Peptide Growth Factors: Role in Epithelial-Lamina Propria Cell Interactions* YASHWANT R. MAHIDA, CAROLINA CIACCI, AND DANIEL K. PODOLSKYb Gastrointestinal Unit and Center for the Study of Znjlammatory Bowel Disease Massachusetts General Hospital Boston, Massachusetts 02114
INTRODUCTION The intestinal mucosa is distinguished by a dynamic population of epithelial cells at its surface which undergoes nearly complete turnover every few days. In adition to the epithelium, the mucosa encompasses an underlying heterogenous population of immune and other cell populations within the lamina propria. A number of observations suggest that dynamic interactions occur between the epithelium and the immune cell populations present in the lamina propria. These interactions may play an important role in both effecting functional modulation of immune responses and restoring epithelial surface integrity following iqjury. The epithelial component of the mucosal surface is remarkable for the spatial organization which results in a continuum from mitotically active cells in the crypt to the differentiated cells in the villus and ultimately senescent cells which are lost from villus tips in the small intestine or mucosal surface in the colon. This sequence of events occur in a vertical orientation, from the crypt to the luminal surface. In addition to this process of constitutive epithelial turnover, there appear to be additional distinct and probably complex processes involved in reconstituting mucosal integrity in response to injury. Fundamental features of the pluripotent stem cell compartment and its regulation in the intestinal mucosa are still unknown. A number of factors have been shown to play a role in the regulation of intestinal epithelial proliferation and differentiation, including components of the extracellular matrix,' the mesenchyme* and p~lyamines.~ Most importantly, many studies suggest that several previously identified peptide factors are involved and it is likely that still more remain to be characterized. Each of the peptide factors which appear to play a role in regulating intestinal epithelial cell proliferation are known to have a wide spectrum of activities among different cell types with considerable overlap in biological properties. Each of the peptides can be produced by a variety of cell types within the mucosa including both the epithelial compartment as well the underlying lamina propria. These include transforming growth factor p (TGFP), a Studies carried out in authors' laboratory were supported by grants from the National Institutes of Health (DK41557and P30 DK43351). Corresponding author. 148
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interleukins 1 and 6 and perhaps others. The complexity of the network is further multiplied by their variable range of action. In addition, the final response of individual cells may depend on the aggregate mixture of peptides interacting with receptors on the cell surface. The lamina propria is made up of a complex mixed population of cells including many which play a role in mediating immunological responses. These include B lymphocytes, CD4 and CD8 positive T lymphocytes, macrophages and dendritic cells. A distinct population of T lymphocytes are closely associated with the surface epithelial cells. These intraepithelial lymphocytes express CD8 molecules and, depending on the species, varying proportions also express y/6 rather than the more common T cell surface alp receptor^.^ The mononuclear cells in the intestinal mucosa exhibit considerable flux; a major route of turnover being entry by migration from the circulation and exit via lymphatic drainage. The original source of migrating cells may be Peyer's patches, thymus or the bone marrow. The destination of cells leaving the lamina propria may include rnesenteric lymph nodes or other sites of mucosa-associated lymphoid tissue. Although it has not yet been demonstrated, it is also possible that the intraepithelial lymphocytes migrate into and out of the lamina p r ~ p r i a . ~ , ~ The predominant immunoglobulin produced in the intestinal mucosa is IgA. Interaction between plasma cell secreted IgA (and IgM) and epithelial cells (via secretory component) is essential for transport into the lumen.6 Epithelial cells as appear to be capable of presenting antigens to intraepithelial suggested by expression of MHC class I1 molecules by normal villus epithelial cells'O and by colonic epithelial cells in association with mucosal inflammation. ' I These observations underscore potentially important functional interactions between epithelial and immune cell populations. In addition to interactions with intraepithelial lymphocytes, close association between epithelial cells and macrophages may occur in the dome region of Peyer's patches where antigens are transported through M cells,'* and can be taken up by macrophages located just below.'3 Whether the M cell is in fact a specialized epithelial cell itself is uncertain, though that appears likely. Although direct interactions between epithelial cells and lymphocyte and macrophage populations are probably important, extensive interactions between epithelial cells and cells of the lamina propria may be mediated by soluble peptide factors. Macrophages are recognized to be capable of secreting many regulatory peptidesI4 and together with mucosal lymphocytes (including intraepithelial lymphocytes) may produce a wide range of peptides which could modulate both their own as well as epithelial cell functional properties. These factors include transforming growth factor p, interleukins I , 2, 3, 6, 8 and 10, tumor necrosis factor a, interferon-y, colony stimulating factors and macrophage inflammatory proteins 1 and 2.
