Myofibroblasts. II. Intestinal subepithelial myofibroblasts

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1 invited review Myofibroblasts. II. Intestinal subepithelial myofibroblasts D. W. POWELL, R. C. MIFFLIN, J. D. VALENTICH, S. E. CROWE, J. I. SAADA, AND A. B. WEST University of Texas Medical Branch at Galveston, Departments of Internal Medicine, Physiology, and Biophysics and Pathology, Galveston, Texas Powell, D. W., R. C. Mifflin, J. D. Valentich, S. E. Crowe, J. I. Saada, and A. B. West. Myofibroblasts. II. Intestinal subepithelial myofibroblasts. Am. J. Physiol. 277 (Cell Physiol. 46): C183 C201, 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- ), 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. interstitial cells of Cajal; cyclooxygenase; chemoprevention; wound repair; fibrosis; electrolyte transport; immunophysiology FUNDAMENTAL BIOLOGICAL PROCESSES such as cell motility, proliferation, differentiation, apoptosis, morphogenesis, tissue repair, inflammation, and the immune response are initiated, maintained, and terminated by local interactions between cells. These interactions are brought about by contact of cells with each other or with the extracellular matrix (ECM) or through the elaboration of and response to soluble mediators. Although myofibroblasts were identified morphologically a century ago, it is now recognized that they constitute a family of paracrine cells that play an important role in the regulation of these fundamental processes [see part I of this review, which appeared in the July issue (163)] (181, 207). This article focuses on intestinal subepithelial myofibroblasts (ISEMF), the most recently recognized myofibroblasts in the intestine, with a brief review of the interstitial cells of Cajal (ICC), the other intestinal myofibroblasts. The ICC are located in the submucosa and muscularis propria in association with the smooth muscle layers of the gut. The ISEMF are located in the lamina propria under the epithelial cells. It is not known if these two myofibroblasts are derived from a common precursor (stem) cell. Both exist as a syncytium, but it has not been determined whether the ICC network is physically connected to the ISEMF network. The ICC was discovered over 100 years ago and 50 years ago was shown convincingly not to be a specialized neuron but a fibroblast-like cell (176). Twenty years ago these cells were proposed to be the electrical pacemakers that control motility of the gastrointestinal tract, and recently a large body of evidence affirms this concept (176). Over 30 years ago Pascal, Kaye, and Lane (148) described a sheath of lamina propria fibroblasts in tight apposition to the crypts in the intestine. This structure was confirmed by others and was found also in other gastrointestinal tissue, such as the gallbladder and stomach (70, 95, 100, 101, 110, 111, 127, 133, 178). This fibroblastic sheath or pericryptal cells (fibroblasts), as they were called then (178), are now known to be a /99 $5.00 Copyright 1999 the American Physiological Society C183

2 C184 syncytium of cells that extend throughout the lamina propria (95). ICC Morphologically, the ICC have the classic appearance of myofibroblasts and exist in several separate locations within the gastrointestinal tract (84, 176). Classically, they are located in the intermuscular space between the circular and longitudinal layers of the muscularis propria in the stomach, small bowel, and colon. However, they also reside, with slight differences in morphology, along the submucosal surface of the circular muscle bundles of the colon, within the deep muscular plexus region of the small intestine, and intramuscularly in the esophagus, stomach, and colon (176, 199). These cells have a stellate appearance and are connected in a syncytium via gap junctions (31, 110). They have all the morphological characteristics of myofibroblasts [see part I of this review (163) as well as Ref. 205]. The biology of ICC has been reviewed excellently and in detail by Sanders (176), a review with many original references not listed here. Antibodies against the receptor c-kit localize ICC in the mouse embryo (220) and suggest that the ICC originate from the mesenchyme of the gut and not from neural crest cells, the precursors of intestinal neurons (10, 201, 229). However, the true origin of these cells is still uncertain. The embryonal cells responsible for the synthesis and secretion of stem cell factor (SCF), the ligand that promotes the growth and differentiation of c-kit-positive ICC, have also not been clearly identified (209). Considerable experimental evidence suggests three major functions of the ICC: 1) they are pacemakers for gastrointestinal smooth muscle motility, 2) they facilitate active propagation of electrical events, and 3) they modulate neurotransmission (176). As myofibroblasts, it is likely, however, that they have other functions [see part I of this review (163)], such as immune modulation, growth, repair, and fibrosis. These additional functions should be investigated in ICC. The mammalian intestine demonstrates a wave of electrical activity with a characteristic frequency somewhere between 6 and 12 cycles per minute, depending on the segment studied (85, 176). These slow waves are accompanied by corresponding peristaltic contractions during the intercibal periods, and this activity is thought to serve a housekeeper function of clearing the intestinal lumen between meals. The pattern is disrupted by eating. Both freshly isolated and cultured ICC demonstrate oscillations of membrane potential due to varying Ca 2 conductivity, i.e., activation or closure of voltage-dependent Ca 2 channels (176). This property constitutes the primary evidence for assigning the pacemaker role to the interstitial cells (see Ref. 176 for original references). The lack of propagation of the slow wave in the bulk of smooth muscle, plus the fact that removing a thin strip of tissue that contains the syncytium of ICC also destroys the intestinal slowwave activity, give strong credence to the idea that the ICC syncytium is the origin of the slow wave. Furthermore, chemical lesions of the ICC also inhibit both the pacemaker potentials and slow-wave propagation. Evidence that ICC are involved in neurotransmission is both anatomic (their intermediate position between neural varicosities and the smooth muscle cells) and physiological. ICC are responsive to a host of enteric neurotransmitters, including acetylcholine, nitric oxide, vasoactive intestinal peptide, ATP, and substance P (176). Nitric oxide and, recently, carbon monoxide have been proposed as inhibitory neurotransmitters generated by the ICC and other myofibroblasts (8, 57, 131, 136). Additional evidence for a pacemaker role of ICC comes from experiments using antibodies directed against c-kit protein (85, 200). When neutralizing c-kit (ACKII) antibodies are injected in neonatal animals for several days following birth, intestinal c-kit immunoreactivity disappears and intestinal smooth muscle activity becomes abnormal (85, 144, 200). Furthermore, the electrical rhythmicity of the embryo and neonate is absent until c-kit-positive cells