 and IL-17 A) and anti-inflammatory (IL-10) cytokines in intestinal biopsy specimens of CeD and IBD patients in comparison to healthy subjects.. Intestinal biopsies were collected from 33 patients with IBD, 47 patients with CeD, and 20 healthy individuals. Total RNA was extracted and mRNA expression levels of IL-8, IL-17 A and IL-10 were assessed by qPCR. P-value < 0.05 was considered statistically significant. The expression levels of IL-8 and IL-17 A were higher in biopsies of IBD (UC and CD) and CeD patients compared to the control group (P < 0.05). IBD patients (UC and CD) had higher IL-8 intestinal level than CeD patients (P < 0.0001 and P = 0.0007, respectively). The expression of IL-10 was significantly down-regulated in intestinal biopsies of CeD and IBD patients compared with controls (P < 0.001). In addition, the expression level of this cytokine was significantly lower in IBD patients (P < 0.001 for UC patients and P < 0.0001 for CD patients) than CeD group.. The three selected pro- and anti-inflammatory cytokines showed a similar expression pattern in both IBD and CeD patients. As IBD and CeD are immune-mediated disorders and are accompanied by inflammatory events, the understanding of the similarities and differences among them can help researchers to find out useful candidate therapeutic protocols. We suggest that larger cohort studies be organized to achieve more insights into this regulation.

Topics: Colitis, Ulcerative; Cytokines; Gene Expression; Humans; Inflammatory Bowel Diseases; Interleukin-10; Interleukin-17; Interleukin-8; Intestinal Mucosa

Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn's disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.

Topics: Animals; Colitis; Endoplasmic Reticulum Stress; Humans; Inflammatory Bowel Diseases; Interleukin-8; Mice; p38 Mitogen-Activated Protein Kinases; Trinitrobenzenesulfonic Acid; Tunicamycin

Bone loss and osteoporosis are commonly described as extra-intestinal manifestations of inflammatory bowel disease (IBD). Jianpi Qingchang Bushen decoction (JQBD) is a prescription used in clinical practice. However, further studies are needed to determine whether JQBD regulates the receptor activator of nuclear factor kappa B (NF-κB) (RANK)/receptor activator of NF-κB ligand (RANKL)/ osteoprotegerin (OPG) pathways and could play a role in treating IBD-induced bone loss.. To evaluate the therapeutic effect of JQBD in IBD-induced bone loss and explore the underlying mechanisms.. An IBD-induced bone loss model was constructed by feeding 12 6-to-8-wk-old interleukin-10 (IL-10)-knockout mice with piroxicam for 10 d. The mice were randomly divided into model and JQBD groups. We used wild-type mice as a control. The JQBD group was administered the JQBD suspension for 2 wk by gavage, while the control and model groups were given normal saline at the corresponding time points. All mice were killed after the intervention. The effect of JQBD on body weight, disease activity index (DAI), and colon length was analyzed. Histopathological examination, colon ultrastructure observation, and micro-computed tomographic scanning of the lumbar vertebrae were performed. The gene expression of NF-κB, tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-8 in the colon was evaluated by real-time polymerase chain reaction. Colon samples were assessed by Western blot for the expression of RANKL, OPG, RANK, and NF-κB proteins.. The model group lost body weight, had a shorter colon, and showed a dramatic increase in DAI score, whereas JQBD had protective and therapeutic effects. Treatment with JQBD significantly improved inflammatory cell infiltration and reduced crypt abscess and ulcer formation. Three-dimensional imaging of the vertebral centrum in the model group revealed a lower bone mass, loose trabeculae, and "rod-shaped" changes in the structure compared to the control group and JQBD groups. The bone volume/total volume ratio and bone mineral density were significantly lower in the model group than in the control group. JQBD intervention downregulated the NF-κB, TNF-α, IL-1β, IL-6, and IL-8 mRNA expression levels. The RANKL and OPG protein levels were also improved.. JQBD reduces inflammation of the colonic mucosa and inhibits activation of the RANK/ RANKL/OPG signaling pathway, thereby reducing osteoclast activation and bone resorption and improving bone metabolism.

Topics: Animals; Body Weight; Humans; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Mice; NF-kappa B; Tumor Necrosis Factor-alpha

Inflammatory bowel disease is a chronic disease of the intestinal tract, which is related to increased levels of various inflammatory mediators. This study aims to explore the anti-inflammatory mechanism of small molecular peptide WLS and its alleviating effect on inflammatory bowel disease (IBD). In TNF-α-induced HT-29 cells, WLS inhibited IL-8 secretion, decreased gene expression of pro-inflammatory cytokines IL-8, IL-6, IL-1β, and TNF-α, and inhibited the activation of MAPK/NF-κB signaling pathways. In the dextran sulfate sodium salt (DSS) induced colitis mouse model, WLS inhibited weight loss and disease activity index scores, increased colon length, improved colon histopathology, inhibited secretion of IL-6 and TNF-α in the colon, and down-regulated gene expression of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, IFN-γ, IL-17A). This study revealed that WLS was a novel small molecule peptide with anti-inflammatory activity and may be a potential candidate for the treatment of inflammatory bowel disease.

Topics: Animals; Anti-Inflammatory Agents; Colitis; Cytokines; Dextran Sulfate; HT29 Cells; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-17; Interleukin-6; Interleukin-8; Mice; Mice, Inbred C57BL; NF-kappa B; Signal Transduction; Tumor Necrosis Factor-alpha

Inflammatory bowel disease (IBD) is an inflammatory condition of the gastrointestinal tract, characterized by chronic and relapsing immune system activation, often diagnosed in adolescence, with a rising incidence in pediatric populations. IBD results from altered interactions between gut microbes and the intestinal immune system which induce an aberrant immune response, thus anti-inflammatory or immunosuppressive therapies are generally used. Recent interest has been given to the identification of integrative and complementary approaches that could be able to restore and preserve the intestinal barrier function.. In this work, we tested the effect of a fixed combination of probiotics and herbal extract (Colikind Gocce. Results obtained in this work demonstrated that CKG is able to prevent the impairment of intestinal barrier function induced by inflammation, ameliorating the transepithelial electrical resistance and the paracellular permeability of the Caco-2 monolayer; moreover, CKG is able to counteract the increased release of TNF-a and IL-8 induced by inflammatory stimulus, thus reducing the intestinal inflammation.. This work underlines the protective effect of CKG on intestinal barrier, reducing the damages induced by inflammatory stimulus. This suggests CKG as an interesting product in the management of intestinal inflammatory conditions.

Topics: Anti-Inflammatory Agents; Caco-2 Cells; Culture Media, Conditioned; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Lipopolysaccharides; Probiotics; THP-1 Cells

We aimed to assess patients with axial spondyloarthritis (axSpA) and inflammatory bowel disease (IBD) for disease activity and serum markers of endothelial dysfunction.. We studied 161 patients (123 males, 38 females) with axSpA: 153 with ankylosing spondylitis and 8 with non-radiographic axSpA, and 30 healthy controls (HC). We collected: age; sex; disease duration; extra-articular symptoms (IBD and acute anterior uveitis), comorbidities; human leukocyte antigen B27 status; and treatment. We measured serum interleukin (IL)-6, interleukin-18, IL-23, vascular endothelial growth factor (VEGF) epidermal growth factor (EGF), asymmetric dimethylarginine (ADMA), endothelin-1 (ET-1), and fetuin-A levels.. IBD was diagnosed in 19 (11.8%) patients with axSpA. Compared to patients with axSpA without IBD, those with IBD had higher serum C-reactive protein (CRP) level (p = 0.05), erythrocyte sedimentation rate (ESR) (p = 0.005), and serum ET-1 levels (p = 0.01). In patients with axSpA and IBD, ET-1 levels correlated positively with CRP level (p = 0.006) and ESR (p = 0.02), and ADMA levels with visual analog scale scores (p = 0.01). Patients with axSpA and IBD had higher serum levels of IL-6 (p = 0.01), IL-18 (p = 0.005), and ADMA (p = 0.01) and lower serum levels of fetuin-A (p = 0.01) than did controls.. Patients with axSpA and IBD had higher levels of disease activity, as assessed by ESR and CRP and ET-1 levels, than did patients with axSpA without IBD. Compared to HC, patients with axSpA and IBD had increased IL-18, ADMA levels and decreased fetuin-A level.

Topics: Adult; Axial Spondyloarthritis; Biomarkers; Blood Sedimentation; C-Reactive Protein; Case-Control Studies; Female; Humans; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Male; Middle Aged

Liver X receptor (LXR) exerts anti-inflammatory effects in macrophages. The aim of this study was to explore the expression and function of LXR in the colonic epithelium under inflammatory conditions.. The expression of LXR was explored by Western blot and immunohistochemistry in colonic biopsies from patients diagnosed with inflammatory bowel disease (IBD) and control patients. In addition, LXR and its target gene expression were analyzed in the colon from interleukin (IL)-10-deficient (IL-10-/-) and wild-type mice. Caco-2 cells were pretreated with the synthetic LXR agonist GW3965 and further challenged with IL-1β, the expression of IL-8 and chemokine (C-C motif) ligand (CCL)-28 chemokines, the activation of mitogen-activated protein (MAP) kinases, and the nuclear translocation of the p65 subunit of nuclear factor kappa B was evaluated. Glibenclamide was used as an ABCA1 antagonist.. We found that LXR expression was downregulated in colonic samples from patients with IBD and IL-10-/- mice. The nuclear positivity of LXR inversely correlated with ulcerative colitis histologic activity. Colonic IL-1β mRNA levels negatively correlated with both LXRα and LXRβ in the colon of IL-10-/- mice, where a decreased mRNA expression of the LXR target genes ABCA1 and FAS was shown. In addition, IL-1β decreased the expression of the LXR target gene ABCA1 in cultured intestinal epithelial cells. The synthetic LXR agonist GW3965 led to a decreased nuclear positivity of the p65 subunit of nuclear factor kappa B, a phosphorylation ratio of the p44-42 MAP kinase, and the expression of CCL-28 and IL-8 in IL-1β-stimulated Caco-2 cells. The pharmacological inhibition of ABCA1 increased the phosphorylation of p44-42 after GW3965 treatment and IL-1β stimulation.. The LXR-ABCA1 pathway exerts anti-inflammatory effects in intestinal epithelial cells and is impaired in the colonic mucosa of patients with IBD and IL-10-/- mice.

Topics: Animals; Anti-Inflammatory Agents; ATP Binding Cassette Transporter 1; Caco-2 Cells; Colitis; Epithelial Cells; Humans; Inflammatory Bowel Diseases; Interleukin-10; Interleukin-8; Liver X Receptors; Mice; NF-kappa B; Orphan Nuclear Receptors; RNA, Messenger

Inflammatory bowel disease (IBD) is an immune disorder that develops due to chronic inflammation in several cells. It is known that colorectal and T cells are mainly involved in the pathogenesis of IBD. Chrysophanol is an anthraquinone family member that possesses several bioactivities, including anti-diabetic, anti-tumor, and inhibitory effects on T cell activation. However, it is unknown whether chrysophanol suppresses the activity of colorectal cells. In this study, we found that chrysophanol did not induce cytotoxicity in HT-29 colorectal cells. Pre-treatment with chrysophanol inhibited the mRNA levels of pro-inflammatory cytokines in tumor necrosis factor-α (TNF-α)-stimulated HT-29 cells. Western blot analysis revealed that pre-treatment with chrysophanol mitigates p65 translocation and the mitogen-activated protein kinase (MAPK) pathway in activated HT-29 cells. Results from the in vivo experiment confirmed that oral administration of chrysophanol protects mice from dextran sulfate sodium (DSS)-induced IBD. Chrysophanol administration attenuates the expression of pro-inflammatory cytokines in colon tissues of the DSS-induced IBD model. In addition, we found that oral administration of chrysophanol systemically decreased the expression of effector cytokines from mesenteric lymph nodes. Therefore, these data suggest that chrysophanol has a potent modulatory effect on colorectal cells as well as exhibiting a beneficial potential for curing IBD in vivo.

Topics: Administration, Oral; Animals; Anthraquinones; Anti-Inflammatory Agents; Cell Survival; Colon; Colorectal Neoplasms; Cytokines; Dextran Sulfate; Female; HT29 Cells; Humans; Inflammatory Bowel Diseases; Interleukin-1beta; Interleukin-8; Lymph Nodes; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; T-Lymphocytes; Tumor Necrosis Factor-alpha

Matrix Metalloproteinase-9 (MMP-9) has been shown to play a key role in mediating inflammation and tissue damage in inflammatory bowel disease (IBD). In patients with IBD, the intestinal tight junction (TJ) barrier is compromised as characterized by an increase in intestinal permeability. MMP-9 is elevated in intestinal tissue, serum and stool of patients with IBD. Previous studies from our laboratory showed that MMP-9 causes an increase in intestinal epithelial TJ permeability and that the MMP-9 induced increase in intestinal permeability is an important pathogenic factor contributing to the development of intestinal inflammation in IBD. However, the intracellular mechanisms that mediate the MMP-9 modulation of intestinal barrier function remain unclear.. The main aim of this study was to further elucidate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ permeability using Caco-2 monolayers as an in-vitro model system.. MMP-9 induced increase in Caco-2 TJ permeability was associated with activation and cytoplasmic-to-nuclear translocation of NF-κB p65. Knocking-down NF-κB p65 by siRNA transfection prevented the MMP-9 induced expression of the NF-κB target gene IL-8, myosin light chain kinase (MLCK) protein expression, and subsequently prevented the increase in Caco-2 TJ permeability. In addition, the effect of MMP-9 on Caco-2 intestinal epithelial TJ barrier function was not mediated by apoptosis or necrosis.. Our data show that the MMP-9 induced disruption of Caco-2 intestinal epithelial TJ barrier function is regulated by NF-κB pathway activation of MLCK.

Topics: Caco-2 Cells; Cell Membrane Permeability; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Matrix Metalloproteinase 9; Models, Biological; Myosin-Light-Chain Kinase; NF-kappa B; Tight Junctions

Endoplasmic reticulum [ER] stress in intestinal epithelial cells [IECs] contributes to the pathogenesis of inflammatory bowel disease [IBD]. We hypothesized that ER stress changes innate signalling in human IECs, augmenting toll-like receptor [TLR] responses and inducing pro-inflammatory changes in underlying dendritic cells [DCs].. Caco-2 cells and primary human colon-derived enteroid monolayers were exposed to ATP [control stressor] or thapsigargin [Tg] [ER stress inducer], and were stimulated with the TLR5 agonist flagellin. Cytokine release was measured by an enzyme immunoassay. ER stress markers CHOP, GRP78 and XBP1s/u were measured via quantitative PCR and Western blot. Monocyte-derived DCs [moDCs] were cultured with the IEC supernatants and their activation state was measured. Responses from enteroids derived from IBD patients and healthy control participants were compared.. ER stress enhanced flagellin-induced IL-8 release from Caco-2 cells and enteroids. Moreover, conditioned media activated DCs to become pro-inflammatory, with increased expression of CD80, CD86, MHCII, IL-6, IL-15 and IL-12p70 and decreased expression of CD103 and IL-10. Flagellin-induced IL-8 production correlated with DC activation, suggesting a common stress pathway. Moreover, there were distinct differences in cytokine expression and basal ER stress between IBD and healthy subject-derived enteroid monolayers, suggesting a dysregulated ER stress pathway in IBD-derived enteroids.. Cellular stress enhances TLR5 responses in IECs, leading to increased DC activation, indicating a previously unknown mechanistic link between epithelial ER stress and immune activation in IBD. Furthermore, dysregulated ER stress may be propagated from the intestinal epithelial stem cell niche in IBD patients.

