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Proinflammatory cytokines and eicosanoids are central players in intestinal inflammation. IL-1, a key cytokine associated with intestinal mucosal inflammation, induces COX-2 expression in human colonic myofibroblasts (CMF) and increased prostaglandin E2secretion is associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC). We have previously demonstrated that IL-1α-induced cyclooxygenase-2 (COX-2) expression is the result of NF-κB- and ERK-mediated transcription, as well as COX-2 message stabilization, which depends on p38, MAPKAPK-2 (MK-2) and human antigen R (HuR) RNA binding protein activation. Lipoxygenase (LOX)-derived hydroxyeicosatetraenoic acids (HETEs) are elevated in IBD and colonic adenomas and “cross talk” has been observed between the COX and LOX pathways. Since COX-2 expression is primarily in CMFs in colonic adenomas, we examined the impact of LOX metabolites, particularly HETEs, on IL-1α-induced COX-2 expression in human CMFs. Although 5(S)-, 12(R)-, and 15(S)-HETEs alone had little to no effect on COX-2 expression, they enhanced IL-1-mediated COX-2 expression 3.6 ± 0.5-fold. Studies utilizing heterogeneous nuclear RNA amplification and 5,6-dichloro-β-d-ribofuranosylbenzimidazole treatment were undertaken to measure COX-2 transcription and message stabilization, respectively. We found that HETEs enhanced IL-1-induced COX-2 mRNA levels in CMF as the result of increased p38, MK-2, and HuR activity, increasing message stability greater than that observed with IL-1 alone. Thus HETEs can act synergistically with IL-1α to induce COX-2 expression in human CMFs. HETEs may play a role in both colonic inflammation and in increasing the risk of CRC in IBD independently and via induction of COX-2-mediated prostaglandin secretion.

Elevated mucosal interleukin-1 (IL-1) levels are frequently seen during acute and chronic intestinal inflammation, and IL-1 neutralization lessens the severity of inflammation. One major effect of IL-1 is the increased release of eicosanoid mediators via induction of cyclooxygenase-2 (COX-2). One site of COX-2-derived prostaglandin synthesis during acute and chronic intestinal inflammation is the intestinal myofibroblast. COX-2 expression has also been documented in these cells in colonic neoplasms. Thus an understanding of the regulation of COX-2 expression in human intestinal myofibroblasts is important. As an initial step toward this goal we have characterized IL-1α signaling pathways that induce COX-2 expression in cultured human intestinal myofibroblasts. IL-1 treatment resulted in a dramatic transcriptional induction of COX-2 gene expression. Activation of nuclear factor-κB (NF-κB), extracellular signal-regulated protein kinase (ERK), p38, and protein kinase C (PKC) signaling pathways was each necessary for optimal COX-2 induction. In contrast to what occurs in other cell types, including other myofibroblasts such as renal mesangial cells, PKC inhibition did not prevent IL-1-induced NF-κB or mitogen activated protein kinase/ stress-activated protein kinase activation, suggesting a novel role for PKC isoforms during this process. The stimulatory effects of PKC, NF-κB, ERK-1/2, and presumably c-Jun NH2-terminal kinase activation were exerted at the transcriptional level, whereas p38 activation resulted in increased stability of the COX-2 message. We conclude that, in intestinal myofibroblasts, IL-1-mediated induction of COX-2 expression is a complex process that requires input from multiple signaling pathways. Each parallel pathway acts in relative autonomy, the sum of their actions culminating in a dramatic increase in COX-2 transcription and message stability.

Because interleukin-1 (IL-1) is an important mediator in the inflamed intestine, its effects on enterocyte-subepithelial myofibroblast (SEMF) interaction were investigated in vitro. Acutely juxtaposing T84 cells with 18Co or P2JF SEMF preincubated with IL-1 alpha significantly enhanced T84 short-circuit current (Isc) responsiveness to secretagogues in comparison to SEMF not activated by IL-1 alpha. The sensitivity of T84 cell Isc to Ca(2+)-dependent, but not adenosine 3',5'-cyclic monophosphate-dependent, secretagogues was augmented by IL-1 alpha-treated SEMF. These effects of IL-1 alpha are directly correlated with SEMF prostaglandin E2 (PGE2) production. Both IL-1 alpha augmentation of Cl secretagogue responsiveness and PGE2 formation were inhibited by IL-1 receptor antagonist. Within 5 h, IL-1 alpha stimulated a 10-fold increase in cyclooxygenase (COX)-2 steady-state mRNA levels in 18Co cells. In contrast, COX-1 message levels increased more slowly to two- to threefold above control levels after 24 h incubation. These results demonstrate that the proinflammatory cytokine IL-1 alpha accentuates intestinal SEMF augmentation of enterocyte responsiveness to Ca(2+)-dependent CI-secretagogues. PGE2 is an important mediator of SEMF-enterocyte interaction. The effects of IL-1 alpha on SEMF PGE2 productions are, at least in part, due to stimulation of COX gene expression.