appear (201). Mutants of c-kit expression clarify the function of ICC (85, 90, 176). The white-spotting (W) locus in mice and a hypopigmentation disorder in humans known as piebaldism are allelic with c-kit. A number of spontaneous mutations of the W locus are available, which either completely or partially block c-kit protein expression. Mutations that completely block expression of c-kit are fatal; homozygotes (W/W) die in utero. However, there are point mutations of the locus, such as W v, that do not completely abolish the receptor expression. W v /W v homozygotes or compound W/W v heterozygotes lose electrical slow-wave rhythmicity and have abnormalities of intestinal smooth muscle function. Humans born with piebaldism often develop congenital megacolon. Mutants of SCF, so-called Steel mutants, also clarify the role of the ICC in gut motility (176). The classic Steel (Sl) mutants represent a complete deletion of the genomic region that codes for SCF, and homozygotes (Sl/Sl) do not survive because of development of severe anemia. However, nonlethal mutants such as Sl-dickie (Sl d ) and compound heterozygotes such as Sl/Sl d retain some c-kit signaling activity (16). The ICC of these animals are histologically abnormal, and their electrical and contractile patterns of the intestinal smooth muscle are abnormal as well (217). The recognized human diseases associated with abnormalities of the ICC are diseases of gastrointestinal motility and inflammation (84): congenital megacolon of piebaldism and Hirschsprung s disease (176, 209), infantile hypertrophic pyloric stenosis (208), intestinal pseudoobstruction (18, 84, 89), possibly achalasia (218), and ulcerative colitis (173). Hirschsprung s is a polygenic disease that results in enteric nervous system aganglionosis and disturbed motility. Mutant genes associated with Hirschsprung s disease include 1) the RET protooncogene, a tyrosine kinase receptor, which prevents ganglion cell migration from the neural crest (151); 2) the endothelin-3 (ET-3) gene (172); and 3) the gene for the ET-3 receptor (172). The ICC in Hirschsprung s disease are present, but morphologically

3 C185 abnormal, indicating that disturbed ICC function accompanies aganglionosis in this polygenic disease (176, 209). Perhaps the presence of ICC is required for the migration and full development of ganglionic neurons (115) (or vice versa) or, alternatively, some defect in the embryological mesenchyme affects both the migration of neural crest cells and the differentiation of the ICC. Infantile hypertrophic pyloric stenosis is a relatively common disease of newborns, characterized by hypertrophy of the muscular region of the pyloric sphincter (208). Various pathological disturbances have been demonstrated, including abnormalities of the enteric nervous system and, more recently, the reduction or lack of the neuronal isoform of nitric oxide synthase. c-kit immunoreactive ICC have been shown to be absent in the longitudinal muscle layer and in most of the hypertrophic muscle of all 26 patients investigated (208). The exact role of the ICC in the muscular abnormality of this disease is unclear. Recently, abnormalities in nitric oxide-dependent inhibitory neurotransmission in the lower esophageal sphincter (LES) of W/W v mutant mice raise the interesting possibility of a role of the ICC in human achalasia, a disease in which the LES fails to relax (218). This work needs to be confirmed and extended in the human disease. Abnormalities of the colonic ICC at an ultrastructural level have been demonstrated in patients with severe ulcerative colitis (173). The role these abnormalities may play in the deranged motility of colonic inflammation remains to be defined. It is possible that the histological abnormalities of the ICC in ulcerative colitis might be secondary to high-dose corticosteroid therapy rather than the inflammation. Glucocorticoids decrease the number of tissue mast cells (also c-kitpositive cells) by downregulating the amount of SCF produced by fibroblasts (55). Therefore, reductions in the amount of available SCF could result in histological abnormalities and even apoptosis of the ICC. ISEMF ISEMF exist in a subepithelial location throughout the gastrointestinal tract from esophagus to anus and in the gallbladder and pancreas, but they are best described in the small intestine and colon (70, 95, 100, 101, 111, 148). Although initially thought of as a sheath of fibroblasts, more dense in the region of crypts than at the surface of the colon or in the villi of the small intestine (70, 100, 127, 148), it is now clear that they exist as a syncytium that extends throughout the lamina propria of the gut, merging with the pericytes surrounding the blood vessels that course through the tissue (95). In the region of the crypts, the myofibroblasts are oval and scaphoid in appearance and appear to overlap like shingles on a roof (100, 137). Even here they are attached one to the other with both gap junctions and adherens junctions, as they are throughout the syncytium. In the upper regions of the colonic crypts and in the small intestinal villi, the ISEMF take on a stellate morphology (Fig. 1) (95, 111, 207). Ultrastructural studies demonstrate close contacts between ISEMF and synaptic vesicle-containing nerve terminals (70). We have found that 18Co colonic myofibroblasts contain carbachol receptors coupled to prostaglandin (PG) E 2 synthesis and reversal of stellate transformation (77, 205). These data suggest that ISEMF can be modulated by cholinergic inputs, and structures resembling dendritic spines on stellate 18Co processes may be the location of these acetylcholine receptors (205). At transmission electron microscopic resolution, ISEMF have the typical appearance of myofibroblasts: a cell membrane with multiple caveolae, a welldeveloped rough endoplasmic reticulum and Golgi complex, and a cytoplasm filled with microfilament bundles (stress fibers) and associated dense bodies [see part I of this review (163) and Ref. 205]. In the small intestinal villus, the processes of the ISEMF are even more attenuated and encircle the capillaries that extend throughout the lamina propria (111). Here ISEMF are essentially indistinguishable from pericytes (35) (and indeed may be one and the same). The basal lamina contains numerous fenestrations, particularly on the upper two-thirds of the villus, and cell processes of the myofibroblasts and/or the epithelium can extend through these fenestrae (100, 111, 202). The myofibroblasts are embedded also in a subepithelial sheet of reticular fibers that also contains fenestrae or foramina through which lymphocytes and macrophages traverse (202). Just below the surface Fig. 1. Scanning electron micrographs of intestinal subepithelial myofibroblasts (ISEMF) in rat intestinal villi. A: lower-power view showing ISEMF syncytium (*) underneath palisading columnar epithelial cells (EP). Arrows indicate apical surface of goblet cells. Bar, 20 µm. [From Desaki et al. (35).] B: higher-power view demonstrating anastomosing syncytium of stellate fibroblast-like cells (FLC), which are the ISEMF. Bar, 5 µm. [From Gannon and Perry (61a).]