Peptide Factors and Intestinal Epithelial Cells Despite continued loss of mature epithelial cells from villus tips and their replacement form the crypts, the integrity of the mucosal barrier and other functions are preserved. It is evident that these processes require precise regulation of proliferation and differentiation. As noted, in addition to peptide factors, components of the extracellular matrix and mesenchyme are also likely to be important. Nontransformed cell lines established from rat small intestinal epithelium and designated IEC-6, IEC-17 and IEC-18 have been useful models to study the contri-
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bution of extracellular matrix and peptide factors in intestinal epithelial cell proliferation and differentiation. These cell lines retain many of the characteristics of crypt ~el1s.l~ It has been reported that the cells can be induced to differentiate into mature epithelial cell types (enterocytes, goblet, endocrine and Paneth cells) by association with fetal rat gut mesenchyme.2In addition, enterocyte differentiation has been reported when IEC-6 cells were grown on a complex extracellular matrix without additional cellular elements. Epidermal growth factor (EGF) a homologue of transforming growth factor a (TGFa) stimulates proliferation of IEC cell lines. In contrast, transforming growth factor p (TGFp; a family of molecules produced by many different cell types which mediates a wide range of biological effects) is a very potent inhibitor of proliferation.16In addition to inhibition of proliferation, TGFpl also induces some features of the differentiated phenotype in IEC cells. Both TGFa and TGFp are produced by the IEC cell lines and their expression appears to be controlled by autocrine mechanisms." Addition of EGF (as a surrogate for TGFa) to subconfluent cells resulted in enhanced expression of TGFa mRNA which peaked at 3-6 hours. Subsequently, TGFa expression declined in parallel with increasing level of expression of TGF& in the IEC cells. In contrast, addition of TGFP to subconfluent IEC cells resulted in both autocrine induction of its own expression and suppression of TGFa expression. These observations suggest that an initial proliferative stimulus is reinforced by autocrine induction of TGFa but is ultimately downregulated through parallel induction of TGFp expression. The physiological relevance of these studies (on the IEC cells) is underscored by the observed expression of TGFa and TGFp mRNA and protein in primary rat intestinal epithelial cells.I8 Expression of EGF could not be demonstrated, suggesting that TGFa may be the physiological ligand for previously described EGF receptors on the basolateral surface of normal intestinal epithelial cells. Interestingly,a gradient of TGFa expression was observed among isolated primary intestinal epithelial cells. Paradoxically, preferential expression was seen in villus cells with progressively lower amounts in the crypts. In contrast, highest levels of TGFP expression were found in the crypts with progressively lower levels in villus cell populations. These gradients of expression may suggest mechanisms for regulation of proliferation but other functions of the peptides cannot be excluded. EGF, and by implication TGFa, can modulate nutrient and electrolyte transport as well as disaccharidase expression in enterocyte~.'~-~' The predominance of TGFP in crypt populations may imply the need for a constitutive brake on proliferation. Recent findings suggest that the insulin family of growth factors which have been shown to be important in mediatinggrowth and differentiation in many tissues may also have a role in the intestinal epithelium. Insulin-like growth factor type I1 (IGF-11) has been previously found to be expressed in late fetal development of the i n t e ~ t i n e .Recent ~ ~ . ~ ~studies in our laboratory, using isolated intestinal epithelial cells and the IEC-6 cell lines suggest that this factor is also present in the mature intestinal epithelium. Studies in the human colon cancer derived