Topics: Adenosine Triphosphate; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Caco-2 Cells; Cell Differentiation; Chemokine CCL20; Colon; Culture Media, Conditioned; Cytokines; Dendritic Cells; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Flagellin; Histocompatibility Antigens Class II; Humans; Inflammation; Inflammatory Bowel Diseases; Integrin alpha Chains; Interleukin-10; Interleukin-12; Interleukin-15; Interleukin-6; Interleukin-8; Intestinal Mucosa; Lactones; Organoids; RNA, Messenger; Sesquiterpenes; Signal Transduction; Toll-Like Receptor 5; Tumor Necrosis Factor-alpha

Glucocorticoids are very effective anti-inflammatory drugs and widely used for inflammatory bowel disease (IBD) patients. However, approximately 20% of IBD patients do not respond to glucocorticoids and the reason for this is largely unknown. Dietary advanced glycation endproducts (AGEs) are formed via the Maillard reaction during the thermal processing of food products and can induce a pro-inflammatory reaction in human cells. To investigate whether this pro-inflammatory response could be mitigated by glucocorticoids, human macrophage-like cells were exposed to both LPS and AGEs to induce interleukin-8 (IL8) secretion. This pro-inflammatory response was then modulated by adding pharmacological compounds interfering in different steps of the anti-inflammatory mechanism of glucocorticoids: rapamycin, quercetin, and theophylline. Additionally, intracellular reactive oxygen species (ROS) were measured and the glucocorticoid receptor phosphorylation state was assessed. The results show that AGEs induced glucocorticoid resistance, which could be mitigated by quercetin and rapamycin. No change in the phosphorylation state of the glucocorticoid receptor was observed. Additionally, intracellular ROS formation was induced by AGEs, which was mitigated by quercetin. This suggests that AGE-induced ROS is an underlying mechanism to AGE-induced glucocorticoid resistance. This study shows for the first time the phenomenon of dietary AGE-induced glucocorticoid resistance due to the formation of ROS. Our findings indicate that food products with a high inflammatory potential can induce glucocorticoid resistance; these results may be of great importance to IBD patients suffering from glucocorticoid resistance.

Topics: Drug Resistance; Glucocorticoids; Glycation End Products, Advanced; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Macrophages; Maillard Reaction; Phosphorylation; Reactive Oxygen Species; Receptors, Glucocorticoid; THP-1 Cells

Inflammatory bowel disease (IBD) characterized by chronic intestinal inflammation and intestinal microbial dysbiosis present a major risk factor in the development of colorectal cancer. Previously, dietary polyphenols from mango (Mangifera indica L.) such as gallotannins and gallic acid have been shown to mitigate intestinal inflammation and carcinogenesis, as well as modulate intestinal microbial composition. To further translate findings from preclinical models, we hypothesized that mango polyphenols possess anti-inflammatory and microbiome-modulatory activities and may improve symptoms of IBD, reduce biomarkers for inflammation and modulate the intestinal microbiome when administered as an adjuvant treatment in combination with conventional medications in patients with mild to moderate IBD. In this study, ten participants received a daily dose of 200-400 g of mango pulp for 8 weeks (NCT02227602). Mango intake significantly improved the primary outcome Simple Clinical Colitis Activity Index (SCCAI) score and decreased the plasma levels of pro-inflammatory cytokines including interleukin-8 (IL-8), growth-regulated oncogene (GRO) and granulocyte macrophage colony-stimulating factor (GM-CSF) by 16.2% (P = .0475), 25.0% (P = .0375) and 28.6% (P = .0485), all factors related to neutrophil-induced inflammation, respectively. Mango intake beneficially altered fecal microbial composition by significantly increasing the abundance of Lactobacillus spp., Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus lactis, which was accompanied by increased fecal butyric acid production. Therefore, enriching diet with mango fruits or potentially other gallotannin-rich foods seems to be a promising adjuvant therapy combined with conventional medications in the management of IBD via reducing biomarkers of inflammation and modulating the intestinal microbiota.

Topics: Adolescent; Adult; Aged; Chemokine CXCL1; Diet; Feces; Female; Fruit; Gastrointestinal Microbiome; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammatory Bowel Diseases; Interleukin-8; Lactobacillus; Male; Mangifera; Middle Aged; Pilot Projects; Polyphenols; Young Adult

Cannabis benefits patients with inflammatory bowel disease (IBD). Cannabinoid receptors are expressed in gut immune cells and in epithelial cells of inflamed guts. Mucosal healing (MH) requires epithelial layer restoration.. To analyze the effects of CB2 agonist on parameters implicated in gut inflammation and MH.. Uninflamed tissue had higher epithelial proliferation (Ki67: 50%↑,. Using ex vivo and in vitro human models, we demonstrated that manipulating the cannabinoid system affects colon cells and secretome characteristics that facilitate MH in IBD.

Topics: Adult; Aged; Apoptosis; Autophagy; Biopsy; Caco-2 Cells; Cannabinoids; Case-Control Studies; Cell Proliferation; Colon; Colonoscopy; Drug Evaluation, Preclinical; Female; Healthy Volunteers; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Ki-67 Antigen; Male; Matrix Metalloproteinase 9; Middle Aged; Permeability; Receptor, Cannabinoid, CB2; Tissue Culture Techniques; Young Adult

Alterations in the gut microbiota composition play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) as specific commensal bacterial species are underrepresented in the microbiota of IBD patients. In this study, we examined the therapeutic potential of three commensal bacterial species,

Topics: Bacteroides; Caco-2 Cells; Chemokine CCL2; Claudin-2; Clostridiales; Electric Impedance; Epithelial Cells; Faecalibacterium prausnitzii; Gastrointestinal Microbiome; HT29 Cells; Humans; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; Lipopolysaccharides; Occludin; Permeability; Tumor Necrosis Factor-alpha

Monocyte chemotactic protein 1-induced protein 1 (MCPIP-1) is highly expressed in activated immune cells and plays an important role in negatively regulating immune responses. However, its role in regulating neutrophil functions in the pathogenesis of inflammatory bowel disease (IBD) is still unclear. Here, we found that MCPIP-1 was markedly increased at both the transcriptional and translational levels in inflamed mucosa of IBD patients compared with healthy controls, which was mainly expressed in neutrophils. Interestingly, MG-132, a proteasome inhibitor reducing the degradation of MCPIP-1, further facilitated neutrophils to express MCPIP-1

Topics: Adult; Animals; Blotting, Western; Chemokine CCL2; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique; Humans; Inflammatory Bowel Diseases; Interleukin-1beta; Interleukin-6; Interleukin-8; Leupeptins; Male; Mice; Mice, Inbred C57BL; Middle Aged; Neutrophils; Real-Time Polymerase Chain Reaction; Ribonucleases; Transcription Factors; Tumor Necrosis Factor-alpha; Young Adult

Topics: Adaptor Proteins, Signal Transducing; Bacterial Translocation; Case-Control Studies; Chemokine CCL2; Cholera Toxin; Extracellular Vesicles; Gram-Negative Bacteria; Haptoglobins; HIV Infections; Humans; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Intestinal Mucosa; Lipopolysaccharides; Microscopy, Immunoelectron; Protein Precursors

Patients presenting with Inflammatory bowel disease have been shown to exhibit an altered microbiome in both Crohn's disease and Ulcerative colitis. This shift in the microbial content led us to question whether several of these microbes are important in inflammatory processes present in these diseases and more specifically whether lipopolysaccharides from the gram-negative cell wall differentially stimulates resident cells. We, therefore, investigated the possible contribution of five major species of gram-negative bacteria found to be altered in presence during disease progression and evaluate their pathogenicity through LPS. We demonstrated that LPS from these different species had individual capacities to induce NF-κB and pro-inflammatory IL-8 production from HEK-TLR4 cells in a TLR4 dependent manner. Additional work using human intestinal colonic epithelial cell monolayers (Caco-2) demonstrated that the cells responded to the serotype specific LPS in a distinct manner, inducing many inflammatory mediators such as TNF-α and IL-10 in significantly altered proportions. Furthermore, the permeability of Caco-2 monolayers, as a test for their ability to alter intestinal permeability, was also differentially altered by the serotype specific LPS modulating trans-epithelial electrical resistance, small molecule movement, and tight junction integrity. Our results suggest that specific species of bacteria may be potentiating the pathogenesis of IBD and chronic inflammatory diseases through their serotype specific LPS responses.

Topics: Caco-2 Cells; Cell Line; Cell Survival; Cytokines; Epithelial Cells; Gram-Negative Bacteria; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Intestines; Lipopolysaccharides; NF-kappa B; Permeability; Species Specificity; Tight Junctions; Toll-Like Receptor 4

In this study we investigated the presence and relative abundance of important genera of the gut microbiota in IBD patients and their role in induction of IL8 in a cell culture model.. Stool samples of IBD patients and healthy controls were collected and relative diversity of thirteen bacterial families was measured using quantitative real-time PCR assay. Moreover, filtrate of the stool samples was used for treatment of HT-29 cell line to analyze involvement of diversity of the fecal bacterial communities in the extent of IL8 induction.. Bacteroides, Faecalibacterium prausnitzii, Prevotella spp., and Methanobrevibacterium were significantly less abundant in IBD patients (UC, N = 22; CD, N = 7) compared with control group (N = 29). Increase in relative amounts of Haemophilus, Streptococcus spp., and H. pylori were detected in IBD patients, which was not statistically significant. Relative decrease in amount of Bacteroides spp., Faecalibacterium prausnitzii, and Prevotella spp. were found in UC patients with disease activity score greater than 4; however, higher levels of Streptococcus and Haemophilus were detected in the patients who were at flares. A relationship between the reduction of Haemophilus spp. and higher BMI was shown in IBD patients. Expression of IL8 was significantly higher in the treated cells by the fecal inoculates of IBD patients. Increase in relative amounts of Enterobacteriacea showed a correlation with the higher level of IL8 induction in both groups.. These results showed that changes in the fecal microbiota composition could affect disease activity, BMI, and IL8 induction.

Topics: Adult; Case-Control Studies; Disease Progression; Feces; Female; Gastrointestinal Microbiome; HT29 Cells; Humans; Inflammatory Bowel Diseases; Interleukin-8; Male; Middle Aged; Young Adult

Plant-derived food consumption has gained attention as potential intervention for the improvement of intestinal inflammatory diseases. Apple consumption has been shown to be effective at ameliorating intestinal inflammation symptoms. These beneficial effects have been related to (poly)phenols, including phloretin (Phlor) and its glycoside named phloridzin (Phldz). To deepen the modulatory effects of these molecules we studied: i) their influence on the synthesis of proinflammatory molecules (PGE

Topics: Anti-Inflammatory Agents; Cell Line; Colon; Diet; Dinoprostone; Fruit; Glycation End Products, Advanced; Humans; Inflammation; Inflammatory Bowel Diseases; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Interleukin-8; Malus; Phloretin; Phlorhizin; Phytochemicals; Phytotherapy; Plant Extracts; Polyphenols; Receptors, CCR2

Inflammatory bowel disease (IBD) is a common disorder of chronic intestinal inflammation that can be caused by the disruption of intestinal immune homeostasis.. We aimed to evaluate the role of enhancer of zeste homolog 2 (EZH2) in the inflammatory response and explore the association between EZH2 and necroptosis in human epithelial colorectal adenocarcinoma cell lines.. In both in vitro and in vivo models, expression of EZH2 in intestinal tissues was verified by histology. The expression of inflammatory cytokines in cell lines treated with EZH2 siRNA with or without stimulus was analyzed by quantitative real-time polymerase chain reaction. An intestinal necroptosis cell model was established to elucidate whether EZH2 is involved in necroptosis.. Our present data indicated that EZH2 expression was decreased in in vitro and in vivo models and in patients with inflammatory bowel disease. EZH2 downregulation increased the expression of inflammatory factors, including TNF-α, IL-8, IL-17, CCL5, and CCL20 in a Caco-2 cell model. The JNK pathway was activated with the reduction of EZH2. In the necroptosis model, downregulation of EZH2 was detected with the upregulation of necroptotic markers RIP1 and RIP3. In addition, EZH2 knockdown with siRNA increased p-JNK and p-c-Jun.. Our data suggest that EZH2 plays an important role in the development of intestinal inflammation and necroptosis. Hence, EZH2 could be a potential therapeutic target for IBD.

Topics: Animals; Caco-2 Cells; Chemokine CCL20; Chemokine CCL5; Colitis; Colitis, Ulcerative; Crohn Disease; Dextran Sulfate; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; Gene Knockdown Techniques; Humans; In Vitro Techniques; Inflammation; Inflammatory Bowel Diseases; Interleukin-17; Interleukin-8; Intestinal Mucosa; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Necroptosis; Nuclear Pore Complex Proteins; Phosphoproteins; Proto-Oncogene Proteins c-jun; Real-Time Polymerase Chain Reaction; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Trinitrobenzenesulfonic Acid; Tumor Necrosis Factor-alpha

Topics: Active Transport, Cell Nucleus; Anthocyanins; Anti-Inflammatory Agents; Caco-2 Cells; Colon; Dose-Response Relationship, Drug; Electric Impedance; Fruit; Glutathione Peroxidase; Humans; Inflammatory Bowel Diseases; Interleukin-1beta; Interleukin-6; Interleukin-8; Permeability; Phytotherapy; Plant Extracts; Plants, Medicinal; Prunus avium; Time Factors; Transcription Factor RelA; Tumor Necrosis Factor-alpha

In IBD, interleukin-23 (IL-23) and its receptor (IL-23R) are implicated in disease initiation and progression. Novel insight into which cells produce IL-23 at the site of inflammation at an early stage of IBD will promote the development of new tools for diagnosis, treatment and patient monitoring. We examined the cellular source of IL-23 in colon tissue of untreated newly diagnosed paediatric patients with IBD.. Colon tissues from IBD and non-IBD patients were analysed by quantitative real-time PCR (qPCR), immunofluorescence confocal microscopy and flow cytometry after appropriate sample preparation. Blood samples from IBD and non-IBD patients and healthy controls were analysed using flow cytometry and qPCR.. We discovered that tissue-infiltrating neutrophils were the main source of IL-23 in the colon of paediatric patients with IBD, while IL-23(+) human leucocyte antigen-DR(+) or IL-23(+)CD14(+) cells were scarce or non-detectable, respectively. The colonic IL-23(+) neutrophils expressed C-X-C motif (CXC)R1 and CXCR2, receptors for the CXC ligand 8 (CXCL8) chemokine family, and a corresponding CXCR1(+)CXCR2(+)IL-23(+)subpopulation of neutrophils was also identified in the blood of both patients with IBD and healthy individuals. However, CXCL8-family chemokines were only elevated in colon tissue from patients with IBD.. This study provides the first evidence of CXCR1(+)CXCR2(+)IL-23-producing neutrophils that infiltrate and accumulate in inflamed colon tissue of patients with IBD. Thus, this novel source of IL-23 may play a key role in disease progression and will be important to take into consideration in the development of future strategies to monitor, treat and prevent IBD.

Topics: Adolescent; Child; Child, Preschool; Colon; Disease Progression; Female; Humans; Inflammatory Bowel Diseases; Interleukin-23; Interleukin-8; Male; Neutrophil Infiltration; Patient Acuity; Receptors, Interleukin

A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Caco-2 Cells; Humans; Ileus; In Vitro Techniques; Inflammatory Bowel Diseases; Interleukin-1beta; Interleukin-6; Interleukin-8; Intestinal Mucosa; Lab-On-A-Chip Devices; Microbiota; Models, Biological; Peristalsis; Probiotics; Tumor Necrosis Factor-alpha

About 60% of infliximab (IFX)-treated patients develop antidrug antibodies (ADA), although their clinical significance remains disputed. The aim of this study was to develop an assay for assessing ADA-neutralizing potential, and clinical significance.. An immune assay was devised in which the inhibition of IFX binding to plated-tumor necrosis factor in the presence of patient sera or controls, was assessed and defined as IFX-tumor necrosis factor binding reduction ratio (ITBR). The assay was compared to a bioassay in which tumor necrosis factor-α-induced interleukin-8 secretion from HT-29 cells was assessed after addition of IFX to ADA-containing sera or control sera.. Both assays detected neutralizing antibodies in 39 of 44 ADA-positive sera. The median ITBR was 3.66 (mean 4.9 ± 3.2) in 29 ADA-positive patients with loss of response (LOR), and 1.3 (mean 1.9 ± 1.3) in 15 patients without LOR (P = 0.001). ADA titers in both groups were similar (median 9.5 and 10.2 μg/mL, respectively P = 0.74). Using an ITBR of 1.65, the sensitivity for LOR detection was 86.2% and the specificity was 66.7%. (positive predictive value 83%; negative predictive value 71.4%; P = 0.001). When early ADA-IFX-sera from IFX-treated patients with or without subsequent LOR were compared, the median ITBRs were 1.1 and 0.57, respectively (P = 0.028).. Detection of neutralizing antibody activity was superior to antibody quantization by enzyme-linked immunosorbent assay with respect to correlation with clinical LOR, and for prediction of subsequent LOR. These findings may assist in optimizing infliximab therapy in patients with inflammatory bowel disease.

