Daniela Vogl

Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa

BACKGROUND & AIMS Transcription factors of the nuclear factor kappaB (NF-kappaB) family play an important role in the regulation of genes involved in inflammation. In inflammatory bowel diseases, proinflammatory cytokines known to be regulated by NF-kappaB are involved. The aim of this study was to investigate the role of NF-kappaB activation during mucosal inflammation in situ. METHODS A monoclonal antibody, alpha-p65mAb, was applied for immunofluorescence and immunohistochemical analysis that recognizes activated NF-kappaB. Electrophoretic mobility shift assay was used to directly demonstrate the presence of active DNA-binding NF-kappaB. RESULTS Using the alpha-p65mAb antibody, activated NF-kappaB could be found in biopsy specimens from inflamed mucosa but was almost absent in uninflamed mucosa. The number of cells showing NF-kappaB activation correlated with the degree of mucosal inflammation but was not significantly different between inflamed mucosa from patients with Crohns disease, ulcerative colitis, and nonspecific colitis or diverticulitis. NF-kappaB activation was localized in macrophages and in epithelial cells as identified by double-labeling techniques. Electrophoretic mobility shift assay with isolated lamina propria mononuclear cells and epithelial cells confirmed these results. CONCLUSIONS This study shows for the first time the activation of NF-kappaB during human mucosal inflammation in situ. In addition to macrophages, epithelial cells contained activated NF-kappaB, indicating an involvement in the inflammatory process.

Imbalance of the interleukin 1 system in colonic mucosa—association with intestinal inflammation and interleukin 1 receptor agonist genotype 2

Background—Interleukin 1 (IL-1) α and β are potent cytokines which play key roles in inflammation. They are controlled by IL-1 receptor antagonist (IL-1ra). Aims—To investigate the influence of mucosal inflammation and IL-1ra genotype on the IL-1ra:IL-1 balance. Patients and methods—IL-1α, IL-1β, and IL-1ra were measured by enzyme linked immunosorbent assay (ELISA) in biopsy specimens taken from inflamed and non-inflamed colon of 60 patients with Crohn’s disease (CD), 34 with ulcerative colitis (UC), 15 inflammatory controls, and 103 non-inflamed controls. IL-1ra genotype was determined by polymerase chain reaction and gel electrophoresis. Results—IL-1α and IL-1β were significantly increased in inflamed mucosa in inflammatory bowel disease (IBD) (CD: 53.5 (22.4) and 409.9 (118.7) pg/mg protein, respectively; UC: 18.9 (6.8) and 214.5 (78.2) pg/mg, respectively) and non-IBD patients (19.2 (7.4) and 281.4 (121.0) pg/mg, respectively; p<0.0001) compared with normal controls (2.8 (0.6) and 30.6 (5.6) pg/mg, respectively). In CD IL-1α and β were also significantly increased in non-inflamed mucosa (6.1 (1.3) pg/mg and 88.7 (17.4) pg/mg, respectively; p<0.0012). IL-1ra:(IL-1α+β) ratios were significantly decreased in inflamed mucosa of patients with CD (182 (45); p<0.0001), UC (425 (136); p=0.0018) and without IBD (221 (76); p<0.0001), and in non-inflamed mucosa in CD (369 (149); p<0.0001) compared with normal controls (1307 (245); p<0.0001). Patients with IL-1ra genotype 2 had slightly but significantly reduced mucosal IL-1ra concentrations (p=0.003). The greatest difference was seen in colonic biopsy specimens from patients with inflamed Crohn’s disease. Conclusion—Mucosal inflammation can modulate the balance of the IL-1:IL-1ra system in colonic mucosa.

Association of humoral markers of inflammation and dehydroepiandrosterone sulfate or cortisol serum levels in patients with chronic inflammatory bowel disease