cell line CaCo2 showed low level of expression of two IGF-11 transcripts at subconfluence. This expression was enhanced upon conversion to the phenotype of differentiated enterocytes. This supports the concept of increasing expression of IGF-I1 during differentiation of epithelial cells. There is increasing evidence that other molecules in addition to the factors described above may be important in regulating epithelial cell proliferation and preservation of mucosal surface continuity. These include a recently identified family of peptides, designated trefoil proteins, with a distinct pattern of disullide
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bonding which yields a cloverleaf-like secondary structure. Members of this family are resistant to proteolytic degradation and are expressed in the gastrointestinal tract and pancreas as well as the skin of Xenopus laevis. The peptide pS2 is expressed in the normal human stomach and porcine pancreatic spasmolytic polypeptide (PSP; as well as the mouse and human homologs mSP and hSP) in the pancreas. The functions of this family of proteins remain to be characterized. PSP has been shown to have growth factor activity and other members of the family may share the same property.24Our laboratory has recently characterized a new member of this family in the rat intestine and designated it intestinal trefoil factor (ITF).25In the gastrointestinal tract, it is expressed by goblet cells only in the small and large intestine. Other groups have demonstrated increased expression of other trefoil factors near areas of intestinal mucosal injury suggesting that they may play an important role in h e a l i ~ ~ g .However, ~ ~ . ~ ’ the full range of biological properties of this group of proteins remains to be determined. Role of TGFP in Epithelial Injury
Important components of tissue repair following injury include recruitment of macrophages and fibroblasts, production of extracellular matrix and neovascularization. Platelet derived growth factor (PDGF) and TGFP have been shown to be chemoattractants for macrophages and fibroblasts in in vivo models of wound repair. These two peptides also induce deposition of extracellular matrix by fibrob l a s t ~ . TGFP ~ ~ - ~ is~ chemotactic for endothelial cells and induces capillary tube formation.29 It is likely that fibroblast growth factor is also involved in the process of wound repair.3’ Both PDGF and TGFP have been shown to accelerate the rate as well as the strength of healing of incisional wounds in rats.28,32 TGFPl is synthesized as a 390-amino acid precursor with a C-terminal bioactive domain, an N-terminal signal sequence and a 249-amino acid pro-peptide which is g l y c ~ s y l a t e d .The ~ ~ *12.5-kD ~~ mature peptide is obtained after proteolytic cleavage from the pro-region peptide and two of these molecules are joined by disulfide bonds to form the active 25-kD TGFP molecule. TGFP, is normally secreted in a latent form which is formed by the cleaved pro-region glycopeptide remaining associated with the active dimer, most likely by noncovalent interaction^.'^*^' The latent form is biologically inactive and since receptors for TGFP are constitutively expressed by most cell types, bioactivation of the peptide may be an important regulatory step. Activation requires dissociation of the active dimer from the preregion glycopeptide. Latent TGFP can be activated in vitro by agents such as acid and urea. The mechanism of activation in vivo remains to be determined, but it has been proposed that this is mediated by plasmin which cleaves the pro-region glycopeptide with the subsequent change in conformation, leading to release of the active d i ~ n e r . ~ ~ - ” Recently we have examined the role of TGFP in epithelial responses to wounding in vitro using the cell line IEC-6. Wounds were created in confluent monolayers of IEC-6 cells with a razor blade and subsequent cell proliferation and “restitution” assessed. Cell proliferation was observed within 24 hours of creation of wounds but this response was ablated if exogenous TGFP was added to the injured monolayers. However, despite inhibition of proliferation, addition of TGFP enhanced “restitution” by accelerating migration of cells into the wounds. When conditioned media from subconfluent (nonwounded) IEC-6 cells was added to wounded monolayers, bioactive TGFP appeared to be present. TGFP bioactivity in the conditioned media was greater when the IEC-6 cells were pretreated with TGFP. This autocrine