Topics: Adolescent; Adult; Antibodies, Neutralizing; Binding Sites, Antibody; Binding, Competitive; Biological Assay; Drug Resistance; Enzyme-Linked Immunosorbent Assay; Female; HT29 Cells; Humans; Inflammatory Bowel Diseases; Infliximab; Interleukin-8; Male; Middle Aged; Predictive Value of Tests; Tumor Necrosis Factor-alpha; Young Adult

Extracellular vesicles (EVs) are membrane-enclosed particles released by cells as a means of intercellular communication. They are potential novel biomarkers, as they are readily isolated from body fluids, and their composition reflects disease pathways. Whether these particles are released from sites of intestinal inflammation in inflammatory bowel disease (IBD) has not previously been determined.. EVs were isolated by ultracentrifugation of colonic luminal fluid aspirates and characterized according to surface proteins, and constituent mRNA and proteins. The effects of EVs on colonic epithelial cells and macrophages in culture were assessed at the transcriptional, translational, and functional levels.. Intestinal luminal aspirates contained abundant EVs, at a mean concentration of 4.3 × 10 particles/mL and with a mean diameter of 146 nm. EVs from patients with IBD with a high endoscopic score (≥1) contained significantly higher mRNA and protein levels of interleukin 6 (IL-6), IL-8, IL-10, and tumor necrosis factor α than EVs from healthy controls. EVs were absorbed by cultured colonic epithelial cells, leading to an increased translation of IL-8 protein by recipient cells when treated with EVs from patients with IBD. EVs and EV-treated epithelial cells induced migration of a significantly greater number of macrophages than epithelial cells alone.. EVs shed from sites of intestinal inflammation in patients with IBD have a distinct mRNA and protein profile from those of healthy individuals. These EVs have proinflammatory effects on the colonic epithelium, in vitro. Their stability in luminal samples and their mRNA and protein content identify them as a potential fecal biomarker that reflects mucosal inflammatory pathways.

Topics: alpha-Defensins; Animals; Antigens, CD; Calgranulin B; Cell Adhesion Molecules; Cell Line; Cell Movement; Colon; Epithelial Cells; Extracellular Vesicles; Flow Cytometry; GPI-Linked Proteins; Humans; Inflammatory Bowel Diseases; Interleukin-8; Interleukins; Leukocyte Common Antigens; Lipopolysaccharide Receptors; Macrophages; Mice; Microscopy, Electron, Transmission; Mucin-1; Mucin-2; Particle Size; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The anti-inflammatory effect of piperine has been largely investigated in macrophages, but its activity on epithelial cells in inflammatory settings is unclear. The present study aimed to investigate the effect of piperine on the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human epithelial-like SW480 and HT-29 cells. Our data showed that although piperine inhibited the proliferation of SW480 and HT-29 cells in a dose-dependent manner, it had low cytotoxicity on these cell lines with 50 % inhibiting concentration (IC50) values greater than 100 μM. As epithelial-like cells, SW480 and HT-29 cells secreted high levels of the chemokine CXCL8 upon LPS stimulation. Importantly, piperine dose-dependently suppressed LPS-induced secretion of CXCL8 and the expression of CXCL8 messenger RNA (mRNA). Although piperine failed to affect the critical inflammatory nuclear factor-κB pathway, it attenuated the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling. Consistent with previous reports, p38 signaling seemed to play a more pronounced role on the CXCL8 expression than JNK signaling since inhibition of p38, instead of JNK, greatly suppressed LPS-induced CXCL8 expression. Collectively, our results indicated that piperine could attenuate the inflammatory response in epithelial cells via downregulating the MAPK signaling and thus the expression of CXCL8, suggesting its potential application in anti-inflammation therapy.

Topics: Alkaloids; Anti-Inflammatory Agents; Benzodioxoles; Cell Line; Cell Proliferation; Cell Survival; Down-Regulation; Epithelial Cells; HT29 Cells; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Piperidines; Polyunsaturated Alkamides; RNA, Messenger

Targeting early molecular events in intestinal inflammation may represent a useful therapeutic strategy for maintaining remission in inflammatory bowel disease. Recently, we established an intestinal organ culture model (LEL model), which allows to study the initiation of an intestinal inflammatory response in human tissue. In this model, EDTA-mediated depletion of epithelial cells of colonic mucosa results in an instantaneous inflammatory response in resident lamina propria cells, which shows features of intestinal inflammation in vivo. Furthermore, activated immune cells emigrate from the lamina propria onto the luminal side of the basement membrane. Here, we standardize the LEL model and explore its suitability for drug testing. To this end, human mucosal punches of defined surface area were prepared, depleted of epithelial cells, and cultured at an optimized ratio of medium volume/punch area. The intra-assay variability of measurements of inflammatory parameters ranged from 13% for cell migration to 19% for secretion and 30% for tissue gene expression, respectively, of the inflammatory mediators IL-8 and IL-6. Importantly, known suppressive effects of dexamethasone, a drug employed for the treatment of inflammatory bowel diseases, on leucocyte migration, IL8, IL6, and TNF-α production as well as CD86 surface expression by myeloid cells were observed in this model. In conclusion, the present results suggest that the LEL model may represent a useful human experimental system not only for studying initial activation mechanisms in intestinal inflammation but also for evaluating drug compounds for the treatment of mucosal inflammation.

Topics: Anti-Inflammatory Agents; B7-2 Antigen; Cell Movement; Colon; Dexamethasone; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Intestinal Mucosa; Myeloid Cells; Organ Culture Techniques; Tumor Necrosis Factor-alpha

The integrity of the epithelial layer in the gastrointestinal tract protects organisms from exposure to luminal antigens, which are considered the primary cause of chronic intestinal inflammation and allergic responses. The common wheat-associated fungal toxin deoxynivalenol acts as a specific disruptor of the intestinal tight junction network and hence might contribute to the pathogenesis of inflammatory bowel diseases.. The aim of the current study was to assess whether defined galacto-oligosaccharides (GOSs) can prevent deoxynivalenol-induced epithelial dysfunction.. Human epithelial intestinal Caco-2 cells, pretreated with different concentrations of GOSs (0.5%, 1%, and 2%) for 24 h, were stimulated with 4.2-μM deoxynivalenol (24 h), and 6/7-wk-old male B6C3F1 mice were fed a diet supplemented with 1% GOSs for 2 wk before being orally exposed to deoxynivalenol (25 mg/kg body weight, 6 h). Barrier integrity was determined by measuring transepithelial electrical resistance (TEER) and intestinal permeability to marker molecules. A calcium switch assay was conducted to study the assembly of epithelial tight junction proteins. Alterations in tight junction and cytokine expression were assessed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, or ELISA, and their localization was visualized by immunofluorescence microscopy. Sections of the proximal and distal small intestine were stained with hematoxylin/eosin for histomorphometric analysis.. The in vitro data showed that medium supplemented with 2% GOSs improved tight junction assembly reaching an acceleration of 85% after 6 h (P < 0.05). In turn, GOSs prevented the deoxynivalenol-induced loss of epithelial barrier function as measured by TEER (114% of control), and paracellular flux of Lucifer yellow (82.7% of prechallenge values, P < 0.05). Moreover, GOSs stabilized the expression and cellular distribution of claudin3 and suppressed by >50% the deoxynivalenol-induced synthesis and release of interleukin-8 [IL8/chemokine CXC motif ligand (CXCL8)] (P < 0.05). In mice, GOSs prevented the deoxynivalenol-induced mRNA overexpression of claudin3 (P = 0.022) and CXCL8 homolog keratinocyte hemoattractant (Kc) (Cxcl1) (P = 0.06) as well as the deoxynivalenol-induced morphologic defects.. The results demonstrate that GOSs stimulate the tight junction assembly and in turn mitigate the deleterious effects of deoxynivalenol on the intestinal barrier of Caco-2 cells and on villus architecture of B6C3F1 mice.

Topics: Animals; Caco-2 Cells; Claudin-3; Epithelial Cells; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Male; Mice; Oligosaccharides; Permeability; Tight Junctions; Trichothecenes

Trp-His, the anti-atherosclerotic dipeptide, exerted an antiproliferative effect on vascular smooth muscle cells by L-type Ca(2+) channel blocker-like effect. The beneficial potential by the blockade of Ca(2+) channels on chronic intestinal inflammation, including inflammatory bowel disease (IBD), is unclear. Trp-His (100 or 250 mg/kg body weight/day) was administered for 14 days to BALB/c mice, and 5% dextran sodium sulfate (DSS) was administered to induce colitis in the last 7 days. Trp-His reduced DSS-induced typical colitis symptoms and cytokine expression in the colon. Trp-His inhibited interleukin (IL)-8 secretion in tumor necrosis factor (TNF)-α-stimulated HT-29 cells. The inhibitory effect of Trp-His, as well as that of Ca(2+) channel blockers, was impaired by the presence of Ca(2+) channel agonist Bay K 8644. The TNF-α-induced activation of mitogen-activated protein kinases (MAPKs) and IκBα were decreased by Trp-His. These results indicated that the anti-inflammatory effect of Trp-His may be involved in the blockade of L-type Ca(2+) channels.

Topics: Animals; Anti-Inflammatory Agents; Calcium Channels, L-Type; Dipeptides; Female; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestines; Mice; Mice, Inbred BALB C; Tumor Necrosis Factor-alpha

Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1β, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1β brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 μM), and IL-1β prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1β also prevented the IL-1β-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level.

Topics: Antioxidants; Betaxanthins; Caco-2 Cells; Cell Membrane Permeability; Cyclooxygenase 2; Enterocytes; Enzyme Activation; Fruit; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-1beta; Interleukin-6; Interleukin-8; Intestinal Absorption; NADPH Oxidase 1; NADPH Oxidases; NF-kappa B; Nitric Oxide Synthase Type II; Opuntia; Pyridines; Reactive Oxygen Species

The aim of the present study was to evaluate the effectiveness of two isoenergetic elemental formulae with different fat content in the rat model of trinitrobenzene sulphonic acid (TNBS) colitis that mimics human inflammatory bowel disease. A total of forty-five male Wistar rats were assigned to five groups: (1) control group; (2) TNBS-induced colitis group; (3) TNBS-induced colitis group fed a long-chain TAG (LCT)-rich diet; (4) TNBS-induced colitis group fed a medium-chain TAG (MCT)-rich diet; (5) TNBS-induced colitis group fed a baseline diet and administered infliximab. Nutritional management lasted 12 d before and 4 d after rectal administration of TNBS. Subsequently, the rats were killed, and colonic tissue samples were collected for the assessment of histology, inflammation and oxidative stress. The MCT-rich diet decreased IL-6, IL-8 and intercellular adhesion molecule-1 (ICAM-1) levels and glutathione S-transferase (GST) activity, while the LCT-rich diet reduced only ICAM-1 levels and GST activity (P<0.05). Neither elemental formula affected IL-10 levels. Infliximab reduced IL-8 and ICAM-1 levels and GST activity and increased IL-10 levels (P<0.05). No significant differences were detected in oxidative stress. Histological damage scores differed significantly only between the control and the TNBS-induced colitis group. A MCT-rich formula seems to exert stronger anti-inflammatory effects than a LCT-rich formula in TNBS colitis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Colon; Dietary Fats; Disease Models, Animal; Fatty Acids; Food, Formulated; Gastrointestinal Agents; Glutathione Transferase; Inflammatory Bowel Diseases; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Intestinal Mucosa; Male; Molecular Weight; Oxidative Stress; Random Allocation; Rats; Rats, Wistar

Campylobacter concisus, a Gram-negative bacterium that colonizes the human oral cavity, has been shown to be associated with inflammatory bowel diseases (IBD). The effects of different C. concisus strains on intestinal epithelial expression of Toll like receptors (TLR) have not been investigated. This study examined the effects of C. concisus strains isolated from patients with IBD and controls on expression of TLR4, its co-receptor myeloid differentiation factor (MD)-2; TLR2, TLR5, cyclooxygenase-2 (COX-2) and interleukin (IL)-8 in HT-29 cells.Fourteen oral and enteric C. concisus strains isolated from patients with IBD and healthy controls were co-incubated with HT-29 cells. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells in response to C. concisus infection was examined by Western blot, flow cytometry analysis and immunofluorescent staining visualized by confocal microscope. Production of IL-8 was evaluated by enzyme-linked immunosorbent assay.Both oral and enteric C. concisus strains upregulated expression of TLR4 in HT-29 cells. The levels of glycosylated TLR4 (Gly-TLR4) and surface TLR4 induced by C. concisus strains isolated from patients with IBD were significantly higher than those induced by C. concisus strains isolated from the healthy controls. Four C. concisus strains isolated from patients with IBD induced more than two-fold increase of surface expression of MD-2. C. concisus did not affect expression of TLR2 and TLR5. All C. concisus strains induced production of IL-8 and COX-2 in HT-29 cells.This study shows that some C. concisus strains, most from patients with IBD, upregulate surface expression of TLR4 and MD-2 in HT-29 cells. These data suggest that a potential role of specific C. concisus strains in modulating the intestinal epithelial responses to bacterial LPS needs to be investigated.

Topics: Campylobacter; Cyclooxygenase 2; Gene Expression; HT29 Cells; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestines; Lymphocyte Antigen 96; Saliva; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptors

The potential use of polyphenols in the prevention and treatment of chronic inflammatory diseases has been extensively investigated although the mechanisms involved in cellular signaling need to be further elucidated. Cyanidin-3-glucoside is a typical anthocyanin of many pigmented fruits and vegetables widespread in the human diet. In the present study, the protection afforded by cyanidin-3-glucoside against cytokine-triggered inflammatory response was evaluated in the human intestinal HT-29 cell line, in comparison with 5-aminosalicylic acid, a well-established anti-inflammatory drug, used in inflammatory bowel disease. For this purpose, some key inflammatory mediators and inflammatory enzymes were examined. Our data showed that cyanidin-3-glucoside reduced cytokine-induced inflammation in intestinal cells, in terms of NO, PGE2 and IL-8 production and of iNOS and COX-2 expressions, at a much lower concentration than 5-aminosalicylic acid, suggesting a higher anti-inflammatory efficiency. Interestingly, cyanidin-3-glucoside and 5-aminosalicylic acid neither prevented IkB-α degradation nor the activation of NF-kB, but significantly reduced cytokine-induced levels of activated STAT1 accumulated in the cell nucleus. In addition, we established that phosphorylated p38 MAPK was not involved in the protective effect of cyanidin-3-glucoside or 5-aminosalicylic acid. Taking into account the high concentrations of dietary anthocyanins potentially reached in the gastrointestinal tract, cyanidin-3-glucoside may be envisaged as a promising nutraceutical giving complementary benefits in the context of inflammatory bowel disease.

Topics: Anthocyanins; Anti-Inflammatory Agents; Cell Line; Cell Nucleus; Cell Survival; Cyclooxygenase 2; Cytokines; Dinoprostone; Enzyme Activation; Glucosides; HT29 Cells; Humans; Inflammation; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Mesalamine; Nitric Oxide; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Phosphorylation; STAT1 Transcription Factor; Transcription Factor RelA

Inflammatory colorectal polyps (ICRPs) in miniature dachshunds were recently recognized as a major cause of large bowel diarrhea in this dog breed in Japan. ICRPs are characterized by the formation of multiple small polyps and/or space-occupying large polyps in the colorectal area and are thought to be a novel form of inflammatory bowel disease (IBD). To explore key mediators in the pathogenesis of ICRPs, we analyzed several pro-inflammatory cytokine (IL-1β, IL-6, TNF-α, IL-8, IL-12p35, IL-12/23p40, and IL-23p19) mRNA expressions in colorectal polyps in ICRP dogs by quantitative PCR. Among these cytokines, IL-8 mRNA expression was markedly up-regulated in large polyps. To examine IL-8 protein expression, we analyzed IL-8 protein level and its location in colorectal mucosal specimens of ICRP dogs by ELISA and immunofluorescence microscopy. IL-8 protein was significantly increased in large polyps and serum in dogs with ICRPs compared to controls. By immunofluorescence microscopy, IL-8 was only localized in macrophages, but not in mucosal epithelial cells or neutrophils. IL-8-positive macrophages were significantly increased in large polyps compared to controls. These results suggest that IL-8 is produced mainly by macrophages and may induce neutrophil infiltration in the colorectal area of ICRP dogs.

