Intestinal myofibroblasts: targets for stem cell therapy.

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).

Mesenchymal cells of the intestinal lamina propria.

The mesenchymal elements of the intestinal lamina propria reviewed here are the myofibroblasts, fibroblasts, mural cells (pericytes) of the vasculature, bone marrow-derived stromal stem cells, smooth muscle of the muscularis mucosae, and smooth muscle surrounding the lymphatic lacteals. These cells share similar marker molecules, origins, and coordinated biological functions previously ascribed solely to subepithelial myofibroblasts. We review the functional anatomy of intestinal mesenchymal cells and describe what is known about their origin in the embryo and their replacement in adults. As part of their putative role in intestinal mucosal morphogenesis, we consider the intestinal stem cell niche. Lastly, we review emerging information about myofibroblasts as nonprofessional immune cells that may be important as an alarm system for the gut and as a participant in peripheral immune tolerance.

Intestinal mesenchymal cells.

The non-white blood cell mesenchymal elements of the intestinal lamina propria are the myofibroblasts, fibroblasts, pericytes, stromal stem cells, muscularis mucosae, and the smooth muscle of the villus core associated with the lymphatic lacteal. We review the functional anatomy of these mesenchymal cells, what is known about their origin in the embryo and their replacement in adults, their putative role in intestinal mucosal morphogenesis, and the intestinal stem cell niche, and we consider new information about myofibroblasts as nonprofessional immune cells. Although our knowledge of the function of mesenchymal cells in intestinal disease is rudimentary, we briefly consider here their roles in cancer and intestinal inflammation.

HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts.

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 E(2) secretion is associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC). We have previously demonstrated that IL-1alpha-induced cyclooxygenase-2 (COX-2) expression is the result of NF-kappaB- 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-1alpha-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-beta-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-1alpha 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.

Epithelial cells and their neighbors I. Role of intestinal myofibroblasts in development, repair, and cancer.

Intestinal myofibroblasts are alpha-smooth muscle actin-positive stromal cells that exist as a syncytium with fibroblasts and mural cells in the lamina propria of the gut. Through expression and secretion of cytokines, chemokines, growth factors, prostaglandins, and basal lamina/extracellular matrix molecules, as well as expression of adhesion molecules and receptors for many of the same soluble factors and matrix, myofibroblasts mediate information flow between the epithelium and the mesenchymal elements of the lamina propria. With the use of these factors and receptors, they play a fundamental role in intestinal organogenesis and in the repair of wounding or disease. Intestinal neoplasms enlist and conscript myofibroblast factors and matrix molecules to promote neoplastic growth, carcinoma invasion, and distant metastases.

Class II MHC-expressing myofibroblasts play a role in the immunopathogenesis associated with staphylococcal enterotoxins.

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.

Intestinal myofibroblasts and immune tolerance.

Stromal cells, such as myofibroblasts and fibroblasts, represent a significant fraction of MHC class II-positive cells in the normal human colonic lamina propria, suggesting they may play an important role in CD4(+) T cell regulation in a tolerogenic environment. The aim of this study was to examine whether human colonic myofibroblasts (CMFs) phenotypically and functionally resemble conventional antigen-presenting cells (APCs). Our results support the hypothesis that intestinal myofibroblasts are a novel, nonprofessional APC phenotype important in modulating mucosal T cell responses. Given their strategic location, we propose that intestinal myofibroblasts play a critical role in mediating tolerance to luminal antigens.

Myofibroblasts. II. Intestinal subepithelial myofibroblasts.

Intestinal subepithelial myofibroblasts (ISEMF) and the interstitial cells of Cajal are the two types of myofibroblasts identified in the intestine. Intestinal myofibroblasts are activated and proliferate in response to various growth factors, particularly the platelet-derived growth factor (PDGF) family, which includes PDGF-BB and stem cell factor (SCF), through expression of PDGF receptors and the SCF receptor c-kit. ISEMF have been shown to play important roles in the organogenesis of the intestine, and growth factors and cytokines secreted by these cells promote epithelial restitution and proliferation, i.e., wound repair. Their role in the fibrosis of Crohn's disease and collagenous colitis is being investigated. Through cyclooxygenase (COX)-1 and COX-2 activation, ISEMF augment intestinal ion secretion in response to certain secretagogues. By forming a subepithelial barrier to Na(+) diffusion, they create a hypertonic compartment that may account for the ability of the gut to transport fluid against an adverse osmotic gradient. Through the paracrine secretion of prostaglandins and growth factors (e.g., transforming growth factor-beta), ISEMF may play a role in colonic tumorigenesis and metastasis. COX-2 in polyp ISEMF may be a target for nonsteroidal anti-inflammatory drugs (NSAIDs), which would account for the regression of the neoplasms in familial adenomatous polyposis and the preventive effect of NSAIDs in the development of sporadic colon neoplasms. More investigation is needed to clarify the functions of these pleiotropic cells.

Myofibroblasts. I. Paracrine cells important in health and disease.

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-beta 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.

Expression and genetic variation of the Aplysia egg-laying hormone gene family in the atrial gland.