Objectives:Dehydroepiandrosterone sulfate (DHEAS) and cortisol are multifunctional adrenal hormones with immunomodulating properties. DHEAS levels were found to be very low in chronic inflammatory diseases. This study aimed to shed more light on the interrelation between DHEAS and cortisol (and humoral markers of inflammation) in chronic inflammatory bowel disease.Methods:DHEAS and cortisol serum levels were measured by ELISA in the serum of 66 normal subjects, 115 patients with Crohns disease (CD) and 64 patients with ulcerative colitis (UC). Humoral markers of inflammation and disease activity scores were assessed by standard techniques.Results:DHEAS was lower in patients with CD (p < 0.005) and UC (p < 0.005) than in controls, which was, in part, dependent on previous corticosteroid treatment (p < 0.01). In CD patients, z-normalized DHEAS was inversely correlated with blood sedimentation rate (p= 0.017). Z-normalized DHEAS was negatively correlated with interleukin-6 (IL-6) in the form of a trend (p= 0.068), and z-normalized DHEAS was significantly positively correlated with hemoglobin (p= 0.001) but not with the Crohns disease activity index. Cortisol, however, was positively correlated with blood sedimentation rate (p= 0.034) and C-reactive protein (p= 0.006). In contrast, in UC patients no such correlation of z-normalized DHEAS or cortisol and parameters of humoral inflammatory activity or Rachmilewitz index exist.Conclusions:DHEAS as a marker of inflammation was low in CD and UC. In CD patients, low DHEAS and high cortisol serum levels were associated with higher humoral inflammatory activity. With respect to humoral inflammatory activity in CD patients, DHEAS and cortisol seem to be inversely regulated, which may have an impact on several immune functions, such as IL-6 secretion.

Human intestinal epithelial cells secrete interleukin-1 receptor antagonist and interleukin-8 but not interleukin-1 or interleukin-6

BACKGROUND There is growing evidence that intestinal epithelial cells (IECs) are involved in the mucosal immune system. AIM To assess the pattern of cytokines secreted by IECs and lamina propria mononuclear cells (LPMNCs). To achieve this, the expression and secretion of interleukin (IL)-1, IL-1 receptor antagonist (IL-1ra), IL-6, and IL-8 in human primary colonic and ileal IECs and LPMNCs from the same patient were studied. METHODS IECs and LPMNCs were isolated from surgical specimens or endoscopic biopsy samples. mRNA expression was investigated by northern blot analysis. Secretion of IL-1β, IL-6, IL-8, and IL-1ra was measured by enzyme linked immunosorbent assay. RESULTS IL-1ra mRNA levels were higher in IECs than in LPMNCs in all probands. IL-8 mRNA was only present in low amounts in the IECs from two controls. In none of the specimens were IL-1β and IL-6 mRNA present in IECs. Transcripts encoding IL-1β, IL-1ra, IL-6, and IL-8 were identified in LPMNC preparations of all specimens. IECs from normal mucosa produced no detectable amounts of IL-1β or IL-6, whereas LPMNCs did. IECs secreted some IL-8 (65 (9) pg/105 cells) but significantly more was generated by LPMNCs (408 (43) pg/105 cells, p<0.0001). However, IECs secreted more IL-1ra than did LPMNCs (120 (12) v 94 (11) pg/105 cells). In acute inflammation, IEC IL-1ra secretion was significantly increased. A correlation between secreted IL-1ra and the macroscopical degree of inflammation was found in Crohns disease (r = 0.64, p<0.0001, n = 36) and ulcerative colitis (r = 0.76, p<0.0001, n = 24). CONCLUSIONS IECs from normal mucosa express and secrete IL-1ra and low amounts of IL-8, but no IL-1 or IL-6. In inflamed mucosa the secretion of IL-1ra by IECs is slightly increased but may be not sufficient to antagonise the greatly increased production of proinflammatory cytokines by LPMNCs and the IECs themselves.

Alterations of the phenotype of colonic macrophages in inflammatory bowel disease

Background: Intestinal macrophages play an important role in mucosal inflammation. In normal colonic mucosa we recently demonstrated a unique macrophage phenotype with attenuated immune functions. Here we present an analysis of the alterations of the phenotype of colonic macrophages in inflammatory bowel disease (IBD). Methods: Intestinal macrophages were isolated from biopsies of patients with IBD (n = 20). Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, HLA‐DR, CD44, CD11b, CD11c and CD3/CD19 expression. Results: In IBD there was an increase in expression not only of CD14 compared to control mucosa (36.0%±13.2% vs. 10.5%±3.8%, P<0.0001) but also of CD16 (28.6%±10.3% vs. 10.1%±3.9%, P<0.0001), HLA‐DR (53.1%±15.9% vs. 27.3%± 9.2%, P<0.0005), CD11b (42.8%±14.2% vs. 17.4%±6.8%, P<0.0001) and CD11c (35.1% ± 15.9% vs. 17.8%±10.4%, P<0.005.). Furthermore, a hitherto undescribed new population of macrophages could be detected by flow cytometry only in patients with ulcerative colitis (CD16++, CD11b++, CD14low, CD33low, CD11c‐) accounting for 5.8% of all cells isolated. Conclusion: In contrast to colonic macrophages from normal mucosa, there is a significantly higher expression of CD14, CD16, HLA‐DR, CD11b and CD11c in IBD, indicating additional macrophage populations in the inflamed mucosa. This may reflect either a recruitment of new cells from the circulation or a change in phenotype of resident cells.