Topics: Animals; Colonic Polyps; Dog Diseases; Dogs; Female; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Intestinal Polyps; Male; Rectal Diseases; RNA, Messenger

The development of new therapeutic approaches, combining efficacy and safety against intestinal inflammation, notably inflammatory bowel disease (IBD), has emerged as an important goal due to the significant side effects and the lack of effectiveness of standard current therapies. Recently, several studies described the health-promoting effects of red wine, including anti-inflammatory properties, but the molecular mechanisms underlying its beneficial role remain largely unknown. Red wine is rich in phenolic compounds and it has been suggested that the positive effect of red wine intake might be attributed not only to the antioxidant properties of these compounds but also to the modulation of signalling cascades in connection with physiological and pathophysiological conditions such as inflammatory processes. This study assesses the potential anti-inflammatory action of a red wine extract (RWE) enriched in polyphenols in a cellular model of intestinal inflammation using cytokines-stimulated HT-29 colon epithelial cells. RWE suppressed cytokines-induced IκB degradation and interleukin-8 production in a dose-dependent manner. Coherently, key inflammatory mediators downstream NF-κB activation; notably cyclooxygenase-2 and inducible nitric oxide synthase were maintained at low levels by RWE in the presence of the cytokines. Additionally, RWE inhibited both the increase of nitric oxide derived from iNOS and of protein tyrosine nitration, a biomarker of nitrosative stress that typically requires the reaction of nitric oxide with the superoxide radical. Taken together, the anti-inflammatory action of RWE, mechanistically supported by the modulation of cascades orchestrated by NF-κB and involving nitric oxide, suggests that RWE (a readily straightforward preparation when compared with the purification of specific compounds) may represent a simple and inexpensive therapeutic strategy in the context of intestinal inflammation.

Topics: Anti-Inflammatory Agents; Cell Survival; Cyclooxygenase 2; Dose-Response Relationship, Drug; Epithelial Cells; HT29 Cells; Humans; I-kappa B Proteins; Inflammation Mediators; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-1; Interleukin-8; Intestinal Mucosa; Intestines; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide; Nitric Oxide Synthase Type II; Polyphenols; Tumor Necrosis Factor-alpha; Wine

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

Topics: Alkaline Phosphatase; Animals; Bacterial Proteins; Cells, Cultured; Disease Models, Animal; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Intestine, Small; Lipopolysaccharides; Mice; Mice, Knockout; Receptors, Purinergic P2; Uridine Diphosphate

Polymorphonuclear leukocytes (PMN) feature prominently in the mucosa, including in crypt abscesses, of patients with inflammatory bowel disease, yet the mediators that are responsible for this migration are unknown. We discovered that CXCR2 chemokines (reportedly elevated in the mucosa) have reduced potency recruiting PMN across epithelial cell monolayers versus acellular filters, so the objective was to determine what molecules modify transepithelial PMN migration to CXCR2 chemokines.. Transwells with T84 colon carcinoma monolayers or no epithelium were used with adolescent patient peripheral blood PMN and CXCL8 (interleukin-8 [IL-8], binds CXCR1 and CXCR2), CXCL5 (epithelial-derived neutrophil chemoattractant-78 [ENA-78]), or CXCL1 (Gro-α, both bind CXCR2) as chemoattractants.. IL-8 was equally potent at recruiting PMN across filters and T84 monolayers growing on the filters. In contrast, ENA-78 and Gro-α were significantly less potent at recruiting PMN across monolayers than across bare filters. Blocking CXCR1 reduced PMN migration across monolayers to IL-8. We ruled out superoxide radicals possibly enhancing migration to IL-8 by using PMN from a patient with chronic granulomatous disease. PMN constitutively produce adenosine, so we added adenosine deaminase to the transwell assays and observed increased migration to ENA-78 across T84 monolayers. The level of migration was further enhanced by pretreating PMN with adenosine before adding the cells to the assay in the presence of the deaminase.. PMN migration mediated by CXCR2 through the epithelium is regulated by adenosine. Adenosine appears to reduce transepithelial migration by influencing β2 integrin use on the PMN.

Topics: Adenosine; Adenosine Deaminase; Adolescent; Adult; CD18 Antigens; Chemokine CXCL1; Chemokine CXCL5; Chemotaxis, Leukocyte; Child; Colonic Neoplasms; Epithelial Cells; Female; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Ligands; Male; Neutrophil Infiltration; Neutrophils; Receptors, Interleukin-8B; Young Adult

Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y(6) receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5'-diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis-like disease and we characterized the signaling events linking P2Y(6) to CXCL8 expression in IEC.. Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real-time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays.. Mutation of the AP-1 site in the CXCL8 core promoter abolished the UDP-stimulating effect. The c-fos/c-jun dimer was identified as the AP-1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y(6) required PKCδ activation upstream of the signaling pathway composed of MEK1/2-ERK1/2 and c-fos. UDP administration to mice suffering from colitis-like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation.. This study not only describes the P2Y(6) signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y(6) to reduce intestinal inflammation.

Topics: Animals; Blotting, Western; Cells, Cultured; Chemokine CXCL1; Chromatin Immunoprecipitation; Epithelial Cells; Flow Cytometry; Fluorescent Antibody Technique; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Luciferases; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neutrophil Infiltration; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptors, Purinergic P2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription Factor AP-1

Single nucleotide polymorphisms (SNP) in the TLR5 gene have been associated with human inflammatory bowel disease (IBD) and animal models of this disease. We recently demonstrated a significant association between three non-synonymous SNPs in the canine TLR5 gene and IBD in German shepherd dogs (GSDs). However, so far, no direct link between these SNPs and a disturbance in TLR5 function was shown. In the present study, we determined the functional significance of the canine TLR5 SNPs by transfecting the identified risk-protective and risk-associated haplotype into human embryonic kidney cells (HEK) and assessed nuclear factor-kappa B (NF-κB) activation and CXCL8 production after stimulation. In addition, a whole blood assay for TLR5 activation was developed using blood derived from carrier dogs of either haplotype. There was a significant increase in NF-kB activity when cells transfected with the risk-associated TLR5 haplotype were stimulated with flagellin compared to the cells expressing the risk-protective TLR5 haplotype. This difference in NFkB activation correlated with CXCL8 expression in the supernatant measured by ELISA. Furthermore, whole blood taken from carrier dogs of the risk-associated TLR5 haplotype produced significantly more TNF after stimulation with flagellin compared to that taken from carriers of the risk-protective haplotype. Thus, we show for the first time a direct functional impact of the canine IBD risk-associated TLR5 haplotype, which results in hyper-responsiveness to flagellin compared to the IBD risk-protective TLR5 haplotype. Our data potentially suggest that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease.

Topics: Animals; Blood; Dogs; Flagellin; Genetic Predisposition to Disease; Haplotypes; HEK293 Cells; Humans; Inflammatory Bowel Diseases; Interleukin-8; Luminescent Proteins; Microscopy, Confocal; NF-kappa B; Polymorphism, Single Nucleotide; Risk Factors; Toll-Like Receptor 5; Tumor Necrosis Factor-alpha

This study aimed at evaluating the anti-inflammatory properties of a pomegranate fruit husk (PomH) polyphenolic extract, rich in punicalagin, using Caco-2 cells, an in vitro model of human intestinal epithelium. Differentiated cells in bicameral inserts were pretreated or not with a PomH extract or punicalagin, as reference, at the apical side, representing the intestinal lumen. Inflammation was then induced with a cocktail of cytokines (Il-1β, TNFα and IFNγ) and LPS. After 24 h incubation, 3 pro-inflammatory markers, i.e., interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1, were assayed both at their gene transcription (qRT-PCR) and secretion (ELISA) levels. As previously described, the pro-inflammatory cocktail significantly stimulated these 3 markers, at the gene transcript and secretion levels. In inflamed cells, a significant down-regulation of the transcription of the genes encoding IL-6 and MCP-1 was observed in the presence of the PomH extract or punicalagin, while IL-8 transcription was unaffected. Both treatments also decreased the amounts of the 3 proteins with dose-response effects, but only in the apical compartment. A lowered ELISA response was also observed when either IL-6, IL-8 or MCP-1 were mixed with punicalagin in a cell-free culture medium, indicating a direct molecular interaction. In conclusion, the punicalagin-rich PomH extract tested showed anti-inflammatory properties in the Caco-2 in vitro intestinal model. It acted both on the pro-inflammatory gene transcription and protein levels, the later phenomenon being possibly due to a direct molecular trapping. These data suggest that pomegranate husk could be an interesting natural source contributing to prevent intestinal chronic inflammation.

Topics: Anti-Inflammatory Agents; Caco-2 Cells; Chemokine CCL2; Cytokines; Down-Regulation; Humans; Hydrolyzable Tannins; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Intestines; Lipopolysaccharides; Lythraceae; Phytotherapy

Inflammatory bowel diseases (IBD) and colorectal cancer (CRC) are disorders that originate from immune disturbances. In our study, we evaluated the association between the -251 T/A interleukin (IL)-8 and the -1112 C/T IL-13 polymorphisms, the risk of IBD, and CRC development. Genotypes were determined by PCR-restriction fragment length polymorphism in 191 patients with CRC, 150 subjects with IBD, and 205 healthy controls. We found an association between CRC and the presence of the -251 TA genotype and A allele of the IL-8 gene (odds ratios [ORs] 2.28 and 1.65). A similar relationship was observed between these polymorphic variants and ulcerative colitis (OR 2.05 for the -251 TA genotype and OR 1.47 for the -251 A allele) as well as Crohn's disease (ORs 3.11 and 1.56, respectively). Our research also revealed that the CT and TT genotypes of the IL-13 -1112 C/T polymorphism may be connected with a higher risk of CRC (ORs 2.28 and 1.65). The same genotypes affected the susceptibility of IBD (ORs 2.26 and 3.72). Our data showed that the IL-8 -251 T/A and IL-13 -1112 C/T polymorphisms might be associated with the IBD and CRC occurrence and might be used as predictive factors of these diseases in a Polish population.

Topics: Adult; Colorectal Neoplasms; Female; Genotype; Humans; Inflammatory Bowel Diseases; Interleukin-13; Interleukin-8; Male; Middle Aged; Poland; Polymorphism, Genetic

Drug formulation screenings for treatment of inflammatory bowel disease (IBD) are mostly conducted in chemically induced rodent models that represent acute injury-caused inflammation instead of a chronic condition. To accurately screen drug formulations for chronic IBD, a relevant model that mimics the chronic condition in vitro is urgently needed. In an effort to reduce and potentially replace this scientifically and ethically questionable animal testing for IBD drugs, our laboratory has developed an in vitro model for the inflamed intestinal mucosa observed in chronic IBD, which allows high-throughput screening of anti-inflammatory drugs and their formulations. The in vitro model consists of intestinal epithelial cells, human blood-derived macrophages, and dendritic cells that are stimulated by the inflammatory cytokine interleukin-1β. In this study, the model was utilized for evaluation of the efficacy and deposition of budesonide, an anti-inflammatory drug, in three different pharmaceutical formulations: (1) a free drug solution, (2) encapsulated into PLGA nanoparticles, and (3) encapsulated into liposomes. The in vitro model of the inflamed intestinal mucosa demonstrated its ability to differentiate therapeutic efficacy among the formulations while maintaining the convenience of conventional in vitro studies and adequately representing the complex pathophysiological changes observed in vivo.

Topics: Animal Testing Alternatives; Anti-Inflammatory Agents; Budesonide; Caco-2 Cells; Cells, Cultured; Coculture Techniques; Dendritic Cells; Humans; Inflammatory Bowel Diseases; Interleukin-1beta; Interleukin-8; Lactic Acid; Liposomes; Macrophages; Microscopy, Confocal; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer

Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.

Topics: Apoptosis; Bacterial Toxins; Cell Line; Colitis; Colon; Dextran Sulfate; Dose-Response Relationship, Drug; Enterotoxins; Enzyme Activation; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Neurotensin; NF-kappa B; Phosphorylation; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins pp60(c-src); Receptor, IGF Type 1; Receptors, Neurotensin; Time Factors; Tyrphostins

It is demonstrated that excessive activation of NF-κB is central to the pathogenesis of inflammatory bowel disease (IBD). Zinc finger protein A20 (A20) is a key player in the negative feedback regulation of NF-κB signaling in response to multiple stimuli and has been described as central gatekeeper in inflammation and immunity. Mice genetically deficient in A20 develop severe intestinal inflammation and have increased susceptibility to dextran sodium sulfate (DSS)-induced colitis. Few studies have been done to explore the role of A20 in the pathogenesis of IBD. To clarify the relationship between intestinal inflammation and the expression level of A20 in IBD patients, the expression level of A20 and a series of inflammatory cytokines, such as NF-κB, IL-6, and IL-8, in children with IBD and controls were examined.. Terminal ileal mucosal samples were obtained via endoscopy. Fifty-seven mucosal samples were divided into 4 groups: normal control group (n = 16), IBD remission group (n = 12), IBD active group (n = 13) and non-IBD enteritis group (n = 16). According to disease activity index scores, the IBD patients were divided into IBD remission group and IBD active group. Normal control group was consisted of patients with functional bowel disorders or intestinal polyps. Non-IBD enteritis was defined as changes in which endoscopy and histological examination showed inflammatory changes but could not be diagnosed as IBD. Real-time PCR was adopted for detecting the mRNA levels of A20, IL-6 and IL-8. Meanwhile immunohistochemistry was performed to measure the expression of A20 and NF-κB.. (1) The expression of A20 and NF-κB were very low in normal control group, but significantly up-regulated in IBD active group and non-IBD enteritis group (P < 0.01 for both); (2) Compared with normal control group, expression of NF-κB [(9.35 ± 4.84)% vs. (0.57 ± 0.44)%, P < 0.01], IL-6 (t' = 1.34, P > 0.05), IL-8 (t = 1.38, P > 0.05) increased in IBD remission group, while the expression of A20 in both mRNA (t = 1.03, P > 0.05) and protein levels [(0.36 ± 0.18)% vs. (0.87 ± 0.29)%, P < 0.01] decreased; (3) Compared with non-IBD enteritis group, although the expression of NF-κB [(24.17 ± 11.27)% vs. (55.29 ± 21.84)%, P < 0.01], IL-6 (t = 2.22, P < 0.05), IL-8 (t = 2.97, P < 0.01) were highly increased in IBD active group, the expression of A20 in both mRNA(t = 2.26, P < 0.05) and protein levels [(29.23 ± 11.70)% vs. (16.81 ± 5.90)%, P < 0.01]significantly decreased; (4) The expression of IL-6, IL-8 were similar in IBD remission group and non-IBD enteritis group (both P > 0.05), but the expression of A20 was much lower in both mRNA (t = 4.42, P < 0.01) and protein levels [(29.23 ± 11.70)% vs. (0.47 ± 0.25)%, P < 0.01] in IBD remission group.. The results demonstrate that there is an excessive inflammatory response but insufficient up-regulation of A20 expression in IBD patients. Low levels expression of A20 may play an important role in the pathogenesis of IBD.