4 C186 epithelium of the colon, the reticular sheet is called the collagen table. Processes of the myofibroblasts extend through this table and abut the basal lamina under the surface epithelial cells with foot processes reminiscent of those in the kidney glomerulus (100, 148). Thus the subepithelial space appears to have two fenestrated barriers: the basal lamina and the subepithelial reticular sheet in the small intestine (or collagen table in the colon), both of which are formed by connective tissue fibrils secreted by the myofibroblasts. This anatomic feature may have functional implications for water transport by the epithelium (see Water and Electrolyte Transport). Antibodies that react against -smooth muscle ( - SM) actin or against glial fibrillary acidic protein will stain ISEMF as well as ICC, hepatic stellate cells, pancreatic stellate cells, and other stellate-type myofibroblasts such as those in Wharton s jelly of the umbilical cord [see part I of this review (163) and Ref. 61]. The identifying cytoskeletal staining characteristic of ISEMF, however, is their reaction to -SM actin antibodies. ISEMF can be distinguished from smooth muscle of the muscularis mucosae, which is also -SM actin positive, by absence of staining for desmin (Fig. 2) (129, 165, 166). Thus both ISEMF and muscularis mucosae stain for -SM actin and variably for myosin, but the ISEMF, in contradistinction to the muscularis mucosae, is negative (normal intestine) or only weakly positive (severely inflamed intestine) for desmin (129, 165, 166, and A. B. West, unpublished observations). Several independent studies in mice reveal the presence of ICC, as determined by c-kit expression, in the embryo from days onward (10, 176, 199, 220). With the use of -SM actin as a marker, ISEMF in the human embryo are detectable in the intestine at 21 wk of gestation (178). The origin of these cells may be from the neural tubes or neural crest stem cells, migrating along the vagus nerve to the gut (14, 92). Alternatively, ISEMF may transdifferentiate from resident fibroblasts or smooth muscle cells [see part I of this review (163)]. The cells are first visible in immediate juxtaposition with the muscularis mucosae at the base of the intestinal crypts (97). Between the 21st and 39th wk, the number of ISEMF increases progressively in the region between the base and the middle of the crypts, in synchrony with the proliferation and differentiation of the epithelial cells. At birth, the ISEMF line the lower two-thirds of the crypts of the colon, and all of the cells avidly express -SM actin. These studies suggest that antibodies against -SM actin are currently the best markers for ISEMF and, furthermore, that ISEMF may originate from the muscularis mucosae or from cells adajcent to them. It is important to note that c-kit antibody staining, which defines the ICC, has not been studied extensively in ISEMF and, conversely, -SM actin staining, which defines ISEMF, has not been explored well in ICC. [ 3 H]thymidine incorporation into primary cultures of ISEMF indicates proliferative responses to plateletderived growth factor (PDGF)-BB, epidermal growth factor (EGF), basic fibroblast growth factor (bfgf), insulin-like growth factors I and II (IGF-I and IGF-II), interleukin-1 (IL-1 ), and tumor necrosis factor- (TNF- ) (94). In this study, synergism between PDGF-BB and IL-1 was demonstrated, and the proliferative response was inhibited by elevation of cellular camp levels. The effect of SCF was not reported. mrna for c-kit can be detected in 18Co cells, human myofibroblasts cultured from a mucosal biopsy of human neonatal colon (Mifflin, Saada, and Powell, unpublished observations). The functional significance of this remains to be determined. These cells also express SCF message and protein (109), as well as message for PDGF- receptor (Mifflin, Saada, and Powell, unpublished observations). The growth factor(s) and receptor set(s) crucial for the development or proliferation of ISEMF remain to be more clearly determined. FUNCTIONS OF ISEMF Mucosal Growth and Development Intestinal mucosal development takes place along two broad perspectives (34, 72, 102, 186): 1) organogenesis (morphogenesis), during which interactions between the embryonic endoderm and the mesenchyme form the tubular foregut, midgut, and hindgut, which then differentiate into crypts and villi; and 2) cytodifferentiation of epithelial stem cells into the four major intestinal cell types (enterocytes, goblet cells, enteroendocrine cells, and Paneth cells). These are both complex processes that involve cell adhesion, proliferation, migration, differentiation, and, finally, apoptotic cell death (36, 73). There is mounting evidence that the intestinal myofibroblasts take part in both of these developmental events (33, 34, 53, 58, 65, 72, 74, 102, 155). Fibroblasts capable of driving either epithelial proliferation or differentiation have been isolated from the gut lamina propria (58, 72). Over 15 years ago, Haffen et al. (72) showed that isolated intestinal fibroblasts were as potent as dissected intestinal mesenchyme in inducing intestinal endoderm. More recently, they have shown that the intestinal mucosa contains different clones of fibroblasts that will drive either epithelial proliferation or differentiation (58). Transforming growth factor- 1 (TGF- 1) was capable of transforming one set of isolated fibroblasts into -SM-actin-expressing myofibroblasts and inhibiting their proliferation. In contrast, proliferation of the second, typical, fibroblast cell line was inhibited by IL-2 but not by TGF-. When the typical fibrocyte cell line was implanted into fetal endoderm, the endoderm formed deep crypts due to proliferation of the epithelial cells. In contrast, similar coculture containing the -SM-actin-expressing myofibroblasts demonstrated differentiation rather than proliferation of the epithelial cells: the endoderm developed villi with mature differentiated enterocytes, goblet cells, and endocrine cells (58). There were differences in the amount of basement membrane proteins (laminin 1 and type IV collagen) produced by the two (myo)fibroblast cells lines, but it is unclear whether matrix or growth factor secretion caused these different growth patterns.