Regulation of migration of human colonic myofibroblasts.

Background and Aim: The migration of mesenchymal cells to areas of mucosal or submucosal tissue damage is an essential factor for wound healing in the intestine. Thus far, neither migration inducing factors nor signal transduction cascades involved in the migration of colonic myofibroblasts (CMF) have been studied in detail. Methods: Primary CMF were isolated from the mucosa of surgical specimens or endoscopic biopsies. Migration assays of CMF were performed in the modified 48-well Boyden chamber. Secreted growth factors were quantified by ELISA. Results: CMF secrete autocrine or paracrine migration stimulating factors. Culture supernatant of CMF collected after 24, 48, and 72 h (=conditioned media) stimulated the migration of CMF ( 48.9 - 4.5; 60.3 - 5.3 and 67.8 - 6.4 cells/hpf, respectively). Heating of conditioned media to 95°C or addition of cycloheximide during the conditioning period abolished migration. Addition of PDGF-AB (2.5-50 ng/ml) or IGF-I (10-300 ng/ml) to CMF conditioned media further increased the migration of CMF to a maximum of 177 and 160%, respectively, when compared to the migration induced by conditioned medium alone. Addition of EGF (2.5-50 ng/ml) or TGF- g 1 (1-50 pg/ml) caused an increased CMF migration up to 139 and 128%, respectively. MCP-1 (5-50 ng/ml) and bFGF (10-200 ng/ml) had no effect on CMF migration. Conclusion: The growth factors PDGF-AB, IGF-I, EGF and TGF- g 1 stimulate the migration of CMF. However, factors secreted by CMF are essential for their ability to migrate in response to these growth factors. The identification of physiologically relevant migration inducing factors may help to elucidate the network of interactions and the complex mechanisms involved in intestinal wound healing or fibrosis.

Inducible CD40 expression mediates NFκB activation and cytokine secretion in human colonic fibroblasts

Background: CD40 has been shown to be a functional activation antigen on a variety of cell types involved in immune responses. As intestinal fibroblasts and myofibroblasts may play a role during mucosal inflammation, we investigated the functional consequences of CD40 induction in primary cultures of human colonic fibroblasts. Methods: Primary colonic lamina propria fibroblasts (PCLF) were isolated from endoscopic biopsies and surgical specimens. Cultures were used between passages 3 and 9. CD40 surface display was determined by FACS analysis and mRNA expression by reverse transcription-polymerase chain reaction. Secretion of cytokines was determined by ELISA. Nuclear factor κB (NFκB) activation was shown by electrophoretic mobility shift assay (EMSA). Results: After priming with interferon γ (IFN-γ) (200 U/ml) for 72 hours, five of eight tested PCLF cultures showed induction of CD40 surface display (up to 10-fold). Induction of CD40 mRNA expression was demonstrated by semiquantitative polymerase chain reaction. In the responder-PCLF cultures, IFN-γ alone caused a 1.5–5-fold increase in interleukin (IL)-8 secretion. Addition of 1 ng/ml CD40L was sufficient to achieve a further increase in IL-8, IL-6, or monocyte chemotactic protein 1 (MCP-1) secretion (2.5–18-fold of controls). Incubation with CD40L alone without priming with IFN-γ had no effect. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN 100 µM) reduced IFN-γ/CD40L mediated cytokine induction, suggesting participation of NFκB, which was directly demonstrated by EMSA. CD4+ T cells induced MCP-1 secretion by PCLF, which was prevented by addition of an excess of CD40-IgG fusion protein. CD40 expression on PCLF could also be demonstrated in vivo by immunohistochemistry. Conclusion: The CD40-CD40L pathway augments mucosal inflammatory responses via mucosal PCLF. CD40-CD40L mediated T cell/PCLF interactions could play an important role during intestinal mucosal inflammation.