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induction of TGFP is consistent with the studies discussed above. Requirement for proteolytic cleavage of the latent molecule to produce bioactive TGFP was suggested by abolition of TGFP bioactivity by the addition of protease inhibitors during the conditioning period. These studies suggest that the healing of in uiuo epithelial wounds may initially involve TGFP-induced rapid migration of epithelial cells to cover the defect. The TGFP may be produced by the epithelial cells and/or cells within the lamina propria, especially macrophages. This formulation is consistent with in uiuo studies of healing following superficial damage to amphibian and mammalian gastric and colonic mucosa which have shown that initial re-epithelialization occurs by a process known as restitution. In this process, epithelial barrier function is re-established by rapid migration of epithelia1cells shouldering the defect. Histologically, wounds are re-epithelialized with flattened, migrating cells in a process that occurs in less than 6 hours and within 8 minutes in some s t u d i e ~ .These ~ ~ , ~processes ~ occur much more rapidly than could be accounted for by cell proliferation which would take more than 6-12 hours. In support, studies of 3Hthymidine incorporation showed that increased proliferation does not occur during the time period of restit~tion.~' Cytochalasin B (which interferes with microfilament formation) inhibits this process4' in a manner consistent with the proposed role of cell migration in restitution.
Interleukin 2 Response in Epithelial Cells As noted above, several peptide factors produced by macrophagesand lymphocytes (also known as cytokines) are capable of affecting epithelial cell function. Interferon-? has been shown to induce expression of MHC class I1 molecules,42 enhance expression of secretory component43and affect permeability of epithelial cells." Interleukin 145 and tumor necrosis factor a* also influence epithelial cells. It is likely that other factors, which can also be produced by lymphocytes and macrophages in the intestinal mucosa, will affect epithelial cells. Indeed, recent studies in our laboratory suggest that interleukin 2 may have potent functional effects on intestinal epithelial cells. Interleukin 2 (IL2) is a peptide produced by T cells which induces growth and differentiation of lymphocyte^.^^ Studies in lymphoid cells have shown that the IL2 receptor is present in three forms, high-, intermediate- and low-affinity M and M,respectively. These with dissociation constants of lo-'' M, binding affinities are mediated by two polypeptide chains IL2Ra (Tac antigen, p55), which binds IL2 with low affinity, and IL2RP (p70-75) with intermediate affinity. Association of both IL2Ra and IL2RP constitutes the high affinity form of the re~eptor.~' In addition to T cells, IL2 receptors are expressed by activated B cells and m o n ~ c y t e sand ~ ~ also intestinal macrophages in active inflammatory bowel disease.48 In the latter study, a sensitive alkaline-phosphatase anti-alkaline phosphatase (APAAP) technique was used but epithelial cells could not be studied because of endogenous alkaline phosphatase activity which is difficult to block and produces nonspecific staining. Our recent studies have shown that epithelial cells can specifically bind and respond to IL2. Scatchard plot analysis on IEC-6 cells indicates the existence of two distinct populations of specific receptors. Studies on proliferative responses demonstrated that the receptors to IL2 are functional as demonstrated by enhanced expression of TGFP and mac-2.
