Sandra N. Leeb

Wound healing and fibrosis in intestinal disease

The mechanisms of wound healing in general have gained interest in recent years, as it has become obvious that the tightly regulated process of tissue repair and regeneration is of great importance for organ homeostasis. Insufficient as well as excessive tissue repair both impair gastrointestinal function. Formation of ulcers and fistulas on the one hand, and of fibrosis and stricture on the other, represent just two sides of one medal. So far, the physiological pathways involved in intestinal wound healing are only partially understood. During acute and chronic intestinal inflammation, macrophages and neutrophils induce local tissue damage by secreting reactive oxygen radicals and tissue-degrading enzymes. This is followed by the release of pro-inflammatory cytokines, as well as chemotactic and cell-activating peptides previously bound to the matrix. If tissue damage is severe, myofibroblasts migrate to the sites of the defect. This migratory function, the ability to contract the wound area and the production of extracellular matrix (ECM) by intestinal myofibroblast cells certainly have important roles in the physiological situation and are altered by chronic inflammation. Available treatments of intestinal strictures, fibrosis and fistulas are insufficient and unsatisfactory. New therapeutic approaches are urgently needed. Future intervention should involve stronger and more selective prevention of the continuous tissue damage and a change in wound healing by modulation of myofibroblast migration and ECM synthesis. Severe mucosal tissue damage requiring efficient wound healing is a main feature of inflammatory bowel disease (IBD), with its two entities Crohn’s disease and ulcerative colitis. During radiation enteritis1,2 or chronic ischaemic enteritis,3,4 the bowel wall is similarly damaged, with subsequent inflammation. Furthermore, cystic fibrosis also may lead to colonic wall thickening, fibrotic colonopathy and stricture formation.5,6,7,8,9,10 In rare cases, recurrent diverticulitis also may cause colonic strictures.11,12 During …

Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes.

Macrophages in lymphoid organs are in close contact to nerve terminals of the sympathetic nervous system. Hence, these cells could be targets of neuronal modulation. We studied sympathetic neurotransmitters as chemoattractants enabling the aggregation of macrophages and nerve terminals. Norepinephrine (NE), neuropeptide Y (NPY), isoproterenol (β‐adrenergic), p‐aminoclonidine (α2‐adrenergic), methoxamine (α1‐adrenergic), and adenosine triphosphate (ATP) were used to study human monocyte and macrophage migration in 48‐well Boyden chambers. NE stimulated chemotaxis of monocytes and macrophages at an optimal concentration of 10−10 M (P < 0.025). Isoproterenol, but not p‐aminoclonidine or methoxamine, induced chemotaxis of monocytes (10−10 M, P < 0.05). In these studies, elevation of cAMP is a critical step in NE‐induced chemotaxis of monocytes. NPY (10−11 M, P < 0.05) stimulated monocyte chemotaxis as well. ATP at 10−4 and 10−5 M stimulated undirected cell mobility (P < 0.05). All tested neurotransmitters of the sympathetic nerve terminal were potent chemoattractants. These findings may explain the close association of nerves and macrophages in tissue and lymphoid organs and may thus be of functional relevance in neuroimmunomodulation. J. Leukoc. Biol. 67: 553–558; 2000.

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.

Evidence for a differential expression of fibronectin splice forms ED-A and ED-B in Crohn’s disease (CD) mucosa

Background and aimsFibronectin (FN) is an essential factor for the induction of migration of primary colonic lamina propria fibroblasts (CLPF). The FN isoform ED-A is an important inducer of migration. Recently, we have shown that CLPF isolated from inflamed Crohn’s disease (CD) mucosa migrated significantly less than control CLPF. We, therefore, investigated changes in FN or integrin expression that could be relevant for CLPF migration.Materials and methodsmRNA of control-CLPF and CLPF isolated from fibrotic mucosa of CD patients was subtractively hybridized. Expression of FN, ED-A, and ED-B in frozen sections from intestinal mucosa was determined by immunohistochemistry. The mRNA expression of the FN isoforms in control, CD, and fibrosis biopsies was quantified by real-time polymerase chain reaction (PCR). Integrin α5β1 protein and mRNA expression was analyzed by fluorescence activated cell sorting (FACS) and PCR, respectively.ResultsSubtractive hybridization indicated differential regulation of FN isoform expression in CD. The immunohistochemical analysis of FN protein revealed a reduction of FN isoforms in inflamed CD mucosa compared to control mucosa. In CD fistulae, the ED-A and ED-B isoforms were virtually absent. In fibrotic mucosa, both proteins were increased. Real-time PCR showed a decrease of FN and ED-A expression during mucosal inflammation in CD in contrast to UC and a significant increase of FN and isoforms in CD fibrosis. No difference was found for protein and mRNA of integrin α5β1 in control, CD, and fibrosis CLPF by FACS and PCR.ConclusionDownregulated expression of migration-inducing FN-isoforms in contrast to unchanged FN receptor expression may contribute to the observed alterations of CD CLPF migration.

Functional expression of the interleukin-11 receptor alpha-chain in normal colonic epithelium and colon cancer

BackgroundInterleukin-11 (IL-11) has been evaluated as an anti-inflammatory and mucosa-protective therapeutic agent in inflammatory bowel diseases (IBDs). Activity of IL-11 requires binding to the α receptor subunit (IL-11Rα) that provides ligand specificity. Recently, we showed that in the intestinal mucosa, IL-11Rα is mainly present on epithelial cells mediating antiapoptotic effects. The aim of this study was to investigate the expression profiling of IL-11Rα and its downstream signaling cascade in colonic adenoma and carcinoma.Materials and methodsThe expression of IL-11Rα in normal colonic mucosa, 11 colonic adenomas, and 10 carcinomas was analyzed by immunohistochemistry. In addition, IL-11Rα-expression and IL-11Rα-induced phosphorylation of signal transducer and activator of transcription (STAT)3 were investigated by Western blot analysis.ResultsImmunohistochemistry revealed significant IL-11-Rα expression in epithelial cells of normal colonic mucosa. In contrast, the expression of IL-11-Rα in colon adenomas and carcinomas was either absent or only detectable in very few scattered epithelial cells. Densitometric analysis of Western blots confirmed these results, showing a decrease of IL-11Rα-protein in cells isolated from adenomas or carcinomas. Reduced STAT3-phosphorylation in carcinoma cells indicated functional consequences of decreased IL-11Rα-protein expression on signal transduction.ConclusionThis study demonstrates a decrease of IL-11-Rα-protein expression in epithelial cells isolated from colon carcinomas and adenomas compared to normal colonic mucosa and a reduced STAT3 signaling. Because of reduced binding and signal transduction, it is unlikely that therapeutically administered IL-11 would contribute to colorectal carcinoma induction and growth.