Topics: Case-Control Studies; Child; Endopeptidases; Female; Humans; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Intestinal Mucosa; Male; NF-kappa B

Resistin is a cysteine-rich protein, which is abundantly expressed at the site of inflammation, and acts as a regulator of the NF-kB-dependent cytokine cascade. The aim of this study was to evaluate resistin levels in relation to inflammatory mediators, disease phenotype and autoantibody status in a spectrum of pathological conditions of the gastrointestinal tract. Resistin levels were measured with an ELISA in sera originated from 227 patients and 40 healthy controls (HC). Fifty patients diagnosed with non-alcoholic fatty liver disease (NAFLD), 53 ulcerative colitis (UC), 51 Crohn's disease (CD), 46 autoimmune hepatitis (AIH) and 27 primary sclerosing cholangitis (PSC) were included. The sera were analysed with respect to biochemical parameters of systemic inflammation and liver function and to the presence of antibodies to nuclear antigens (ANA), mitochondria (AMA) and smooth muscle (SMA). Compared with HC, resistin levels were raised in AIH (P = 0.017) and PSC (P = 0.03); compared with NAFLD, levels were elevated in CD (P = 0.041), AIH (P < 0.001) and PSC (P < 0.001). Patients with elevated levels of resistin were more often treated with corticosteroids, but no difference was found between active disease and clinical remission. Resistin levels were significantly higher in ANA-positive individuals compared with ANA-negative (P = 0.025). Resistin levels were directly correlated with IL-6 (r = 0.30, P = 0.02) and IL-8 (r = 0.51, P < 0.001). Elevated levels of resistin were prominent in patients with hepatobiliary inflammation and were associated with breach of self-tolerance, i.e. ANA positivity. Thus, we propose that resistin may be an important marker of disease severity in autoantibody-mediated gastrointestinal inflammatory diseases.

Topics: Adult; Aged; Antibodies, Antinuclear; Biomarkers; Cholangitis, Sclerosing; Disease Progression; Fatty Liver; Gene Expression Regulation; Hepatitis; Humans; Immune Tolerance; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Male; Middle Aged; Non-alcoholic Fatty Liver Disease; Resistin

To investigate the association of interleukin 8 (IL-8) gene polymorphisms with the risks of inflammatory bowel disease (IBD).. Single nucleotide polymorphisms (SNPs) of IL-8 gene at -845 T/C, -738 T/A, -353 A/T, -251 T/A and +678 T/C were analyzed in 183 IBD patients. They included Crohn's disease (CD, n = 41), ulcerative colitis (UC, n = 142) and healthy controls (n = 160). The methods of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction-sequence specific primers (PCR-SSP) were employed.. No association was observed between any of these five SNPs in IL-8 gene with the occurrence of IBD. A specific haplotype AAT (-353 A/T, -251 T/A & +678 T/C) was over-represented in UC cases when compared with controls (31.0% vs 23.7%, P = 0.046). But the distributions of this haplotype did not show significant difference between CD cases and controls.. Our data support a significant but modest association between the AAT haplotype of IL-8 gene and UC (OR = 1.441, 95%CI 1.007 - 2.063).

Topics: Adult; Asian People; Case-Control Studies; Female; Genetic Predisposition to Disease; Genotype; Humans; Inflammatory Bowel Diseases; Interleukin-8; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Young Adult

The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC(-/-)/PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and in vivo, topical treatment with activated PC led to mucosal healing and amelioration of colitis. Taken together, these findings demonstrate that the PC pathway is a unique system involved in controlling intestinal homeostasis and inflammation by regulating epithelial barrier function.

Topics: Animals; Anticoagulants; Antigens, CD; Caco-2 Cells; Cells, Cultured; Colitis; Endothelial Protein C Receptor; Epithelial Cells; Gene Expression; Humans; Immunohistochemistry; Inflammatory Bowel Diseases; Interleukin-6; Interleukin-8; Intestinal Mucosa; Intestines; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phosphoproteins; Protein C; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tight Junctions; Zonula Occludens-1 Protein

TNF-alpha is a major cytokine involved in inflammatory bowel disease (IBD). In this study, water extract of Grifola frondosa (GFW) was evaluated for its protective effects against colon inflammation through the modulation of TNF-alpha action. In coculture of HT-29 human colon cancer cells with U937 human monocytic cells, TNF-alpha-induced monocyte adhesion to HT-29 cells was significantly suppressed by GFW (10, 50, 100 micg/ml). The reduced adhesion by GFW correlated with the suppressed expression of MCP-1 and IL-8, the major IBD-associated chemokines. In addition, treatment with GFW significantly suppressed TNF-alpha-induced reactive oxygen species production and NF-kappaB transcriptional activity in HT-29 cells. In differentiated U937 monocytic cells, LPS-induced TNF-alpha production, which is known to be mediated through NF-kappaB activation, was significantly suppressed by GFW. In an in vivo rat model of IBD, oral administration of GFW for 5 days (1 g/kg per day) significantly inhibited the trinitrobenzene sulfonic acid (TNBS)-induced weight loss, colon ulceration, myeloperoxidase activity, and TNF-alpha expression in the colon tissue. Moreover, the effect of GFW was similar to that of intra-peritoneal injection of 5-aminosalicylic acid (5-ASA), an active metabolite of sulfasalazine, commonly used drug for the treatment of IBD. The results suggest that GFW ameliorates colon inflammation by suppressing production of TNF-alpha as well as its signaling through NF-kappaB leading to the expression of inflammatory chemokines, MCP-1 and IL-8. Taken together, the results strongly suggest GFW is a valuable medicinal food for IBD treatment, and thus may be used as an alternative medicine for IBD.

Topics: Animals; Cell Adhesion; Cell Extracts; Chemokine CCL2; Coculture Techniques; Colon; Grifola; HT29 Cells; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Monocytes; NF-kappa B; Peroxidase; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stomach Ulcer; Transcription, Genetic; Trinitrobenzenesulfonic Acid; Tumor Necrosis Factor-alpha; U937 Cells; Weight Loss

In inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for this proinflammatory capacity of IECs in IBD remain unclear. The CD40/CD40L interaction activates various pathways in immune and nonimmune cells related to inflammation and was shown to be critical for the development of IBD. Here we demonstrate CD40 expression within IECs during active IBD. Endoscopically obtained biopsies taken from Crohn's disease (n = 112) and ulcerative colitis patients (n = 67) consistently showed immunofluorescence staining for CD40 in IECs of inflamed ileal or colonic mucosa. In noninvolved mucosa during active disease, tissue obtained during Crohn's disease or ulcerative colitis in remission and biopsies from healthy controls (n = 38) IECs almost entirely lacked CD40 staining. Flow cytometry and RT-PCR analysis using different intestinal epithelial cell lines (HT29, SW480, and T84) showed IFN-gamma to effectively induce CD40 in IECs. Cells were virtually unresponsive to LPS or whole E. coli regarding CD40 expression. In addition, a moderate induction of CD40 was found in response to TNF-alpha, which exerted synergistical effects with IFN-gamma. CD40 ligation by CD40L-transfected murine fibroblasts or soluble CD40L increased the secretion of IL-8 in IFN-gamma pretreated HT29 cells. Our findings provide evidence for the epithelial expression and modulation of CD40 in IBD-affected mucosa and indicate its involvement in the proinflammatory function of IECs.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Biopsy; CD40 Antigens; CD40 Ligand; Epithelial Cells; Female; Fibroblasts; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-8; Intestinal Mucosa; Male; Mice; Middle Aged; Remission Induction

Inflammatory bowel disease (IBD) is a polygenetic disorder. Our group previously showed that a variant within the CXCL9 gene is associated with pediatric Crohn's disease. As CXCL9, CXCL10, and CXCL11 are the 3 ligands to the receptor CXCR3, the aim of this study was to investigate the colonic transcriptional activity of the CXCR3 axis and to perform SNP genotyping of a CXCL11 polymorphism in a large pediatric and adult IBD cohort.. mRNA expression of CXCR3, CXCL9, CXCL10, CXCL11, and IL8 was analyzed in colonic biopsies using real-time PCR. CXCL11 rs6817952 nucleotide substitution was determined in 501 German individuals with IBD (336 CD, 165 UC) including 258 children and 243 adults as well as in 231 controls by a TaqMan SNP genotyping assay.. CXCR3 axis genes were significantly overexpressed in inflamed colonic tissue of pediatric CD and UC patients. The prevalence of hetero- and homozygous variants of the rs6817952 genotype was higher in pediatric but not in adult CD patients compared with that in controls (P = 0.04). Moreover, carriers of the hetero- and homozygous genotype variants of rs6817952 were at increased risk for UC in all age groups (P = 0.009).. Our study provides evidence of the significant overexpression of the CXCR3 axis in active IBD, suggesting it has a role in IBD pathogenesis. The rs6817952 A variant is a risk allele for pediatric CD and UC in all age groups. Therapeutic studies will have to show whether the blockade of chemokine receptors such as CXCR3 can modulate intestinal inflammation in a clinical application.

Topics: Adolescent; Adult; Aged; Biopsy; Chemokine CXCL10; Chemokine CXCL11; Chemokine CXCL9; Child; Child, Preschool; Cohort Studies; Colon; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Genotype; Germany; Humans; Infant; Inflammatory Bowel Diseases; Interleukin-8; Male; Middle Aged; Polymorphism, Single Nucleotide; Prevalence; Receptors, CXCR3; Risk Factors; Young Adult

Growing evidence indicates that innate immunity, including toll-like receptor (TLR) signalling, plays a role in inflammatory bowel disease (IBD). This may also apply in the case of TLR-8, which has recently been shown to reverse the immunosuppressive function of regulatory T cells. However, the role of TLR-8 in IBD is currently unknown, and therefore we investigated the expression of TLR-8 and its natural antagonist, Tollip, in normal and inflamed human gut, and examined whether the receptor is functionally active.. TLR-8 and Tollip mRNA expression were measured in colonic epithelial cells (CEC) and lamina propria mononuclear cells (LPMNC) by quantitative polymerase chain reaction. TLR-8 protein expression was visualized in whole biopsy specimens by indirect immunofluorescence microscopy. Cellular localization of TLR-8 protein was assessed by immuno-electron microscopy. IL-8 secretion was measured by ELISA after stimulation with TLR-8 ligand.. TLR-8 mRNA and protein expression were substantially up-regulated in CEC from inflamed mucosa from patients with ulcerative colitis (approximately 350-fold, p<0.01) and Crohn's disease (approximately 45-fold, p<0.05) compared to controls. TLR-8 proteins resided on the luminal surface membrane and in intracellular organelles. Tollip was not increased in CEC from IBD patients. CEC from normal mucosa responded to TLR-8 stimulation by secreting IL-8. TLR-8 was expressed only on the mRNA level in LPMNC with no differences between IBD patients and controls.. Expression of TLR-8, but not Tollip, is highly up-regulated in the colonic epithelium from patients with active IBD. Since the receptor is functionally active, our data suggest that TLR-8 signalling is important in the pathogenesis of IBD.

Topics: Adolescent; Adult; Aged; Colitis, Ulcerative; Crohn Disease; Epithelial Cells; Female; Gene Expression; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; Toll-Like Receptor 8; Up-Regulation

Lymphocyte recruitment is a key pathogenic event in inflammatory bowel disease (IBD). Adhesion of T cells to human intestinal microvascular endothelial cells (HIMEC) is mediated by ICAM-1, VCAM-1 and fractalkine (FKN), but the signaling molecules that orchestrate this process have yet to be identified. Because MAPK play an important role in the response of many cell types to pro-inflammatory stimuli, we assessed the functional role of p38 MAPK, p42/44 MAPK and JNK in the regulation of lymphocyte adhesion to and chemotaxis across the microvasculature in IBD. We found that the MAPK were phosphorylated in the bowel microvasculature and human intestinal fibroblasts of patients with IBD but not of healthy individuals. Stimulation of HIMEC with TNF-alpha triggered phosphorylation of the MAPK, and up-regulation of VCAM-1, FKN and ICAM-1. Blockade of p38 decreased the expression of all MAPK by 50% (p<0.01), whereas inhibition of p42/44 decreased the expression of ICAM-1 and FKN by 50% (p<0.01). Treatment of human intestinal fibroblasts with TNF-alpha elicited production of IL-8 and MCP-1, which was reduced (p<0.05) by blockade of p38 and p42/44. Finally, blockade of p38 and p42/44 reduced lymphocyte adhesion to (p<0.05) and transmigration across (p<0.05) HIMEC monolayers. These findings suggest a critical role for MAPK in governing lymphocyte influx into the gut in IBD patients, and their blockade may offer a molecular target for blockade of leukocyte recruitment to the intestine.

Topics: Cell Adhesion; Cell Movement; Chemokine CCL2; Chemokine CX3CL1; Fibroblasts; Humans; Inflammatory Bowel Diseases; Intercellular Adhesion Molecule-1; Interleukin-8; MAP Kinase Kinase 4; Microvessels; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; T-Lymphocytes; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Effects of dietary taurine on the experimental colitis induced by dextran sulfate sodium (DSS) were studied. C57BL/6 mice administrated taurine or placebo for 5 days were given 3% DSS to induce acute. The colitis was as-sessed using indices such as diarrhea/bleeding scores, colon length change, histological score and tissue myeloperoxidase (MPO) activity. Further, tissue mRNA levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2, were determined by real-time PCR. Taurine supplementation significantly attenuated the severity of diarrhea, colon shortening, histological score, MPO activity elevation and abnormal MIP-2 gene expression, indicating that taurine prevents DSS-induced colitis. Taurine also inhibited the TNF-alpha-induced secretion of IL-8 (a human homologue of MIP-2) from human intestinal epithelial Caco-2 cells. Inhibition of chemokine secretion from intestinal cells may be involved in the mechanisms underlying the cytoprotective function of taurine in the intestinal epithelium.

Topics: Animals; Dextran Sulfate; Diet; Disease Models, Animal; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Mice; Peroxidase; Taurine

IBD is characterized by a chronic, dysregulated immune response to intestinal bacteria. Past work has focused on the role of T cells and myeloid cells in mediating chronic gastrointestinal and systemic inflammation. Here, we show that circulating and tissue B cells from CD patients demonstrate elevated basal levels of activation. CD patient B cells express surface TLR2, spontaneously secrete high levels of IL-8, and contain increased ex vivo levels of phosphorylated signaling proteins. CD clinical activity correlates directly with B cell expression of IL-8 and TLR2, suggesting a positive relationship between these B cell inflammatory mediators and disease pathogenesis. In contrast, B cells from UC patients express TLR2 but generally do not demonstrate spontaneous IL-8 secretion; however, significant IL-8 production is inducible via TLR2 stimulation. Furthermore, UC clinical activity correlates inversely with levels of circulating TLR2+ B cells, which is opposite to the association observed in CD. In conclusion, TLR2+ B cells are associated with clinical measures of disease activity and differentially associated with CD- and UC-specific patterns of inflammatory mediators, suggesting a formerly unappreciated role of B cells in the pathogenesis of IBD.

Topics: Adult; Aged; B-Lymphocytes; Female; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Interleukin-8; Lymphocyte Activation; Male; Middle Aged; T-Lymphocytes; Toll-Like Receptor 2

Apple procyanidins (ACT) is a natural biologically active compound extracted from apple. Our recent studies have shown that ACT ameliorates the symptoms of atopic dermatitis and inhibits food-allergen-induced oral sensitization. The aim of this study was to investigate the potential protective effect and mechanism of action of ACT in a murine model of inflammatory bowel disease. We investigated the preventive effects of ACT in experimental models of colitis induced by dextran sulfate sodium (DSS) or oxazolone. Oral administration of ACT before DSS treatment attenuated the DSS-induced mortality rate and decreased body weight loss. ACT also prevented the body weight loss associated with oxazolone-induced colitis. Next we examined the effect of ACT on intraepithelial lymphocytes (IEL), which is a major T cell population in the intestine. Oral administration of ACT increased the proportions of TCRgammadelta and TCRalphabeta-CD8alphaalpha T cells in IEL and suppressed interferon gamma synthesis in stimulated IEL. In addition, ACT inhibited phorbol 12-myristate 13-acetate-induced secretion of interleukin 8 (IL-8) in intestinal epithelial cells. The combined anti-inflammatory and immunomodulatory effects of ACT on intestinal epithelial cells and IEL suggest that it may be an effective oral preventive agent for inflammatory bowel diseases.