T-cell co-stimulatory molecules are upregulated on intestinal macrophages from inflammatory bowel disease mucosa

BACKGROUND AND AIMS Macrophages play an important role during mucosal inflammation in inflammatory bowel disease (IBD). As the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) play an integral role in the activation of T cells by antigen-presenting cells (APC) we investigated the surface expression of B7-1 and B7-2 on colonic macrophages from normal and IBD mucosa. METHODS Intestinal macrophages were isolated from biopsies of 13 control persons and 14 patients with IBD (seven with Crohns disease (CD); and seven with ulcerative colitis (UC)). Cells were characterized by triple fluorescence flow cytometrical analysis using CD33 as macrophage marker. RESULTS The expression of B7-1 (CD80) (9.2% +/- 4.2%) and B7-2 (CD86) (15.1% +/- 7.3%) was low on colonic macrophages from normal mucosa, indicating only a low antigen presenting potential. However, on macrophages from IBD colon there was a significant increase in the expression of co-stimulatory molecules (CD80, 33.8% +/- 8.9%, P = 0.00005 vs. control; CD86, 39.9% +/- 8.8%, P = 0.00002). There was no significant difference between CD and UC in the expression of CD80 (CD, 31.3% +/- 6.7%; UC, 34.4% +/- 13.3%) and CD86 (CD, 41.9% +/- 3.8%; UC, 35.6% +/- 13.8%). While in normal mucosa only 10.6% +/- 4.9% of the macrophages expressed CD14, more than 90% of the CD86/CD80 positive cells of the inflamed mucosa were positive for CD14. CONCLUSION Colonic macrophages from normal mucosa rarely express the co-stimulatory molecules CD80 and CD86. In IBD a new macrophage population is found with high expression of co-stimulatory molecules presumably responsible for the perpetuated immune response.

Autocrine Fibronectin-Induced Migration of Human Colonic Fibroblasts

OBJECTIVES:A central event during wound repair is the migration of activated fibroblasts to the wound area. Thus far, the mechanisms inducing migration of colonic lamina propria fibroblasts (CLPF) have not been studied in detail. Previously, we have shown that CLPF secrete factors that are essential to their ability to migrate in response to different growth factors.METHODS:Primary human CLPF were obtained from endoscopic biopsies or surgical specimens taken from normal mucosa areas of patients undergoing surveillance colonoscopy or surgery for colorectal carcinoma. Migration assays of CLPF were performed in the modified 48-well Boyden chamber.RESULTS:Conditioned medium of CLPF collected after 24-h stimulated migration of CLPF (22 ± 2 cells/hpf). Filtration of conditioned medium through a 300-kDa filter reduced the migration-inducing potential in subsequent migration assays to 2 ± 1 cells/hpf, filtration through a 100-kDa filter abolished migration of CLPF completely, indicating that large molecules such as extracellular matrix components could be responsible for the induction of CLPF migration. Enzyme-linked immunosorbent assays revealed the presence of fibronectin in conditioned medium (17.3 μg/ml). Immunoprecipitation of fibronectin in conditioned medium of CLPF reduced the migration-inducing potential by 63%. Addition of fibronectin to fibronectin-depleted conditioned medium reconstituted the migration. Dose-response assays with fibronectin (1–100 μg/ml) diluted in nonconditioned medium induced migration of CLPF in a dose-dependent manner. Maximum migration was induced with 25 μg/ml fibronectin (37 ± 5 cells/hpf).CONCLUSIONS:Fibronectin is an autocrine and paracrine factor essential for intestinal fibroblast migration. Fibronectin induces migration of intestinal fibroblasts and is essential for their ability to migrate in response to different growth factors. A detailed understanding of the regulation of the migration of intestinal fibroblasts is necessary to gain further insights in the pathophysiology of stricture and fistula formation.

Monocyte differentiation in intestine-like macrophage phenotype induced by epithelial cells

Macrophages in normal colonic mucosa show a specific and distinct phenotype with low expression of the typical monocyte/macrophage surface antigens CD14, CD16, and CD11b and T‐cell costimulatory molecules. A method for the in vitro induction of a macrophage phenotype similar to this intestinal phenotype is presented. Multicellular spheroids (MCSs) of intestinal epithelial cell (IEC) and control cell lines were cocultured with elutriated monocytes. Surface antigen expression was analyzed by immunohistochemistry and flow cytometry. Interleukin (IL)‐1β mRNA was measured by quantitative PCR. Monocytes adhered and infiltrated the MCSs within 24 h. In the MCSs of all IEC lines, the typical monocyte/macrophage surface antigens CD14, CD16, CD11b, and CD11c, which are detectable after 24 h of coculture by immunohistochemistry and flow cytometry, were down‐regulated after 7 days (e.g., for CD14 at 24 h, expression was 86% of CD33+ cells; at day 7, it was 11%). A clear decrease of lipopolysaccharide (LPS)‐stimulated IL‐1β transcription in monocytes cocultured with IEC MCSs could be observed during the 7‐day period. For the first time an intestine‐like macrophage‐phenotype could be induced in vitro. Interactions with IECs play an essential role during this differentiation, which is of functional relevance, e.g., for LPS‐induced cytokine secretion.