5 C187 Fig. 2. A: normal human colonic mucosa stained for -smooth muscle ( - SM) actin. Note weak staining of ISEMF and strong staining of muscularis mucosae. B: muscularis mucosae stains strongly for desmin but ISEMF do not. C: cross section of human colon of a patient with radiation colitis. Note that ISEMF are activated and strongly express -SM actin. D: note lack of ISEMF desmin staining of the same tissue as in C. Coculture of fibroblasts or myofibroblasts with epithelial cells will cause the epithelial cell to proliferate and differentiate (33, 74). Foreskin and lung fibroblasts (74) and primary cultured ISEMF (33) are capable of inducing cellular differentiation of HT-29 and T84 human colon cancer cells. When grown in collagen gels without (myo)fibroblasts, T84 cells formed round, ball-shaped colonies. When these (myo)fibroblasts were included, a basal lamina formed around the T84 colonies, they differentiated into one-cell-layer hollow balls with a lumen, and the epithelial cells changed from thin, cuboidal cells with occasional microvilli to columnar enterocyte-like epithelial cells with well-developed microvilli (74). In separate culture studies, it was shown that hepatocyte growth factor (HGF) caused proliferation but not differentiation of the T84 cells, whereas TGF- had the opposite effect (74). Furthermore, antibodies against TGF- or against the TGF- latencyassociated protein abrogated the differentiating effect of fibroblasts cocultured with the T84 cells (74). Thus (myo)fibroblast-derived HGF appears to be a proliferative growth factor for the intestinal epithelial stem cell, whereas (myo)fibroblast-secreted TGF- appears to promote epithelial cell differentiation. The studies detailed above suggest that phenotypically different lamina propria fibroblasts are capable of

6 C188 causing either proliferation or differentiation of the intestinal epithelium, that basement membrane formation is an important ingredient in differentiation, and, finally, that HGF is a proliferating growth factor, whereas TGF- is a differentiating growth factor for the intestinal epithelium. Studies of keratinocyte growth factor (KGF) and KGF receptor expression during fetal development suggest that this factor, also of fibroblast origin, is equally important in mediating the morphogenic mesenchymal-epithelial interactions (53). A third axis of function and differentiation is a proximal-distal gradient from duodenum to distal colon. Plateroti et al. (155) have shown that myofibroblasts from proximal jejunum, distal ileum, and proximal colon secrete different and characteristic amounts of HGF, TGF- 1, and epimorphin and have differing effects on endodermal growth in coculture. These studies suggest that the ISEMF may be important in proximal-distal gut differentiation as well as cryptvillus morphogenesis (65). There is evidence that myofibroblasts, like intestinal epithelial cells, proliferate, migrate, and differentiate along the crypt-villus axis. In [ 3 H]thymidine pulselabeling experiments of rabbit colon and adult mouse jejunum, both pericryptal myofibroblasts and crypt epithelial cells took up thymidine after a single pulse administration, i.e., both were proliferating (127, 148). Both the labeled cell populations appeared to migrate up the basement membrane over the course of 2 4 days, disappearing at the tip of the small intestine villi or the surface of the colon through the processes of exfoliation and/or apoptosis (73). These experiments suggest that myofibroblasts differentiate as they move from a discoid morphology at the base of the crypt to the stellate shape as they move toward the intestinal lumen. Perhaps maturation and differentiation of the myofibroblasts drive the maturation and differentiation of the epithelial cells that are moving pari passu up the basement membrane. It is also conceivable that there are signals from the epithelial cells that drive myofibroblast differentiation. It should be noted, however, that this finding of crypt myofibroblast labeling and migration could not be confirmed in studies with mouse small intestine and colon (142). Protection and Wound Healing Functions of ISEMF Contraction. ISEMF (95), as well as many other myofibroblasts, contain smooth muscle myosin isoforms in addition to smooth muscle actin. Thus they have the requisite machinery for contraction and/or motility. Histological studies of various intestinal disease states show that the small intestinal villus shortens dramatically after injury. This has been most clearly demonstrated with a highly reproducible model of intestinal epithelial injury in guinea pig ileum (133). This process is energy dependent and greatly facilitated by the presence of the enteric nervous system, i.e., villus shortening is significantly reduced if the tissue is depleted of ATP or if the tissue is functionally denervated by a neurotoxin such as TTX (133). Because the small intestine villus contains typical smooth muscle fibers, presumably originating from the muscularis mucosae, which emanate up through the core of the villus, it is possible that this villus shortening is due to contraction of these conventional smooth muscle elements. However, the extensive organization of the ISEMF network and its apparent innervation by the enteric nervous system make it a likely candidate as well (70, 205). It can be speculated also that the ISEMF network alone, or through its connections to the smooth muscle cells of the muscularis mucosae that extend up the villus core, is responsible for the rhythmic villus contractions observed in the small intestine (224). This phenomenon was first described over 150 years ago but has received little study in the past two decades. Almost certainly the ISEMF syncytium accounts for the mucosal contractions in the stomach, which lead to expulsion of gastric acid from the gastric pits in a jetlike fashion (79). Although these cells were described as smooth muscle cells originating from the muscularis mucosae, their anatomic location in the gastric mucosa suggests that they are the gastric counterpart of the ISEMF (194). Intestinal myofibroblasts display receptors for contractile and relaxing agonists such as endothelin and atrial natriuretic peptide (59, 60, 205). Of interest, ISEMF fail to respond to other guanylate cyclase stimulants such as guanylin, heat-stable