Topics: Adjuvants, Immunologic; Administration, Oral; Animals; Cell Line; Colon; Dextran Sulfate; Disease Models, Animal; Humans; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-8; Malus; Mice; Mice, Inbred C57BL; Oxazolone; Proanthocyanidins; T-Lymphocytes

Mucosally adherent E. coli are found in inflammatory bowel disease (IBD) and colon cancer. They promote release of the proinflammatory cytokine interleukin-8 (IL-8). We explored mechanisms for this release and its inhibition by drugs.. IL-8 release from colon epithelial cells in response to mucosal E. coli isolates from IBD, colon cancer, and controls was characterized at the cellular and molecular level.. IL-8 response of HT29 cells was greater with Crohn's disease (689 +/- 298 [mean +/- SD] pg IL-8/mL at 4 hours, n = 7) and colon cancer isolates (532 +/- 415 pg/mL, n = 14) than with ulcerative colitis (236 +/- 58 pg/mL, n = 6) or control isolates (236 +/- 100 pg/mL, n = 6, P < 0.0001). Bacterial supernatants contained shed flagellin that triggered IL-8 release. For whole bacteria the IL-8 response to E. coli that agglutinate red blood cells (548 +/- 428 pg IL-8/mL, n = 16), a function that correlates with epithelial invasion, was greater than for nonhemagglutinators (281 +/- 253 pg/mL, n = 17; P < 0.0001). This was particularly marked among E. coli that, although flagellate, could not release IL-8 from TLR5-transfected HEK293 cells. IL-8 release was mediated by extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and inhibited by mesalamine, but not hydrocortisone, at therapeutic concentrations.. Mucosa-associated E. coli shed flagellin that elicits epithelial IL-8 release but this may only become relevant when the mucosal barrier is weakened to expose basolateral TLR5. Adherent and invasive IBD and colon cancer E. coli isolates also elicit a flagellin-independent IL-8 response that may be relevant when the mucosal barrier is intact. The IL-8 release is MAPK-dependent and inhibited by mesalamine.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Case-Control Studies; Cells, Cultured; Colonic Neoplasms; Escherichia coli; Flagellin; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; MAP Kinase Signaling System; Mesalamine

Dysregulation of interleukin-8 (IL-8) production has been proposed to contribute to intestinal inflammation in inflammatory bowel disease (IBD) patients. Previous studies, which evaluate adult patients with long-standing or steroid-modulated disease, have reported conflicting results regarding the role of IL-8 in IBD pathogenesis. The present study evaluates IL-8 in colonic organ cultures and sera of newly and previously diagnosed pediatric IBD patients with various degrees of histopathologic activity. Colon and terminal ileum biopsies were obtained from 26 patients with Crohn's disease, 12 with ulcerative colitis, 4 with indeterminate colitis, and 12 age-matched normal controls. IBD patients were additionally characterized as newly or previously diagnosed. Supernatants from organ-cultured lamina propria biopsies and sera were evaluated by ELISA for IL-8 protein. IL-8 increased with degree of histologic inflammation regardless of diagnosis (no pathologic diagnosis, 62.6 ng/ml, interquartile range [IQR] 30.4-94.6 ng/ml; mild, 92.0 ng/ml, IQR 21.9-170.0 ng/ml; moderate, 676.2 ng/ml, IQR 46.4-2967.7 ng/ml; severe, 585.6 ng/ml, IQR 149.7-1602.2 ng/ml; P < 0.01). Lamina propria IL-8 was significantly elevated in moderately and severely inflamed tissue segments (603.26 ng/ml; IQR, 72.15-2240.4 ng/ml) compared to noninflamed and mildly inflamed segments (67.70 ng/ml; IQR, 30.38-124.1 ng/ml; P = 0.0009). There was no significant trend in IL-8 concentration when compared by clinical diagnosis. No significant difference was found in IL-8 concentrations in organ cultures from newly diagnosed patients versus those from previously diagnosed patients. There was no significant correlation between serum IL-8 concentration and organ culture IL-8 concentration. We conclude that higher concentrations of IL-8 are found in more histologically inflamed tissue segments from pediatric IBD patients. IL-8 does not appear to be associated with clinical IBD subtype. IL-8 appears to be an integral part of both early and established mucosal inflammation in pediatric IBD patients. These findings suggest that IL-8-specific therapies may universally modify inflammatory activity in IBD patients.

Topics: Adolescent; Adult; Case-Control Studies; Child; Child, Preschool; Colitis, Ulcerative; Colon; Crohn Disease; Enzyme-Linked Immunosorbent Assay; Female; Humans; Ileum; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Linear Models; Male; Mucous Membrane; Organ Culture Techniques; Philadelphia; Severity of Illness Index

Angiogenesis is a novel component in inflammatory bowel disease (IBD) pathogenesis. We have previously shown that immune-nonimmune interactions through the CD40-CD40-ligand (CD40L) pathway might sustain gut inflammation, although their effect on regulating inflammation-driven angiogenesis is unknown. The present study evaluated the role of the CD40-CD40L interaction in the promotion of immune-mediated angiogenesis in IBD.. Human nonimmune cells of colonic origin-namely, human intestinal fibroblasts (HIFs) and human intestinal microvascular endothelial cells (HIMECs)-were activated with either soluble CD40L (sCD40L), or CD40(+) D1.1 cells or CD40L-activated lamina propria T (LPT) cells before measuring pro-angiogenic cytokine release. Blocking antibodies to either CD40 or CD40L were used to disrupt the CD40-CD40L interaction. The dextran sodium sulphate (DSS) model of experimental colitis in CD40 and CD40L knockout mice was established to assess whether the CD40-CD40L pathway was implicated in controlling inflammation-driven angiogenesis in vivo.. Engagement of CD40 on HIFs promoted the release of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and hepatocyte growth factor (HGF). LPT cells were potent inducers of pro-angiogenic cytokine secretion by HIFs. Supernatants from sCD40L-activated HIFs induced migration of HIMECs and tubule formation, both of which were inhibited by blocking antibodies to either VEGF, IL-8 or HGF. Both CD40- and CD40L-deficient mice were protected from DSS-induced colitis and displayed a significant impairment of gut inflammation-driven angiogenesis, as assessed by microvascular density.. The CD40-CD40L pathway appears to be crucially involved in regulating inflammation-driven angiogenesis, suggesting that strategies aimed at blocking CD40-CD40L interactions might be beneficial in acute and chronic intestinal injury.

Topics: Animals; CD40 Antigens; CD40 Ligand; Cell Line; Cells, Cultured; Chemotaxis, Leukocyte; Colitis; Colon; Disease Models, Animal; Endothelial Cells; Fibroblasts; Hepatocyte Growth Factor; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Mice; Mice, Knockout; Neovascularization, Pathologic; Vascular Endothelial Growth Factor A

Exclusive enteral nutrition using polymeric formula (PF) is a well-established therapeutic option for active Crohn's disease; however, its mechanisms of action are unknown. We investigated the anti-inflammatory effects of PF in an in vitro model of epithelial cell inflammation. PF did not affect cell viability over a range of dilutions, but when PF was added to the culture medium the interleukin (IL)-8 response to proinflammatory stimuli was significantly reduced. This effect was due to PF acting directly on the cells as the IL-8 response was still reduced when PF was separated from the proinflammatory stimuli in a 2-compartment system. In the presence of PF, nuclear factor (NF)-kappaB nuclear migration was not inhibited; however, IkappaBalpha degradation was delayed. PF has direct anti-inflammatory effects upon immortalized colonic enterocytes. Therefore PF may, in part, modulate gut inflammation by directly reducing the inflammatory response of the intestinal epithelium.

Topics: Blotting, Western; Cell Survival; Cells, Cultured; Culture Media; Drug Combinations; Enterocytes; Enzyme-Linked Immunosorbent Assay; HT29 Cells; Humans; Immunoglobulin G; Inflammatory Bowel Diseases; Interleukin-1alpha; Interleukin-8; Lipopolysaccharides; NF-kappa B; Transcription Factor RelA; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Inflammatory bowel disease (IBD) is characterized by an exaggerated immune response that involves pro-inflammatory cytokines including IL-8. Production of these pro-inflammatory cytokines is triggered by pathogen-associated molecular patterns (PAMP). Butyrate, a product of bacterial fermentation of carbohydrates, has been reported to modulate inflammation in IBD, possibly by regulating production of pro-inflammatory cytokines. However, this effect of butyrate is controversial. In this study, we used Pam3CSK4 (Pam3CysSerLys4), the acylated NH2-terminus of the bacterial lipoprotein (a PAMP), to mimic in vivo infection of pathogens. Butyrate transiently down-regulated expression of IL-8 stimulated by Pam3CSK4. Treatment of cells with butyrate before Pam3CSK4, however, enhanced production of IL-8. Furthermore, butyrate induced expression of A20, a negative regulator of the nuclear factor-kappaB pathway. Over-expression of A20 inhibited Pam3CSK4-triggered IL-8 expression. Our data suggest that the inflammatory modulation of butyrate in IBD is mediated by A20 and a short pulse rather than continuous administration of butyrate may provide a protective effect on IBD.

Topics: Anti-Inflammatory Agents; Butyrates; Caco-2 Cells; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gastrointestinal Agents; Humans; I-kappa B Proteins; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Intestines; Intracellular Signaling Peptides and Proteins; Lipopeptides; NF-kappa B; NF-KappaB Inhibitor alpha; Nuclear Proteins; Organ Culture Techniques; Peptides; Phosphorylation; Time Factors; Transfection; Tumor Necrosis Factor alpha-Induced Protein 3

Few data exist on the molecular events causing intestinal epithelial destruction during inflammatory processes, such as inflammatory bowel disease (IBD). In this work, we analyzed the potential implication of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in these inflammatory lesions.. TRAIL and TRAIL-receptor expression were analyzed in normal, inflammatory ileum/colon and human intestinal epithelial cell (IEC) lines (HIEC), Caco-2, and HT-29 using RNase protection assay, real-time and reverse-transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. TRAIL-induced activation of NF-kappaB was determined by electrophoretic mobility shift assay. Caspase-recruitment domain (CARD)15 expression and interleukin-(IL)8 production were studied by RT-PCR and enzyme-linked immunosorbent assay. Apoptosis was monitored using Annexin-V/caspase-3 assays.. Normal mature IEC expressed low TRAIL levels, whereas, in inflammatory lesions, TRAIL messenger RNA and protein were markedly up-regulated in IEC and lamina propria lymphocytes at levels comparable with trinitrobenzene sulfonic acid-induced colitis. Interferon-gamma and TNF-alpha potently induced TRAIL in IEC. In vitro analyses revealed a dual biologic effect of TRAIL on HIEC: Under noninflammatory conditions, TRAIL up-regulated via nuclear factor-kappaB CARD15 and IL-8, whereas, under inflammatory conditions, TRAIL became a potent inducer of apoptosis in HIEC, which was confirmed ex vivo using ileal organ cultures. TNF-alpha markedly increased the expression of the proapoptotic receptor TRAIL-R2. TRAIL-induced IEC apoptosis required a functional caspase cascade.. TRAIL is a new inflammatory mediator implicated in the homeostasis of intestinal epithelial barrier functions. TRAIL is highly up-regulated in IEC in inflammatory ileum and colon. It may augment in an auto-/paracrine fashion the elimination of IEC via apoptosis.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Base Sequence; Blotting, Western; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Female; Flow Cytometry; Gene Expression Regulation; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-8; Male; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; Sampling Studies; Sensitivity and Specificity; Statistics, Nonparametric; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Angiogenesis is a critical component of neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory bowel disease (IBD) has yet to be established. We assessed mucosal vascularization, expression of endothelial alphaVbeta3 integrin, angiogenic factors, and their bioactivity in Crohn's disease (CD) and ulcerative colitis (UC) mucosa.. Mucosal endothelium was immunostained for CD31 and factor VIII and quantified by digital morphometry. alphaVbeta3 expression was studied in vivo by confocal microscopy and in vitro by flow cytometric analysis of human intestinal microvascular endothelial cells (HIMECs). Vascular endothelial growth factor (VEGF), interleukin (IL)-8, and bFGF levels were measured in mucosal extracts and cells and angiogenic bioactivity shown by induction of HIMEC migration and the corneal and chorioallantoic membrane angiogenesis assays.. Microvessel density was increased in IBD mucosa. Endothelial alphaVbeta3 was strongly expressed in IBD but only sporadically in normal mucosa and was up-regulated in HIMECs by VEGF, tumor necrosis factor alpha, and bFGF. IBD mucosal extracts induced a significantly higher degree of HIMEC migration than control mucosa, and this response was mostly dependent on IL-8 and less on basic fibroblast growth factor or vascular endothelial growth factor. Compared with normal mucosa, IBD mucosal extracts induced a potent angiogenic response in both the corneal and chorioallantoic membrane assays.. These results provide morphological, phenotypic and functional evidence of potent angiogenic activity in both CD and UC mucosa, indicating that the local microvasculature undergoes an intense process of inflammation-dependent angiogenesis. Thus, angiogenesis appears to be an integral component of IBD pathogenesis, providing the practical and conceptual framework for anti-angiogenic therapies in IBD.

Topics: Biopsy, Needle; Case-Control Studies; Cells, Cultured; Chemotaxis; Endothelial Cells; Female; Fibroblast Growth Factor 2; Flow Cytometry; Humans; Immunohistochemistry; Inflammation Mediators; Inflammatory Bowel Diseases; Integrins; Interleukin-8; Intestinal Mucosa; Male; Microcirculation; Neovascularization, Pathologic; Sampling Studies; Sensitivity and Specificity; Vascular Endothelial Growth Factor A

To elucidate the molecular mechanisms involved in the therapeutic effects of granulocyte/monocyte adsorption apheresis, changes were investigated in the cytokine responses of peripheral blood mononuclear cells (PBMC) before and after granulocyte/monocyte adsorptive apheresis in ulcerative colitis (UC) patients. Four patients with active UC were enrolled. All patients responded to granulocyte/monocyte adsorptive apheresis. A total of 20 sessions of four patients were analyzed. Peripheral blood mononuclear cells were isolated from peripheral venous blood within 5 min before and after each session of granulocyte/monocyte adsorptive apheresis. The cells were stimulated with interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha for 24 h, and the secreted IL-8 and IL-6 levels were determined by enzyme-linked immunosorbent assay (ELISA). IL-1beta-induced IL-8 and IL-6 secretion was significantly decreased after granulocyte/monocyte adsorptive apheresis. TNF-alpha-induced IL-8 secretion was also significantly decreased after apheresis, but there was no significant difference in TNF-alpha-induced IL-6 secretion (P = 0.052). In conclusion, granulocyte/monocyte adsorptive apheresis down-regulates the IL-1beta- and TNF-alpha-induced inflammatory responses in PBMC. The induction of hyporesponsiveness to pro-inflammatory cytokines may be an important factor mediating the clinical effects of granulocyte/macrophage adsorptive apheresis in UC patients.

Topics: Adult; Blood Component Removal; Colitis, Ulcerative; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Granulocytes; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-6; Interleukin-8; Male; Middle Aged; Monocytes; Treatment Outcome; Tumor Necrosis Factor-alpha

In acute or chronic inflammatory bowel disease (IBD) interferon gamma (IFNgamma) is still considered to be an important pro-inflammatory mediator. In the present study we investigated the impact of IFNgamma on interleukin-8 (IL-8) production as a read-out for cell activation in intestinal epithelial cell (IEC) lines and primary human colonic epithelial cells (CEC).. Primary cultures of human CEC were established from the mucosa of patients without inflammatory disease. CEC, HT-29 or Caco-2 cells were incubated with either IFNgamma, tumor necrosis factor (TNF)alpha or IL-10. IL-8 and IL-1Ra secretion was determined by ELISA. Competicon PCR was used for quantification of IL-8mRNA. Apoptosis was quantified by propidium iodine incorporation and fluorescence activated cell sorting (FACS) analysis.. In contrast to HT-29 cells in primary human CEC 100 U/ml IFNgamma inhibited IL-8 secretion significantly to 70+/-15% of unstimulated primary CEC (p<0.005) more effectively than IL-10 (87+/-21% versus unstimulated cells, n.s.). In HT-29 cells, IL-8 secretion was induced to 405+/-101% of unstimulated cells. In Caco-2 cells, IFNgamma had no significant effect on IL-8 secretion. The effect in HT-29 and CEC was concentration dependent. In primary CEC, 200 U/ml IFNgamma further reduced IL-8 secretion to 48+/-18% of unstimulated CEC (p<0.05). Whereas IL-8 mRNA was strongly upregulated in HT-29 cells, no upregulation or even a downregulation was found in CEC. Pre-incubation with 100 U/ml IFNgamma did not increase the susceptibility to apoptosis mediated by anti-Fas antibody (CH-11) in primary CEC, whereas HT-29 cells showed increased rates of apoptosis after priming with IFNgamma.. In contrast to HT-29, IFNgamma downregulated IL-8 secretion and did not induce IL-8 mRNA expression in primary human CEC. This effect was not due to induction of apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Cell Culture Techniques; Colon; Down-Regulation; Epithelial Cells; Humans; Inflammation; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-8; Intestinal Mucosa

Recruitment of polymorphonuclear leucocytes (PMN) across the intestinal epithelium is dependent on specific adhesion molecules and chemoattractants diffusing from the intestinal lumen. The present understanding is that in response to fMLP, PMN migration across a T84 colon carcinoma monolayer is dependent on the beta(2) integrin, Mac-1 (CD11b/CD18). To further understand PMN transepithelial migration, we sought to determine whether migration to C5a, IL-8 and LTB(4) was similarly Mac-1-, or even CD18-dependent. T84 epithelial cell monolayers growing on Transwell filters were used in combination with radiolabelled peripheral blood PMN. The number of migrated PMN was established by the amount of radioactivity recovered from the well after the migration period. Monoclonal antibodies were used to block integrin function. Whereas essentially all migration to fMLP across T84 monolayers was prevented by anti-CD18 antibody, significant migration to C5a, IL-8 or LTB(4) persisted despite anti-CD18 antibody, indicating PMN are capable of beta(2) integrin-independent transepithelial migration. An antibody to CD11b but not CD11a blocked migration to an extent similar as with anti-CD18. CD18-independent PMN migration to C5a occurred only in the basolateral-to-apical direction across epithelial cells. Co-stimulation of PMN with C5a and fMLP or IL-8 plus LTB(4) and fMLP still resulted in CD18-independent migration. Thus CD18 use during PMN migration across this model epithelium is a function of the chemoattractant inducing migration. The finding of CD18-independent migration mechanisms needs to be considered when developing antiadhesion molecule strategies to reduce or reverse intestinal inflammation.