We have screened an Aplysia atrial gland cDNA library using an egg-laying hormone (ELH) precursor probe and have isolated and characterized five different clones, four of which are full-length and approximately 0.8 kb in size. The characterization of these cDNA clones firmly established the genetic variation of the ELH-related precursors expressed in the atrial gland and provided a rational basis for their revised nomenclature proposed herein. The five precursor ELH-related cDNA sequences obtained predicted the following genetically distinct polypeptide precursors designated as: A, [Asp143]A, [Glu94,Gln139]A, [Pro25]B, and [Phe96,Asp107]BT. The [Phe96,Asp107]Br cDNA sequence predicted a truncated form of a B-type precursor. Northern blot analysis of atrial gland RNA identified two transcripts of about equal intensity of 0.9 kb and 1.1 kb. Polymerase chain reaction of genomic DNA, together with DNA sequence analysis, resolved previously reported discrepancies between genomic and cDNA sequences of the ELH-related precursors. Taken together the results obtained identified the expression of five ELH-related precursor genes in the atrial gland of Aplysia from at least two genetic loci per haploid genome.

trans-activation of the alpha 1-acid glycoprotein gene acute phase responsive element by multiple isoforms of C/EBP and glucocorticoid receptor.

alpha 1-Acid glycoprotein (AGP) is a major acute phase protein synthesized primarily by the liver. The AGP gene is transcriptionally activated in hepatocytes during the acute phase response to bacterial lipopolysaccharide. In this study, we analyzed an acute phase responsive element (APRE) located between nucleotide residues -127 to -104 relative to the transcription initiation site of the mouse AGP gene. Binding studies show that several trans-acting factors interact with the APRE. Using monospecific antibodies we demonstrate that three isoforms of the CCAAT/enhancer-binding protein (C/EBP) family, namely C/EBP alpha, C/EBP beta, and C/EBP delta, bind to the APRE. Furthermore, with liver nuclear protein from control animals, C/EPB alpha is the predominant form that binds to the APRE, whereas with nuclear proteins from acute phase-induced animals, C/EBP alpha is replaced by C/EBP beta. The mechanism of activation of the AGP gene during the acute phase response appears to involve an exchange of C/EBP alpha by C/EBP beta. C/EBP delta does not play a role in this reaction. Interestingly, the C/EBP binding site of the APRE partially overlaps a functional glucocorticoid responsive element. We present evidence that both purified C/EBP alpha and glucocorticoid receptor bind strongly to the APRE. By site-specific mutation, we have identified the C/EBP and glucocorticoid receptor binding sites in the APRE. These mutants were used in expression vectors to demonstrate that both C/EBP and glucocorticoid receptor are essential for maximal response to interleukin-6 and dexamethasone. These results demonstrate that the APRE is a composite binding site for multiple factors that are responsible for the transcriptional control of the mouse AGP. Finally, functional analyses indicate that C/EBP alpha, C/EBP beta, and C/EBP delta are strong transcriptional trans-activators of the AGP APRE in hepatoma cells. These data suggest that the regulatory activity of the C/EBP with the APRE in the liver may require interactions with adjacent proteins.

Coupled transcription-polyadenylation in a cell-free system.

To investigate the relationship between transcription and polyadenylation, an in vitro system has been developed in which endogenously transcribed pre-mRNAs containing functional polyadenylation sites are rapidly and accurately cleaved in a HeLa nuclear extract. Cleavage of endogenously transcribed substrates differed from that of exogenous substrates in that a proximal 3' terminus was not required, the reaction was more tolerant of increased Mg2+ levels, and endogenous substrates were cleaved more efficiently. A promoter dependence for this reaction was suggested by the observation that substrates transcribed by bacteriophage T7 RNA polymerase in the presence of nuclear extract were not cleaved. In addition, analysis of the bovine growth hormone poly(A) site indicated that it is highly efficient in vitro which agrees with previous in vivo data. The availability of an in vitro system in which transcription and polyadenylation are coupled should facilitate analysis of the relation between 3' end processing and RNA polymerase II transcription termination as well as the promoter requirements for polyadenylation.

Genetic analysis of L-ethionine-mediated induction of alpha-fetoprotein in mice.

Two genetic loci regulate hepatic alpha-fetoprotein (AFP) mRNA levels in adult mice. The raf locus controls basal levels and the Rif locus determines the degree of induction during liver regeneration. We have investigated the function of each locus during L-ethionine-mediated AFP induction using adult female mice with different Rif/raf genotypes. A single intraperitoneal injection of L-ethionine (0.5 mg/g body weight) resulted in significant triglyceride accumulation in hepatic parenchymal cells and increased AFP synthesis 48-96 h following injection. Hepatic AFP mRNA levels in Balb/cJ mice (high basal level/high induction level during regeneration) were 10- to 30-fold higher than Balb/cCRBL or C3H/He mice (low basal level/high induction level) following ethionine injection, indicating that raf-mediated differences persisted throughout the course of acute ethionine poisoning. The magnitude of this induction was similar to that seen during carbon tetrachloride-induced regeneration. In contrast, C57BL/6 mice (low basal level/low induction level during regeneration) contained hepatic AFP mRNA levels similar to Balb/cCRBL and C3H/He mice following ethionine injection. Thus, Rif-dependent differences seen during liver regeneration were not seen during acute ethionine poisoning. This leads us to conclude that (1) hepatic AFP mRNA induction by ethionine may not be mediated by the Rif locus if Rif is a transcriptional inducer, or (2) if Rif is a transcriptional repressor, it is inactivated equally in all strains during acute ethionine poisoning, unlike during liver regeneration. Hepatic albumin mRNA levels were not affected by ethionine treatment in vivo. L-Ethionine elevated AFP mRNA levels in primary mouse hepatocyte cultures; however, ethionine treatment also increased albumin mRNA levels in vitro.