Escherichia coli enterotoxin (Sta), or sodium nitroprusside, suggesting that these receptors and/or the specific guanylate cyclases are absent from the ISEMF cells (205). The role of the ISEMF, which have the anatomic appearance of pericytes, in the autoregulation of lamina propria blood flow remains to be determined. Restitution. The rapid migration of epithelial cells over a denuded basement membrane is an important intestinal repair response to minor/moderate injury (214). It is likely that ISEMF play an important role in this process, both because their location under the basement membrane is ideal for paracrine action and because myofibroblasts secrete the agents that thus far have been shown to enhance epithelial cell migration in experimental disease states in vivo and in wounding models in vitro. Factors promoting restitution include PGE 2 (in synergism with prostacyclin) (13), IL-1 (37), interferon- (IFN- ) (37), TGF- (37), EGF (37, 157, 170), acidic FGF (afgf) and bfgf (39), TGF- (especially TGF- 3) (25, 130), and HGF (143, 195). The most compelling evidence for a role of ISEMF in restitution comes from the laboratories of Mahida and Podolsky (130). They have shown that the active factor in conditioned media from primary cultures of ISEMF that promote IEC-6 or T84 epithelial cell motility is TGF- 3. The PGs may have their action by virtue of stimulating HGF secretion, since anti-hgf antibodies abrogate PG acceleration of gastric epithelial cell restitution in a coculture model of gastric epithelial cells and fibroblasts (195). Similarly, anti-tgf- antibodies block the restitution-promoting effects of IL-1, IFN-, TGF-, EGF, afgf, and bfgf (37, 39). Thus these agents may not act directly on the epithelial cell to promote cell motility; rather, they may augment the secretion of

7 C189 and/or the expression of receptors for TGF- and HGF. Of interest, TGF- may also enhance the barrier integrity of newly formed tight junctions between epithelial cells and protect against the barrier-destroying effects of IFN- (154). Trefoil peptides also stimulate intestinal epithelial restitution (106), but these factors are secreted by epithelial mucus cells (goblet cells), not by ISEMF (24). It is of interest that PDGF, an agent that stimulates myofibroblast and smooth muscle motility, was without effect on the motility (restitution property) of the intestinal epithelial cell (37). Protection and repair. Tissue repair is a complex, coordinated event [see part I of this review (163)], in which there is release of various lipid mediators such as eicosanoids, gases such as nitric oxide, cytokines such as TNF-, IL-1, IL-6, IL-2, and IL-15, and the various growth factors mentioned in the previous section. Many of these factors activate myofibroblasts, resulting in myofibroblast motility and the release of ECM proteins and other growth factors. Remodeling of intestinal tissue (villous atrophy/crypt hyperplasia) is also an important response to gut injury (121). There is evidence that myofibroblasts take part in this process through the secretion of matrix metalloproteinases and other proteases (32, 121), as well as secretion of TGF- and KGF (5). In a cascade fashion, growth factors induce angiogenesis and epithelial proliferation and differentiation. The process is terminated by myofibroblast apoptosis [see part I of this review (163)]. Myofibroblasts take part in the healing of gastrointestinal ulcerations. Myofibroblasts are identified as -SMactin-positive cells at the base of gastric and duodenal ulcers (140, 141) and in the colon after experimental lesions (211). In vitro studies of primary ISEMF cultures suggest that the important factors inducing myofibroblast activation and proliferation are TNF-, IL-6, EGF, bfgf, IGF-I and IGF-II, PDGF-BB, and IL-1 (the latter two synergistically) (94). Many of the factors secreted by the activated myofibroblasts, as well as their respective receptors, are upregulated in the intestine in various disease states. Examples include PGs via cyclooxygenase (COX)-2 activity (104, 132, 196), TGF- (4, 38, 191), EGF (117), TGF- (4, 38), bfgf (86), HGF (107, 180), and KGF (23, 54, 107) in experimental colitis, small bowel injury (38), or gastric ulcer models or disease (86, 107, 180), and also in naturally occurring inflammatory bowel disease (4). Furthermore, animals lacking TGF- are more susceptible to colonic injury, and this susceptibility is ameliorated by exogenous administration of the factor (48). In fact, the exogenous administration of many growth factors such as EGF (156, 170), bfgf (87), IGF-I (152), KGF (230), and IL-II (118, 145) results in amelioration of experimental or natural gastrointestinal injury. Not only are these growth factors upregulated in intestinal disease or injury, they have proliferative effects on normal or transformed epithelial cell lines in culture and cause epithelial proliferation in vivo. ET-1 (184), IL-2 (184), TGF- (96), IL-15 (167), afgf (39), bfgf (39), KGF (39, 107), and HGF (107) all have mitogenic activity in epithelial cell culture models. Administration of EGF and TGF- (81, 158), IGF-I and II (158), and KGF (82) to animals causes intestinal epithelial cell and, in some cases, hepatocellular proliferation. Transgenic animals overproducing IGF-I have predominantly muscle layer proliferation (215), suggesting that this factor may be more important in muscle repair. A protective role for nitric oxide cannot be overlooked. Inducible nitric oxide synthase-deficient mice have poor wound healing that is corrected by exogenous administration of nitric oxide (226). Furthermore, administration of nitric oxide donors prevents gastrointestinal injury from nonsteroidal anti-inflammatory drugs (NSAIDs) (50). While other myofibroblasts (e.g., the renal mesangial cell) are significant producers of nitric oxide (112), it is unclear whether ISEMF or ICC are significant sources in the intestine (131, 225). Fibrosis Our understanding of fibrosis in the intestine is less advanced than in liver or skin, but intestinal smooth muscle cells and/or myofibroblasts have been incriminated in the process (see Ref. 66 for overview and original references). Fibrotic strictures of the small intestine, particularly the ileum, are often the outcome of repeated bouts of inflammation due to Crohn s disease (regional enteritis). Sections