Topics: Analysis of Variance; Antibodies, Monoclonal; CD18 Antigens; Cell Adhesion; Cell Count; Cell Line, Tumor; Cell Movement; Complement C5a; Epithelial Cells; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils

We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-kappaB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism of immune-epithelial cell cross-talk on Gram-negative enteric bacteria-induced NF-kappaB signalling and pro-inflammatory gene expression in IEC using HT-29/MTX as well as CaCO-2 transwell cultures Interestingly, while differentiated HT-29/MTX cells are unresponsive to non-pathogenic Gram negative bacterial stimulation, interleukin-8 (IL-8) mRNA accumulation is strongly induced in Escherichia coli- but not Bacteroides vulgatus-stimulated IEC cocultured with peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC). The presence of PBMC triggered both E. coli- and B. vulgatus-induced mRNA expression of the Toll-like receptor-4 accessory protein MD-2 as well as endogenous IkappaBalpha phosphorylation, demonstrating similar capabilities of these bacteria to induce proximal NF-kappaB signalling. However, B. vulgatus failed to trigger IkappaBalpha degradation and NF-kappaB transcriptional activity in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation, PBMC from patients with active ulcerative colitis and Crohn's disease differentially trigger epithelial cell activation in response to E. coli and E. coli-derived LPS. In conclusion, this study provides evidence for a differential regulation of non-pathogenic Gram-negative bacteria-induced NF-kappaB signalling and IL-8 gene expression in IEC cocultured with immune cells and suggests the presence of mechanisms that assure hyporesponsiveness of the intestinal epithelium to certain commensally enteric bacteria.

Topics: Animals; Antigens, Bacterial; Bacteroides; Caco-2 Cells; Cells, Cultured; Coculture Techniques; Epithelial Cells; Escherichia coli; Gene Expression Regulation; Gram-Negative Bacteria; Humans; Immunity, Mucosal; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Leukocytes, Mononuclear; NF-kappa B; Phosphorylation; RNA, Messenger; Signal Transduction

The immunomodulatory and anti-inflammatory effects of thalidomide are associated with inhibition of TNF-alpha levels. However, the mechanism by which thalidomide reduces TNF-alpha production remains elusive. NF-kappaB is known to play a central role in regulating inflammatory responses in patients with inflammatory bowel disease (IBD). We tested whether thalidomide acts through inhibiting NF-kappaB activity. HT-29 cells were stimulated with LPS (1 microg/ml) alone, or after pretreatment with thalidomide (100 microg/ml), and NF-kappaB activity was determined by gel mobility shift assays. RT-PCR was used to measure expression of the proinflammatory cytokine genes TNF-alpha, IL-1beta and IL-8. The level of TNF-alpha mRNA was also analyzed by real-time quantitative RT-PCR, and TNF-alpha protein was measured by ELISA. Thalidomide pretreatment did not affect NF-kappaB activity in HT-29 cells stimulated with LPS but production of TNF-alpha was depressed. Thalidomide was found to accelerate the degradation of TNF-alpha mRNA, but had little effect on IL-1beta or IL-8. These observations suggest that the immunomodulatory effect of thalidomide in colonic epithelial cells is associated with inhibition of TNF-alpha. However, it does not act by inhibiting NF-kappaB but rather by inducing degradation of TNF-alpha mRNA.

Topics: Cell Line; Colon; Epithelial Cells; Gene Expression Regulation; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-8; Lipopolysaccharides; NF-kappa B; RNA, Messenger; Thalidomide; Tumor Necrosis Factor-alpha

Platelets circulate in an activated state in patients with inflammatory bowel disease (IBD), but their role in the pathogenesis of IBD is unclear. The recent demonstration that activated platelets express CD40 ligand (L) provides a mechanism of interaction with CD40-positive endothelial cells, inducing them to produce proinflammatory mediators. We investigated whether platelets from patients with IBD express enhanced levels of CD40L and induce human intestinal microvascular endothelial cells (HIMEC) to up-regulate cell adhesion molecule (CAM) expression and secrete chemokines.. CD40L expression was assessed in resting and thrombin-activated platelets by flow cytometry and in mucosal microthrombi by confocal microscopy. Platelet-HIMEC cocultures were used to study CAM up-regulation, and interleukin (IL)-8 and RANTES production by HIMEC.. IBD platelets expressed significantly higher CD40L levels than those of healthy subjects, and CD40L-positive platelets were detected in IBD-involved mucosa. Activated platelets up-regulated expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 as well as production of interleukin 8 by HIMEC in a CD40-dependent fashion. High levels of RANTES were present in platelet-HIMEC cocultures and platelets were identified as the source of this chemokine, which mediated T-cell adhesion to HIMEC.. These results show that platelets can actively contribute to mucosal inflammation and represent a previously unrecognized component of IBD pathogenesis.

Topics: Blood Platelets; CD40 Antigens; CD40 Ligand; Cell Adhesion; Cells, Cultured; Chemokine CCL5; Coculture Techniques; Endothelium, Vascular; Humans; Inflammatory Bowel Diseases; Intercellular Adhesion Molecule-1; Interleukin-8; MAP Kinase Signaling System; Microcirculation; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Platelet Aggregation; T-Lymphocytes; Up-Regulation; Vascular Cell Adhesion Molecule-1

Intestinal myofibroblasts are known to respond to inflammatory signals and may play a role in Crohn's disease-associated fibrosis. However, putative involvement by myofibroblasts in innate immune responses as part of intestinal host defense has not been characterized. We therefore analyzed expression and regulation of toll-like receptors (TLRs) in colonic human myofibroblasts (CCD-18) and primary human colonic myofibroblasts in comparison with human lung myofibroblasts (CCD-37).. Expression of TLRs (1-10) and NOD 1 and 2 was assessed before and after stimulation with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by using a custom microarray, reverse-transcription polymerase chain reaction, Northern blot and Western blot analysis, and immunohistochemistry. Activation of signaling pathways, translocation of p65, and secretion of interleukin (IL)-8 were determined.. Messenger RNAs encoding for TLR1-9, as well as NOD1 and NOD2, were amplified from cultured and primary human intestinal myofibroblasts. After stimulation with LPS or LTA, a 1.5-4.2-fold up-regulation of TLRs (2, 3, 4, 6, 7) and elements of the signaling cascade (MyD88, TIR domain-containing adapter protein [TIRAP]) was observed. CCD-18 and CCD-37 cells expressed TLR 2 and 4 protein, which were located primarily on the cell membrane. Stimulation with LTA or LPS resulted in activation of the mitogen-activated protein kinases pathway, nuclear translocation of p65, and significantly increased IL-8 secretion.. Bacterial components directly activate intestinal myofibroblasts expressing TLRs. These cells may therefore participate in innate immune responses by sensing and responding to bacterial products that have penetrated into the subepithelial compartment.

Topics: Cells, Cultured; Colon; Fibroblasts; Humans; Immunity, Innate; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Lipopolysaccharides; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teichoic Acids; Toll-Like Receptor 1; Toll-Like Receptor 2; Toll-Like Receptors

Balance between pro-and anti-inflammatory mediators plays a key role in the pathogenesis and treatment of inflammatory bowel disease. Glutamine can modulate cytokine production by intestinal mucosa in healthy subjects, but studies in inflammatory states are still limited. The aim of this work was to evaluate the effects of glutamine on IL-1beta-induced cytokine production by human gut.. Duodenal biopsies from healthy volunteers were stimulated in vitro by IL-1beta in the presence of increasing glutamine concentrations. Cytokine production was assessed in culture media by ELISA and cytokine mRNA expression in biopsies by RT-PCR. Results, in pg/mg of tissue, (median [range]), were compared by non-parametric paired tests.. IL-1beta stimulation increased IL-6 and IL-8, but did not affect IL-4 and IL-10 production. IL-8 and IL-6 production from stimulated biopsies significantly decreased with increasing glutamine concentration from 0.5 to 10mM, (2543 [828-3634] to 1499 [282-2617] for IL-8, 62 [22-117] to 24 [12-99] for IL-6, both P<0.05), whereas IL-10 production was increased (0.7 [0.2-1.6] to 1.2 [2.6-0.5],P<0.05). Glutamine also increased IL-10 mRNA level in biopsies (P<0.05). IL-4 production was not affected by glutamine.. Glutamine was shown in human intestinal mucosa to reduce the production of the pro-inflammatory cytokines IL-6 and IL-8, and enhance the production of the anti-inflammatory cytokine, IL-10.

Topics: Adult; Cytokines; Duodenum; Female; Glutamine; Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-10; Interleukin-4; Interleukin-6; Interleukin-8; Intestinal Mucosa; Male; Organ Culture Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.

Topics: Antineoplastic Agents; Cells, Cultured; Chemokine CCL2; Colon; Enzyme Inhibitors; Fibroblasts; Gene Expression; Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-17; Interleukin-6; Interleukin-8; Mitogen-Activated Protein Kinases; NF-kappa B; Proline; Protein Synthesis Inhibitors; RNA, Messenger; Thiocarbamates; Tosylphenylalanyl Chloromethyl Ketone; Tumor Necrosis Factor-alpha

Na+/H+ exchangers (NHEs) are integral transmembrane proteins found in all mammalian cells. There is substantial evidence indicating that NHEs regulate inflammatory processes. Because intestinal epithelial cells express a variety of NHEs, we tested the possibility that NHEs are also involved in regulation of the epithelial cell inflammatory response. In addition, since the epithelial inflammatory response is an important contributor to mucosal inflammation in inflammatory bowel disease (IBD), we examined the role of NHEs in the modulation of disease activity in a mouse model of IBD. In human gut epithelial cells, NHE inhibition using a variety of agents, including amiloride, 5-(N-methyl-N-isobutyl)amiloride, 5-(N-ethyl-N-isopropyl)- amiloride, harmaline, clonidine, and cimetidine, suppressed interleukin-8 (IL-8) production. The inhibitory effect of NHE inhibition on IL-8 was associated with a decrease in IL-8 mRNA accumulation. NHE inhibition suppressed both activation of the p42/p44 mitogen-activated protein kinase and nuclear factor-kappaB. Finally, NHE inhibition ameliorated the course of IBD in dextran sulfate-treated mice. Our data demonstrate that inhibition of NHEs may be an approach worthy of pursuing for the treatment of IBD.

Topics: Amiloride; Animals; Colitis; Dextran Sulfate; Enterocytes; Humans; Inflammatory Bowel Diseases; Interleukin-8; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphorylation; RNA, Messenger; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured

Although ãlpha-chemokines, such as interleukin (IL)-8 and epithelial neutrophil-activating peptide 78, are implicated in the pathogenesis of inflammatory bowel disease (IBD), little information is currently available on the expression and cellular source of growth-related gene product-alpha (GROalpha) and its functional relationship to other ãlpha-chemokines in the intestinal mucosa of patients with IBD.. The contents of IL-8 and GROalpha in organ cultures, the expression of IL-8 and GROalpha mRNA, and the modulatory effects of inflammatory mediators on IL-8 and GROalpha-producing cells were examined using colonic mucosal tissues. In vitro stimulatory effects of IL-8 and GROalpha on neutrophils were investigated in terms of chemotactic migration and superoxide anion generation.. The contents of IL-8 and GROalpha in organ cultures were elevated in patients with IBD, especially in those with active ulcerative colitis (UC). Both IL-8 and GROalpha contents increased according to an increase in histological disease activity in patients with UC, but not in those with Crohn disease. In contrast, no significant correlation was found between the contents of these alpha-chemokines and clinical disease activity. In situ hybridization detected increased expression of IL-8 and GROalpha mRNA in macrophages, pericrypt myofibroblasts, and the epithelium of tissue specimens with active lesions of IBD. The secretion of IL-8 and GROalpha from macrophages and myofibroblasts obtained from control patients was upregulated by inflammatory cytokines and bacterial products. The concentrations of recombinant (r)-IL-8, which covered the levels of activity detected in individual organ cultures or cell cultures of fractionated mucosal cells, could induce chemotactic migration and superoxide anion generation in neutrophils in vitro, and r-GROalpha had synergistic effects on r-IL-8-induced neutrophil activation.. A coordinate upregulation of IL-8 and GROalpha may be involved in the tissue injury in patients with IBD through their stimulatory effects on neutrophils.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Cell Culture Techniques; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Female; Growth Substances; Humans; In Situ Hybridization; Inflammatory Bowel Diseases; Intercellular Signaling Peptides and Proteins; Interleukin-8; Intestinal Mucosa; Male; Middle Aged; Molecular Sequence Data; Neutrophils; Up-Regulation

Alpha1-proteinase inhibitor (alpha1-PI), an anti-inflammatory protein thought to play a role in the intestinal inflammation, is synthesised by and released from the intestinal epithelial cells. IL-1beta is a key proinflammatory cytokine in the abnormal immune response that occurs in inflammatory bowel disease. Butyrate is a normal luminal constituent in the colon, known to be of benefit in preventing inflammatory bowel disease. Direct modes of action of butyrate in intestinal inflammation have been poorly studied so far. The aim of this study was to investigate the effects of butyrate on cytokine-mediated alpha1-PI release in intestinal epithelial cells.. Differentiated Caco-2 cells were incubated with IL-1beta in the presence or absence of 2 mM butyrate. Alpha1-PI expression in the cells was evaluated by Western blot analysis and alpha1-PI release by ELISA.. Treatment with butyrate alone had no effect on alpha1-PI expression in differentiated Caco-2 cells. However, treatment of the cells with 2 mM butyrate significantly reduced the alpha1-PI level in IL-1beta-treated cells. In the cell culture medium, the presence of butyrate impaired the IL-1beta-induced alpha1-PI release to 17-35%. The treatment induced no change in the number of detached cells or the percentage of viable cells.. Our data show that butyrate inhibits alpha1-PI release from Caco-2 colonocytes treated with IL-1beta. It is therefore likely that anti-inflammatory actions of butyrate occur via a mechanism that does not involve direct regulation of cytokine-induced anti-inflammatory protein expression in intestinal epithelial cells.