The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA+) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA+subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).

Food poisoning due to staphylococcal enterotoxins (SEs) affects hundreds of thousands of people each year. Little is known about how SEs initiate immune responses and cause pathogenesis. Here, we demonstrate that cultured human intestinal myofibroblasts (IMFs) bind SEs in an MHC class II-dependent fashion. IMFs respond to SE exposure with increased secretion of IL-6, IL-8, and TNF-alpha. A significant proliferative T cell response was observed when MHC class II-expressing IMFs were pulsed with SEA and cocultured with human CD4(+) T cells. In conclusion, our findings support the hypothesis that IMFs may play an important role in pathology associated with staphlococcocal enterotoxigenic disease.

Context.—Myofibroblasts are distinct cells with characteristics of both smooth muscle cells and fibroblasts. Through their ability to secrete cytokines, chemokines, prostaglandins, growth factors, and matrix components, they are thought to play critical roles in inflammation, growth, repair, and neoplasia. Objective.—The goal of this study was to identify the distinct cell populations of the lamina propria of normal colon and colorectal polyps. Design.—We studied the expression of α-smooth muscle actin (αSMA), smooth muscle myosin (SMM), desmin, vimentin, and c-kit by intestinal mesenchymal (stromal) cells in the normal colonic mucosa (n = 5), as well as in hyperplastic polyps (n = 5), sporadic colorectal adenomas (n = 47), and adenomas from patients with familial polyposis (n = 36). Results.—In the normal colonic mucosa, the pericryptal stromal cells were αSMA+, SMM+, desmin−, and vimentin+, defining them as myofibroblasts. In contrast, cells of the muscularis mucosae were αSMA+, SMM+, desmin+, and vimentin−, defining them as smooth muscle cells. α-Smooth muscle actin also highlighted direct connections between the muscularis mucosae and the pericryptal myofibroblasts, and vimentin immunostaining showed a network of connections between the αSMA+ pericryptal myofibroblasts and the αSMA− fibroblasts in the interstitium. In all hyperplastic polyps and adenomatous polyps, the interstitial stromal cells (fibroblasts) now also express αSMA and form a syncytium of αSMA+ networklike connections throughout the lamina propria. Stromal cells of sporadic adenomas demonstrated the same immunohistochemical staining characteristics displayed by adenomas from patients with familial polyposis and by hyperplastic polyps. Conclusions.—These findings indicate that in normal colon, αSMA− fibroblasts are the predominant cell type in the lamina propria. However, the pericryptal (subepithelial) stromal cells are a distinct cell type (αSMA+ myofibroblast) that is immunophenotypically different from muscularis mucosae smooth muscle cells and are connected to the interstitial, nonpericryptal fibroblasts with which they exist as a network throughout the lamina propria of the normal colon. Furthermore, in both hyperplastic and neoplastic polyps, there are changes in nonpericryptal fibroblasts from vimentin+, αSMA−, and SMM− to vimentin+, αSMA+, and SMM+; thus, the interstitial fibroblasts are replaced by myofibroblasts. The factors that cause these changes and the origin of the myofibroblasts need to be determined to clarify the biology of colorectal tumorigenesis.

Myofibroblasts are a unique group of smooth-muscle-like fibroblasts that have a similar appearance and function regardless of their tissue of residence. Through the secretion of inflammatory and anti-inflammatory cytokines, chemokines, growth factors, both lipid and gaseous inflammatory mediators, as well as extracellular matrix proteins and proteases, they play an important role in organogenesis and oncogenesis, inflammation, repair, and fibrosis in most organs and tissues. Platelet-derived growth factor (PDGF) and stem cell factor are two secreted proteins responsible for differentiating myofibroblasts from embryological stem cells. These and other growth factors cause proliferation of myofibroblasts, and myofibroblast secretion of extracellular matrix (ECM) molecules and various cytokines and growth factors causes mobility, proliferation, and differentiation of epithelial or parenchymal cells. Repeated cycles of injury and repair lead to organ or tissue fibrosis through secretion of ECM by the myofibroblasts. Transforming growth factor-β and the PDGF family of growth factors are the key factors in the fibrotic response. Because of their ubiquitous presence in all tissues, myofibroblasts play important roles in various organ diseases and perhaps in multisystem diseases as well.