of Crohn s disease stricture show expansion of the muscularis mucosae from their usual three to five cell layers and accumulation of collagen around those cells. In addition, there is development of islands of smooth-muscle-like cells in the submucosa with invaginations of collagen septa into the circular and longitudinal muscle layers of the muscularis propria. Immunohistological studies and in situ hybridization studies show marked increases in type III collagen and RNA transcripts for procollagens I, III, and V (66). Gut bacterial flora may be important in this fibrosis; experimental injection of bowel flora into the bowel wall initiates intestinal TGF- 1 and collagen synthesis (135). A major question regarding intestinal fibrosis is whether the increase in matrix production emanates from activated resident fibroblasts, smooth muscle cells, or myofibroblasts. Normally, intestinal smooth muscle expresses -SM actin, desmin, and tropomyosin. The smooth muscle cells isolated and cultured from Crohn s disease bowel demonstrate increased amounts of -SM actin, -enteric actin, and desmin (66). In situ, normal ISEMF are desmin negative, but staining characteristics can change during culture or disease states, and in situ immunohistochemical studies of Crohn s disease strictures are not available. We have found increased expression of desmin in ISEMF in Crohn s disease intestine (West, unpublished observations). The intestinal smooth-muscle-like cells responsible for intestinal fibrosis are more stellate in appearance, have more prominent stress fibers, and contain

8 C190 more extensive, dilated endoplasmic reticulum than conventional intestinal smooth muscle cells (66), all characteristics of myofibroblasts [see part I of this review (163)]. We believe, therefore, that the cells responsible for fibrosis in Crohn s disease are activated myofibroblasts (either ISEMF or ICC) and not conventional intestinal smooth muscle. Furthermore, Western analysis demonstrates that cultured ISEMF express several ECM proteins, including type IV collagen and 1- and 1-laminin and fibronectin (122). Perhaps this is semantics, because it is entirely possible that myofibroblasts transdifferentiate from the smooth muscle cells of the muscularis mucosae [see part I of this review (163)]. There are a number of interesting characteristics of the Crohn s disease, collagen-producing, smooth-musclelike cells. The mesenchymal cells isolated from the lamina propria of Crohn s strictures produce more total collagen and more type III collagen than mesenchymal cells isolated from nonstrictured or normal intestine (66). TGF- stimulates (67), while IL-1 and PDGF-BB downregulate, procollagen secretion but not its synthesis (69). In contrast to the similar effect of IL-1 and PDGF on collagen formation and secretion, IL-1 caused a marked concentration-dependent increase in collagenase mrna levels, whereas PDGF had no effect (69). IL-1 and dexamethasone inhibit collagen synthesis in dermal fibroblasts, yet they increase procollagen synthesis by intestinal smooth muscle cells (128). At certain concentrations in vitro, glucocorticoids actually increase procollagen gene expression by human intestinal smooth muscle cells (68), a finding with potentially important therapeutic implications for the treatment of inflammatory bowel disease with corticosteroids. Collagenous colitis is a disease that appears to be due to abnormalities of or activation of the subepithelial myofibroblasts of the colon (1, 231). This is a form of inflammatory bowel disease, causing watery diarrhea predominantly in middle-aged or older women, who often have other manifestations of autoimmune diseases. The pathological abnormality in collagenous colitis is defined entirely at the microscopic level, where it is characterized by an expansion of the normal reticular band (collagen table) that exists in the subepithelial region of the surface epithelium of the colon, i.e., in the location of the ISEMF (231). Along with the collagen band, there is an increased number of the mononuclear inflammatory cells in the lamina propria. The possibility of an abnormality in collagen type or secretion in this disease has been previously studied with somewhat controversial findings (1). It has been suggested that in this disease there is a normal distribution of type IV collagen, increased type III collagen, and reduced or abnormal type I collagen production (1). Normal type IV collagen distribution in this disease indicates that the band is not an expanded basement membrane. More recent studies suggest increases in type VI collagen and tenascin in the collagen band under the surface epithelium, with focal increases in types I, III, and VI collagen in the area of the crypts (1). However, no increase in type VI collagen mrna was found in the myofibroblasts around and entrapped in the collagen table. These studies suggest that the accumulation of type VI collagen is not caused by increased collagen synthesis by the myofibroblasts but rather by abnormalities in the degradation of the intracellular matrix, e.g., decreased secretion of metalloproteinases or increased secretion of tissue inhibitors of metalloproteinases (1). Granulomas of the intestine are basically of two types: accumulations of only macrophages as seen often in Crohn s disease or -SM-actin-positive myofibroblasts surrounding macrophages and/or giant cells, which are thought to be of either macrophage or myofibroblast origin, as commonly seen in sarcoidosis or foreign body reactions (174). A model for granuloma formulation in the intestine is the injection of sterile bacterial peptidoglycan-polysaccharide polymers into the bowel wall (232). The bacterial cell wall polymers increased both TGF- immunostaining and collagen deposition, as well as collagen -1 mrna in the granulomas (211). Furthermore, TGF- directly stimulated collagen type I, TGF- 1, IL-1, and IL-6 mrna in myofibroblasts cultured from the granulomas (211). Thus granuloma myofibroblasts represent another potential origin for the intestinal fibrosis of Crohn s disease. Immunology and Inflammation Immune cell functions, such as secretion of cytokines and expression of adhesion proteins by epithelial cells and other nontraditional immune cells, have recently been a topic of much investigation (56). Cytokines produced by ISEMF (20, 56, 