Topics: alpha 1-Antitrypsin; Butyrates; Caco-2 Cells; Cytokines; Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-6; Interleukin-8; Intestinal Mucosa; Tumor Necrosis Factor-alpha

P-selectin, a membrane glycoprotein which is expressed on activated platelets and endothelial cells, plays a crucial role in the inflammatory response. The main action is adhesion of leukocytes, facilitation of diapedesis and induction of cytokine production from monocytes (MCP-1 and IL-8), mediated via RANTES released from activated platelets. An abnormal platelet activity has been reported in patients with ulcerative colitis (UC) and Crohn's disease (CD), jointly referred to as inflammatory bowel disease (IBD), which could have an aggravating influence on the inflammatory response. In addition, an up-regulation of platelet IL-8 receptors among patients with IBD has been reported. To reveal a presumptuous platelet dysfunction we analysed the expression of platelet surface P-selectin at resting state and after stimulation with thrombin, collagen, epinephrine and interleukin 8 (IL-8), and plasma levels of soluble P-selectin, neuropeptide Y (NPY) and RANTES in patients with IBD.. Blood from twelve healthy subjects (control group) and twenty-one patients with IBD who had not taken any anti-platelet drugs or steroids were analysed.. Patients were sub-grouped according to disease entity, disease activity and 5ASA medication. Surface P-selectin expression on isolated human platelets and plasma P-selectin, NPY and RANTES were analysed with ELISA. All values are presented as mean +/- standard error of the mean (SEM). Mann-Whitney U test and Wilcoxon matched rank test were used for statistical analyses.. Patients with IBD in remission (n = 9) had higher basal P-selectin expression, 0.38+/-0.04, compared to the control group (n = 12), 0.22+/-0.03,p < 0.01. UC patients (n = 16) showed down-regulation of P-selectin expression after stimulation with IL-8, 0.26+/-0.03 to 0.22+/-0.02, p < 0.05. No significant differences could be observed concerning soluble P-selectin and NPY in plasma. Patients with 5ASA (n = 12) had lower levels of plasma RANTES, 2.39+/-0.06 microg/l, compared to the control group (n = 12), 3.29+/-0.19 microg/l, p < 0.01, and patients without 5ASA (n = 9), 2.90+/-0.17 microg/l, p < 0.05.. Patients with IBD in remission have higher basal platelet surface P-selectin expression. An exaggerated platelet activity with increased expression of platelet P-selectin and release of inflammatory mediators such as RANTES, which is chemotactic and induce chemokine production, could have a reinforcing and aggravating influence on the inflammatory response and increase the susceptibility to IBD. In addition IL-8 has a down-regulating effect on platelet surface P-selectin expression and 5ASA medication seems to lower plasma RANTES. If 5ASA is responsible for lowering the concentration of RANTES this could be one of the beneficial outcomes of 5ASA medication.

Topics: Adult; Aged; Antigens, CD; Aspirin; Blood Platelets; Chemokine CCL5; Female; Humans; Inflammatory Bowel Diseases; Integrin beta3; Interleukin-8; Male; Middle Aged; Neuropeptide Y; P-Selectin; Platelet Membrane Glycoproteins

Cytokine-mediated impairment of viability and metabolic function of epithelial cells has been suggested as a possible early pathogenic event in the development of inflammatory bowel disease (IBD). It is currently unknown whether pro-inflammatory cytokines have a direct effect on human nontransformed colonic epithelial cells. We investigated the effects of TNFalpha, IFNgamma and IL-1beta on viability, short chain fatty acid (butyrate) oxidation and IL-8 secretion in human colonic epithelial cell cultures in vitro obtained from macroscopically normal mucosa from IBD patients and controls. Colonic crypts were isolated from endoscopical biopsies by ultra-short (10 min) EDTA/EGTA treatment, and exposed to TNFalpha, IFNgamma and IL-1beta for 24 hours. The combination of TNFalpha+IFNgamma induced a significant decrease in cell viability as judged by methyltetrazoleum (MTT) metabolism which decreased to median 68% of unexposed cultures (P < 0.01). This effect was more pronounced than that observed after addition of TNFalpha (median 88%) (P < 0.05), but not IFNgamma alone (median 78%), whereas IL-1beta had no significant effect. Cells from IBD patients were significantly less sensitive to TNFalpha + IFNgamma exposure (median 74%) compared to cells from controls (median 58 %) (P < 0.05). Butyrate oxidation, as measured by entrapment of 14CO2, was not inhibited in cells exposed to TNFalpha + IFNgamma, neither from controls (median 112%) nor from IBD patients (median 108%), suggesting a relative increase of this specific metabolic function in living cells in response to immunoinflammatory stress. IL-8 levels in cell supernatants were increased by TNFalpha + IFNgamma, supporting the role of the epithelium in signalling between luminal factors and mucosal immune cells. In conclusion, we report that TNFalpha and IFNgamma damage and influence human colonic epithelial cell function in vitro and that such mechanisms, if operative in vivo, also may be involved in the pathogenesis of IBD.

Topics: Adult; Aged; Butyrates; Cell Survival; Cells, Cultured; Colon; Colonoscopy; Epithelial Cells; Female; Humans; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-1; Interleukin-8; Intestinal Mucosa; Male; Middle Aged; Oxidation-Reduction; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-gamma protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-gamma ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-kappaB via an IkappaB-alpha-dependent mechanism. Moreover, thiazolidinedione ligands for PPAR-gamma markedly reduce colonic inflammation in a mouse model of IBD. These results suggest that colonic PPAR-gamma may be a therapeutic target in humans suffering from IBD.

Topics: Animals; Caco-2 Cells; Colitis; Cytokines; DNA-Binding Proteins; Epithelium; Gene Expression; HT29 Cells; Humans; I-kappa B Proteins; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Ligands; Mice; Microbodies; NF-kappa B; NF-KappaB Inhibitor alpha; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors

Nuclear factor kappB (NFkappaB) is a transcription factor that controls several genes important for immunity and inflammation. The aim of this study was to assess if activation of NFkappaB plays a role in the pathogenesis of inflammatory bowel disease (IBD), and whether steroid treatment affects NFkappaB activation. Activation of NFkappaB was analysed in colon biopsy samples of 13 patients with active IBD (8 Crohn's colitis, 5 ulcerative colitis) by electrophoretic mobility-shift assays, under basal conditions and 3 weeks after treatment with 0.75 mg kg(-1) day(-1) prednisolone. The presence of interleukin-8 mRNA in biopsies was assessed by RT-PCR. A specific NFkappaB band was present in all nuclear extracts from inflamed mucosa, whereas the band was barely detectable in uninflamed colonic mucosa. NFkappaB bands were super-shifted by antibodies against p50 subunit, whereas antibodies against p65, p52, c-Rel, or Rel B did not modify the mobility of the band. Increased interleukin-8 mRNA was detected at the same sites of NFkappaB activation. Steroid-induced healing of colonic inflammation was associated with disappearance of NFkappaB from nuclear extracts. These results support the notion that NFkappaB plays an important role in the pathogenesis of IBD, and that blockade of NFkappaB activation is one of the mechanisms by which steroids suppress the inflammatory cascade in IBD.

Topics: Adult; Anti-Inflammatory Agents; Electrophoresis, Polyacrylamide Gel; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; NF-kappa B; Polymerase Chain Reaction; Prednisolone; RNA, Messenger

Inflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta, have been implicated as primary mediators of intestinal inflammation in inflammatory bowel disease.. To investigate the in vitro effects of oxpentifylline (pentoxifylline; PTX; a phosphodiesterase inhibitor) on inflammatory cytokine production (1) by peripheral mononuclear cells (PBMCs) and (2) by inflamed intestinal mucosa cultures from patients with Crohn's disease and patients with ulcerative colitis.. PBMCs and mucosal biopsy specimens were cultured for 24 hours in the absence or presence of PTX (up to 100 micrograms/ml), and the secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 determined by enzyme linked immunosorbent assays (ELISAs).. PTX inhibited the release of TNF-alpha by PBMCs from patients with inflammatory bowel disease and the secretion of TNF-alpha and IL-1 beta by organ cultures of inflamed mucosa from the same patients. Secretion of TNF-alpha by PBMCs was inhibited by about 50% at a PTX concentration of 25 micrograms/ml (IC50). PTX was equally potent in cultures from controls, patients with Crohn's disease, and those with ulcerative colitis. The concentrations of IL-6 and IL-8 were not significantly modified in PBMCs, but IL-6 increased slightly in organ culture supernatants.. PTX or more potent related compounds may represent a new family of cytokine inhibitors, potentially interesting for treatment of inflammatory bowel disease.

Topics: Adult; Colitis, Ulcerative; Colon; Crohn Disease; Culture Techniques; Cytokines; Female; Humans; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-6; Interleukin-8; Intestinal Mucosa; Leukocytes, Mononuclear; Male; Middle Aged; Pentoxifylline; Phosphodiesterase Inhibitors; Tumor Necrosis Factor-alpha

In this study, our purpose was to determine whether locally generated interleukin-8 (IL-8) is involved in neutrophil migration and binding to extracellular matrix, using the colonic mucosal specimens obtained from patients with inflammatory bowel disease (IBD).. Levels of IL-8 secreted in the organ cultures were measured by enzyme-linked immunosorbent assay. Chemotactic activity and binding capacity of neutrophils were induced by the organ culture supernatants.. Significantly higher levels of IL-8 were secreted in patients with IBD, and its elevation was more prominent in patients with active ulcerative colitis. The organ culture supernatants induced higher chemotactic activity and binding capacity of neutrophils in patients with IBD, especially in those with active ulcerative colitis, compared with controls. These effects were inhibited significantly when the supernatants were submitted to preincubation with neutralizing anti-IL-8 antibody.. Increased mucosal generation of IL-8 may attract neutrophils from the circulation into the inflammatory site and induce binding of neutrophils in the interstitial tissue, contributing to accumulation and activation of neutrophils in the affected mucosa with IBD.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cell Adhesion; Chemotaxis, Leukocyte; Colon; Extracellular Matrix; Female; Humans; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Male; Middle Aged; Neutrophils; Organ Culture Techniques

Secretion of chemokines by epithelial cells may represent a crucial event in the pathogenesis of inflammatory bowel disease (IBD). Expression of the chemokine epithelial neutrophil-activating peptide 78 (ENA-78) was monitored in patients with IBD and normal controls.. In situ hybridizations were performed on 41 tissue specimens from 15 patients with IBD and 10 controls to detect ENA-78 messenger RNA (mRNA). Immunofluorescence stainings were used to localize ENA-78 protein.. Intestinal epithelial cells expressing ENA-78 mRNA at detectable levels are found at comparable frequencies in patients with Crohn's disease and ulcerative colitis. Tissue specimens with mild to moderate histological signs of disease activity show slightly higher frequencies of ENA-78 mRNA-expressing epithelial cells than areas with signs of severe disease activity (P = 0.14). Immunofluorescence stainings showed presence of the ENA-78 protein in > 90% of preserved epithelial cells in IBD, in control tissues, ENA-78 mRNA was not detectable, and ENA-78 protein was detectable in 0%-30% of epithelial cells.. The observations are in agreement with a role of the C-X-C chemokine ENA-78 in the pathogenesis of IBD.

Topics: Acute Disease; Adult; Amino Acid Sequence; Appendicitis; Chemokine CXCL5; Chemokines; Chemokines, CXC; Female; Fluorescent Antibody Technique; Humans; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Male; Middle Aged; Reference Values; RNA, Messenger; Tissue Distribution

Mast cells can serve as a possible important source of cytokine production in inflamed tissue which can be regulated by stimuli different from those activating other immune system cells. To study the expression of specific genes in mast cells derived from small human colonic mucosal endoscopic biopsies, we first modified a previously reported procedure to achieve a significantly enriched mast cell fraction. Then, by using single-cell RT-PCR analysis the expression of the IgE Fc receptor (Fc epsilonRI) and c-kit mRNA was determined. It was observed that the Fc epsilonRI-positive cells also expressed c-kit. This observation provided further evidence that Fc epsilonRI-positive cells are indeed mast cells. Analysis of biopsies from 12 patients (four control and eight patients with inflammatory bowel disease (IBD)) was carried out, revealing that all of the Fc epsilonRI-positive cells expressed IL-3, while the expression of IL-4 was detected only in some of these positive cells. TNF alpha was not detected in these cells. Therefore, it would seem that most intestinal mast cells produce IL-3. Since it has been reported that IL-3 synthesis was down-regulated in steroid-treated cells, the expression pattern of IL-3 in intestinal mast cells derived from steroid-treated IBD patients was then determined. IL-3 mRNA was detected in only two out of 24 Fc epsilonRI-positive cells derived from these steroid-treated patients. These results lend strong support to the idea that the down-regulation of IL-3 in mast cells derived from steroid-treated IBD patients occurs in vivo and could be an important mechanism for immunomodulation in IBD.

Topics: Biopsy; Cells, Cultured; Colon; DNA Primers; Gene Expression Regulation; Glucocorticoids; Humans; Inflammatory Bowel Diseases; Interleukin-3; Interleukin-4; Interleukin-8; Interleukins; Intestinal Mucosa; Mast Cells; Oligonucleotide Probes; Polymerase Chain Reaction; Receptors, IgE; Reference Values; Transcription, Genetic

Neutrophils are important cellular mediators in inflammatory bowel disease (IBD). Interleukin (IL)8, a powerful neutrophil chemoattractant, is found in increased quantities in inflamed mucosa, but the cells of origin are uncertain. IL8 gene expression was studied by in situ hybridisation in uninflamed intestinal tissue resected for colon carcinoma (n = 7) and in inflamed colonic tissue resected for IBD (n = 11). Immunohistochemistry was used to assess the phenotype of IL8 expressing macrophages and the production of IL8 protein. Macrophages isolated from intestinal resections and lipopolysaccharide stimulated peripheral blood monocytes treated with 5-aminosalicylic acid, hydrocortisone, and cyclosporin A were examined for IL8 mRNA by northern blotting and IL8 secretion by enzyme linked immunosorbent assay (ELISA). In all cases IL8 mRNA was detected by in situ hybridisation in macrophages and neutrophils adjacent to ulceration in inflamed bowel, but not detected in uninflamed mucosa from carcinoma resections. Recently recruited CD14 positive macrophages were responsible for some of this IL8 expression. IL8 protein was present in the same distribution as mRNA. Epithelial cells in normal and inflamed tissue showed neither mRNA nor protein. IL8 mRNA was expressed significantly more commonly by macrophages from IBD affected than from normal mucosa, and IL8 secretion by IBD but not normal colon macrophages was augmented significantly by lipopolysaccharide treatment. IL8 expression and production by lipopolysaccharide treated blood monocytes was inhibited by the therapeutic agents tested. These results show that neutrophils and recently recruited macrophages are responsible for production of IL8 in IBD, suggesting a mechanism for a continuing cycle of neutrophil attraction. Agents used therapeutically in these diseases may be effective in part by disrupting this cycle.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Inflammatory Bowel Diseases; Interleukin-8; Lipopolysaccharide Receptors; Middle Aged; Neutrophils; Phagocytes; RNA, Messenger

Inflammatory bowel disease (IBD) is characterized by T-cell activation and mucosal influx of inflammatory cells partly mediated by increased local release of cytokines and chemokines. Increased levels of activated platelets are reported in IBD. Activated platelets induce endothelial cells in vitro to secrete several cytokines and growth factors and to express adhesion molecules. This study investigates the expression of interleukin-1 (IL-1), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors on circulating platelets from patients with IBD and healthy controls and assesses the in vitro effect of various concentrations of IL-1 beta, IL-8 and GM-CSF on platelet activation in healthy controls. Flow cytometry was performed to quantify the percentage of platelets binding phycoerythrin (PE) labeled recombinant human IL-1 beta, IL-8 and GM-CSF. Platelet activation was assessed using fluorochrome labeled anti-GMP-140, an activation-dependent antigen. Results are expressed as percentage cytokine receptor expressing platelets (median and interquartile range IQR). Platelets from patients with IBD expressed significantly more cytokine receptors compared to healthy controls: IL-1R [8.7% (5.5-18.2) vs 3.1% (2.4-4.8), p < 0.05], IL-8R [22.5% (18.1-27.9) vs 8% (4.5-9.2), p < 0.001)], GM-CSFR [25.9% (16.1-39.2) vs 3.9% (2.7-3.9), p < 0.001]. The percentage of activated platelets was significantly increased after in vitro stimulation with IL-1 beta, IL-8 and GM-CSF. We conclude that cytokines and chemokines modulate platelet activation through specific, functional receptors which are upregulated in IBD.

Topics: Blood Platelets; Endothelium; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; In Vitro Techniques; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-8; Platelet Activation; Receptors, Cytokine; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Receptors, Interleukin; Receptors, Interleukin-1; Receptors, Interleukin-8A