Subepithelial myofibroblasts are located at the interface between the epithelium and lamina propria in most mucosal tissues. Their biological functions are largely unknown because a long-term cell culture model for these cells has not been available. In this report, we define the phenotypic properties of a human colonic cell line (18Co) that exhibits most of the known characteristics of intestinal subepithelial myofibroblasts in situ. These characteristics include 1) a cell shape that can be reversibly interconverted between a flattened discoid and stellate morphology, 2) intracellular organelles reminiscent of myofibroblasts and smooth muscle cells in situ, 3) expression of alpha-smooth muscle actin, 4) plasma membrane receptors for endothelins and natriuretic peptides, and 5) regulation of epithelial sensitivity to calcium-dependent secretagogues by paracrine secretion of prostaglandins. 18Co cells provide an exploitable model to begin defining the physiological and pathophysiological functions of intestinal subepithelial myofibroblasts at the molecular level.

BACKGROUND: The B7 co-stimulator PD-L1 is critical in suppression of activated T cell responses in peripheral tolerance. However, overexpression of PD-L1 may contribute to progression of chronic inflammatory diseases. The role of PD-L1 and the phenotype of PDL1 expressing cells in inflammatory bowel diseases is unclear. Human colonic myofibroblasts and fibroblasts (CMFs) are major PD-L1 expressing cells in normal colonic mucosa and may suppress activated CD4+T cell proliferation and production of the Th1 acute inflammatory cytokine IFN-γ in a PD-L1 dependent manner. We noted a significant increase in PD-L1 surface expression on CMFs isolated from ulcerative colitis (UC). Thus, we hypothesize that overexpression of PD-L1 by CMFs may have a critical role in the chronic inflammatory responses during UC pathogenesis. METHODS: PD-L1 expression in colonic tissue from normal (N), UC and Crohn's Disease (CD) patients was determined by immunostaining followed by confocal microscopy. PD-L1, Tbet and GATA3 expression was analyzed by realtime RT-PCR, Western blot and flow cytometry. Lymphoproliferation assays and ELISA were used to evaluate the role of PD-L1 in the regulation of Th cell activity. RESULTS: Confocal microscopy analysis revealed strong upregulation of PD-L1 in inflamed colonic mucosa from UC when compared to CD or normal controls. The PD-L1 upregulation in UC colon was mostly associated with cells bearing fibroblast/myofibroblast phenotype (CD90+). PD-L1 on CMFs isolated from UC (UC-CMFs) was functional: UC-CMFs were capable of suppressing CD3/CD28-activated Th cell proliferation in a PD-L1 dependent manner. Both Nand UCCMFs inhibited expression of a Th1 transcriptional factor, Tbet, in Th cells. PD-L1 blocking led to increased expression of Tbet in Th cells, but did not affect expression of Th2 transcriptional factor GATA3. Treatment of N-CMFs with IFN-γ (10-1000 U/mL) for 7 days resulted in a dose dependent upregulation of PD-L1. Treatment of N-CMFs with IL-13 (25 ng/mL), the major cytokine associated with the UC atypical Th2 response, also upregulate PD-L1. Our data suggest that increased secretion of IFN-γ by activated T cells during acute intestinal inflammation may contribute to an increase in PD-L1 expression on CMFs resulting in the negative feedback on the Th1 responses, in turn, promoting IL-13 production by Th2 and NK(T) cells, which will further upregulate PD-L1 expression on CMFs. CONCLUSIONS: Overexpression of PD-L1 on CMFs in the UC colonic mucosa may play an important immunoregulatory role promoting the chronicity of the Th2 inflammatory responses during progression of UC. Supported by NIDDK, CCFA and Sealy foundation.

immunohistochemically examined for the expression of enteroglial markers at several different passages. All 5 cell lines expressed robust GFAP, S-100 and vimentin immunoreactivities, but no cell line expressed the mesenchymal markers desmin (smooth muscle cell differentiation marker) or Thy-l.1 (expressed on rodent fibroblasts). To further confirm these findings, dot blot analysis of the cytosolic and membrane protein fraction of each cell line was performed showing expression of GFAP and S-100 in the cytosolic fraction, while no Thy-l.1 expression could be detected. These findings indicate that immortalized enteroglial cells display the same phenotype as cells from primary cultures, suggesting that they are physiologically resembling primary cells. In summary, we are presenting the first immortalized enteroglial cell lines retaining several of the properties that are typical for primary EGC. These cell lines appear to be useful models for detailed investigations of the functional properties of EGC in vitro. Supported by DFG Ru 528/5.