147, 171, 193) in response to stimulation by inflammatory cytokines such as IL-1 and/or TNF- include 1) inflammatory/immune regulators (TNF-, IL-1, IL-6, IL-10), 2) growth factor/ differentiation factors (TGF-, TGF-, PDGF, SCF, HGF, and KGF), and 3) chemotactic factors (IL-8, melanoma growth-stimulating activity, MIP-1, epithelial neutrophil-activating peptide-78). Adhesion protein expression, such as intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and integrins, by myofibroblasts has been documented also in response to inflammation (56, 147). At the electron microscopic level, it is possible to find close apposition of ISEMF and lymphocytes, suggesting that the myofibroblasts may take part in the growth and development of the T cell (202). Fiocchi (56), Toyoda et al. (202), and Roberts et al. (171) have shown that experimentally induced proliferative responses in T cells are enhanced by coculture with intestinal fibroblasts or with smooth muscle cells. PGs derived from either constitutive (COX-1) or inducible (COX-2) forms of cyclooxygenase (PGH 2 synthase) have fundamental regulatory roles in gastrointestinal barrier function, inflammation, the immunophysiology of intestinal electrolyte transport and motility, and gastrointestinal neoplasia (13, 21, 26, 42, 46, 159, 210). The cellular sources of PG in normal and diseased intestine are poorly defined. It does seem likely, however, that the epithelium is the least contribu-

9 C191 tor. When the intestinal mucosa is separated with a combination of sharp dissection and Ca 2 chelation into epithelial and subepithelial (lamina propria, submucosa, and muscularis propria) fractions, 95 99% of rat mucosal PG synthesis emanates from the subepithelial fractions (27, 28, 114). Myofibroblasts are but one of several tissue elements in the subepithelium capable of producing PGs (11, 77, 104, 122, 197, 205); white blood cells of various types, smooth muscle cells, endothelial cells, and conventional fibroblasts are among the other PG-producing constituents (159). The relative proportions of PGs secreted in normal or inflamed states by intestinal myofibroblasts compared with these other elements remain to be determined. Studies of nonstimulated intestine show COX-1 to be the predominant isoform producing basal secretion of PGs in the gastrointestinal tract (98). In the experiments noted above, in which intestine was dissected into epithelial and subepithelial fractions, it is likely that COX-2 is activated by the trauma of the experimental procedures. Immunohistochemical studies show constitutive expression of COX-1 in the crypt epithelium of the small intestine (26) and predominantly in mucous neck cells of the gastric glands (88). However, epithelial COX-2 expression can be induced. Experimental injury (injection of endotoxin) (52) and bacterial (Salmonella) invasion (47), as well as natural disease states such as Crohn s colitis, ulcerative colitis (76, 187), and colonic adenocarcinoma (45), result in expression of COX-2 in intestinal epithelial cells. Furthermore, COX-2 has been cloned from nontransformed rat intestinal epithelial (RIE-1) cells, a cryptlike intestinal epithelial cell line (43). ISEMF also express COX-1 and COX-2 (Fig. 3) (77, 104, 122, 205). IL-1 increased COX-1 mrna in 18Co ISEMF after 24 h exposure, but the threefold increase in message was not accompanied by detectable increases in protein, as determined by Western analysis (175). In contrast, IL-1 induces an exponential increase in COX-2 mrna and protein levels in 18Co cells. Under these conditions, COX-2 accounts for 95% of the total PGE 2 synthesis (77). Immunocytochemical studies localize COX-2 to the lamina propria of the inflamed stomach and intestine (52, 168, 187). This subepithelial localization of COX-2 is usually ascribed to either macrophages or blood vessels, and expression in ISEMF has not been sought or clearly demonstrated. In contrast, COX-2 expression has been definitively located to lamina propria interstitial mesenchymal cells (probably ISEMF) in the early stages of polyp formation in the adenomatous polyposis coli (APC) knockout mouse (146). Thus the relative roles of epithelial, white blood cell, endothelial, and ISEMF PG production, as well as the relative roles of COX-1 vs. COX-2 enzymatic PG production, in intestinal barrier function, inflammation, immunophysiology, and neoplasia remain to be determined. Most studies suggest COX-1 is the predominant enzyme responsible for barrier function and protection against damage, whereas COX-2 expression is increased in gastrointestinal inflammation (13, 26, 29, 46, 47, 52, 210, 212). Whether COX-2 inhibition is sufficient to alleviate pain and inflammation is still a topic of debate (168, 213). Nevertheless, this concept of differential functions of COX-1 and COX-2 has been the theoretical basis for the development of COX-2-selective NSAIDs (210). This concept has been challenged by experiments that show that COX-1 contributes to the inflammatory responses of the gastrointestinal mucosa and that inhibition of COX-2 can exacerbate inflammation in the colon (168, 213). To date, initial reports suggest that selective COX-2 inhibition does, indeed, cause less gastrointestinal mucosal damage than conventional NSAIDs and is effective in symptom relief (75, 120, 185). Water and Electrolyte Transport Secretion. The close proximity of ISEMF to intestinal epithelial cells, being separated only by the basement membrane, makes it reasonable to propose that these cells might be paracrine regulators of intestinal electrolyte transport. To test this hypothesis, we cocultured or separately cultured and then acutely juxtaposed piglet small intestine (P2JF) myofibroblasts or human colonic (18Co) myofibroblasts with T84 cells, a human colon carcinoma cell line that maintains the ability to secrete Cl in response to many of the secretagogues known to affect normal human colon (11, 77, 206). T84 cell Fig. 3. Northern analysis of 18Co total RNA after stimulation with interleukin-1 (IL-1 ) for 5 and 24 h. A: 2.7-kb cyclooxygenase (COX)-1 hybridization shows constitutive expression of message and a 3-fold increase only after 24 h of incubation with IL-1. B: 4.2-kb COX-2 message is increased from barely detectable levels to 10-fold by within only 5hofincubation with IL-1. [From Hinterleitner et al. (77).]

10 C192 Fig. 4. Short-circuit current response (I sc ) of T84 colon carcinoma cells mounted in an Ussing chamber either alone (A) or cocultured with neonatal piglet jejunal myofibroblasts (P2JF) (B), or of separately cultured T84 and P2JFs that were acutely juxtaposed (C). Note augmentation of I sc in response to H 2 O 2 when myofibroblasts are present. Indomethacin (INDO) inhibits augmented I sc (B and C). [From Powell and Berschneider (162).] monolayers were cultured on permeable supports and then mounted in Ussing chambers to determine the Cl secretory response, as measured by the short-circuit current (I sc ) generated by the addition of various secretagogues. The responses of T84 cells alone were compared with those of either cocultured or acutely juxtaposed cultures of T84 cells with the myofibroblast cell lines in both the presence and absence of PG synthesis inhibitors such as indomethacin. As shown in Fig. 4A, T84 cells alone had either a small or no Cl secretory response (increase in I sc ) to agonists such as H 2 O 2, and this response was not altered by the presence of indomethacin. However, when the T84 cells were either cocultured (Fig. 4B) or acutely juxtaposed (Fig. 4C) with the myofibroblasts, these same agonists elicited a much greater Cl secretory response, and that response was almost entirely inhibited by indomethacin. Similar responses were observed when bradykinin, serotonin, histamine, or carbachol (77) was used as an agonist. Augmented Cl secretion is a result of both a camp signal generated in T84 colonocytes by PGs (principally PGE 2 ) released from myofibroblasts and a subsequent Ca 2 signal elicited by Ca 2 -mobilizing secretagogues (7, 206). Furthermore, 24 h of incubation with IL-1 greatly upregulates not only COX-2 protein and PGE 2 secretion from the myofibroblast but also the I sc response by acutely juxtaposed T84 cells (Fig. 5) (77, 175). From these studies, we concluded that the underlying myofibroblasts have a paracrine action, modulating intestinal Cl and water secretion. Furthermore, in various inflammatory states where IL-1 is expressed, this cytokine acts as a thermostat. By regulating PG production, the underlying ISEMF is able to augment the level of Cl and water secretion elicited by inflammatory mediators. Preliminary evidence suggests that the myofibroblasts also augment Cl secretion brought about by activated neutrophils (44). In contrast, using a different reporter, Cl -secreting cell line HCA-7, ISEMF-conditioned media downregulated bradykinininduced I sc (9). Thus it seems likely that ISEMF play a fundamental role in modulating the secretory response of many immune-mediated or inflammatory diarrheas. The studies above were performed in a reductionist model: cell culture of epithelium and myofibroblasts. Fig. 5. A: preincubation (24 h) of human colonic myofibroblasts (18Co) with IL-1 augments I sc response of T84 cells acutely juxtaposed with 18Co cells and then stimulated with histamine or carbachol. B: PGE 2 production from 18Co cells is correspondingly increased by IL-1 preincubation. Both augmented I sc response and PGE 2 production induced by IL-1 are prevented by simultaneous incubation with IL-1 receptor antagonist (IL-1ra). [From Hinterleitner et al. (77).]

11 C193 There is, however, evidence from studies of native tissues that PG release is part of immune mediator, neurotransmitter, and cholera toxin-induced Cl secretion (7, 21, 93, 103, 123, 153, 159, 162, 189, 219). These studies also raise the question whether some of the abnormal electrolyte secretory responses in the intestine of W/W v mice are due to loss of ISEMF rather than loss of mast cells (216). Some of these abnormal electrolyte transport responses (e.g., the cholera toxin response) were not reconstituted by bone marrow transplantation that replaced mast cells in these c-kitnegative animals (216). Absorption. Recently, Naftalin and colleagues (137, 138) have published intriguing experiments that suggest ISEMF also have a critical function in intestinal water and electrolyte absorption. These investigators had previously suggested that colonic crypts act as suction devices to dehydrate feces in the distal segment of the mammalian colon (150, 160). Such a process requires that the colon absorb fluid hypertonically (which it is known to do) (12), but the mechanism of hypertonic absorption by an epithelium has heretofore not been evident (161). Naftalin and colleagues showed that the ISEMF sheath represents a diffusion barrier to Na transport that creates a significantly hypertonic middle compartment between the epithelial tight junctions and vascular system. It has been shown experimentally that such a three-compartment model will permit hypertonic transport in keeping with the Curran- MacIntosh model of water movement across epithelial tissues (30, 137, 138). Furthermore, they demonstrated proliferation of the ISEMF sheath in response to dietary Na depletion, which would account for the increase in Na and water absorption in high ANG II-high aldosterone states. ANG II (and/or aldosterone) has been demonstrated to cause proliferation of cardiac myofibroblasts and to increase their secretion of ECM fibrils (19). ISEMF may be responsible for differentiation of epithelial cells from a secretory to an absorptive phenotype. When T84 colon cells, a secretory cell line, are cocultured with 18Co human myofibroblasts, fluidfilled domes develop under the T84 cells (205). Dome formation in epithelial cell culture systems is usually thought to result from Na and water transport from the culture media to the space between the epithelium and the plastic culture dish. Intestinal Polyps and Neoplasia There is a growing body of evidence for a role of myofibroblasts in the pathogenesis of intestinal inflammatory (hyperplastic) polyps, stromal tumors, hamartomatous tumors, adenomas, and colon cancer. 1) Myofibroblasts themselves can undergo neoplastic transformation (78, 105, 139, 188). The resulting mesenchymal tumors likely constitute a significant proportion of the neoplasms previously called leiomyomas, leiomyosarcomas, fibrosarcomas, histiocytomas, spindle cell tumors, and desmoid tumors. These tumors are now often lumped under a single encompassing term of Fig. 6. -SM actin (A) and desmin (B) staining of a human colonic tubular adenoma. Note the activated ( -SM actin positive, desmin negative) myofibroblasts in the lamina propria of this benign neoplasm. gastrointestinal stromal tumors (GISTs). 2) Myofibroblasts are the primary mesenchymal element in hyperplastic and adenomatous polyps (Fig. 6) (108, 113, 178, 181). 3) Myofibroblasts appear to be the primary mesenchymal element in the hamartomatous polyps seen in juvenile polyposis coli (83, 91) and in Peutz-Jeghers syndrome (108) and perhaps account for the mesenchymal elements in the hamartomatous inflammatory polyps seen in chronic ulcerative colitis (192). 4) Myofibroblasts are responsible for the desmoplastic (fibrotic) reactions seen in many tumors of the gastrointestinal tract, e.g., carcinoid tumors (198, 228). 5) They play a key role in the growth of intestinal neoplasms and in the promotion and/or prevention of metastases (15, 17, 51, 129, 150, 165, 166). 6) Myofibroblasts are a prominent subepithelial element in familial adenomatous polyposis (FAP), and they may well play a key role in many of the extraintestinal manifestations of the FAP variant known as Gardner s syndrome (62, 149). 7) It is unclear if it is the ISEMF (146) or the epithelial cell itself (179, 182, 203) that is the key to NSAID-induced regression of colonic polyps and NSAID prevention of