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Potential Influence of Epithelial Cell-Derived Peptide Factors on Lamina Propria Cells
As noted, a number of peptide factors produced by epithelial cells are known to have significant functional effects on types of cells present in the lamina propria. Most notable among these is TGFP, which has been shown to inhibit T and B cell p r o l i f e r a t i ~ n . In ~ ~addition, .~~ this factor can selectively suppress IgG and IgM production. It is also a potent deactivator of macrophages in picomolar concentrat i o n ~ . ~A' large proportion of intestinal macrophages have been shown to be downregulated in their capacity to produce oxygen radicals,52and it is conceivable that this effect is mediated by TGFP produced by the epithelial cells. Products of activated macrophages can be toxic to surrounding cells, and deactivation of the cells by molecules such as TGFP could be a physiological mechanism for protection against such injury. TGFP is also a potent chemotactic agent for monocytess3and polymorphonuclear leukocyte^^^ and thus is likely to play an important role in recruiting these cell populations into the intestinal mucosa following injury. The elicited monocytes would be capable of producing a variety of peptide factors, including TGFP, which can subsequently influence epithelial cell function. Since redundancy is an important feature of the network of peptide factors, it is likely that a number of molecules are involved in responses to injury and probably a different spectrum of peptides in the subsequent healing process. Interestingly, a number of studies suggest that TGFP may make a significant contribution in all the stages from onset to resolution of injury.55Although the mechanisms involved in these processes are likely to be complex, a model of epithelial lamina propria interaction which can play a role in mucosal response to injury can be outlined. Induction of lymphocyte activation and the subsequent release of IL2 may enhance synthesis of TGFP by the epithelial cells as noted above. The increased TGFP levels may facilitate repair of epithelial wounds as well as elicit monocytes and fibroblasts for more extensive wound repair. It could also provide a feedback mechanism to inhibit T cell proliferation and subsequent production of IL2. REFERENCES 1.
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INTRODUCTION The intestinal mucosa is distinguished by a dynamic population of epithelial cells at its surface which undergoes nearly complete turnover every few days. In adition to the epithelium, the mucosa encompasses an underlying heterogenous population of immune and other cell populations within the lamina propria. A number of observations suggest that dynamic interactions occur between the epithelium and the immune cell populations present in the lamina propria. These interactions may play an important role in both effecting functional modulation of immune responses and restoring epithelial surface integrity following iqjury. The epithelial component of the mucosal surface is remarkable for the spatial organization which results in a continuum from mitotically active cells in the crypt to the differentiated cells in the villus and ultimately senescent cells which are lost from villus tips in the small intestine or mucosal surface in the colon. This sequence of events occur in a vertical orientation, from the crypt to the luminal surface. In addition to this process of constitutive epithelial turnover, there appear to be additional distinct and probably complex processes involved in reconstituting mucosal integrity in response to injury. Fundamental features of the pluripotent stem cell compartment and its regulation in the intestinal mucosa are still unknown. A number of factors have been shown to play a role in the regulation of intestinal epithelial proliferation and differentiation, including components of the extracellular matrix,' the mesenchyme* and p~lyamines.~ Most importantly, many studies suggest that several previously identified peptide factors are involved and it is likely that still more remain to be characterized. Each of the peptide factors which appear to play a role in regulating intestinal epithelial cell proliferation are known to have a wide spectrum of activities among different cell types with considerable overlap in biological properties. Each of the peptides can be produced by a variety of cell types within the mucosa including both the epithelial compartment as well the underlying lamina propria. These include transforming growth factor p (TGFP), a Studies carried out in authors' laboratory were supported by grants from the National Institutes of Health (DK41557and P30 DK43351). Corresponding author. 148
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interleukins 1 and 6 and perhaps others. The complexity of the network is further multiplied by their variable range of action. In addition, the final response of individual cells may depend on the aggregate mixture of peptides interacting with receptors on the cell surface. The lamina propria is made up of a complex mixed population of cells including many which play a role in mediating immunological responses. These include B lymphocytes, CD4 and CD8 positive T lymphocytes, macrophages and dendritic cells. A distinct population of T lymphocytes are closely associated with the surface epithelial cells. These intraepithelial lymphocytes express CD8 molecules and, depending on the species, varying proportions also express y/6 rather than the more common T cell surface alp receptor^.^ The mononuclear cells in the intestinal mucosa exhibit considerable flux; a major route of turnover being entry by migration from the circulation and exit via lymphatic drainage. The original source of migrating cells may be Peyer's patches, thymus or the bone marrow. The destination of cells leaving the lamina propria may include rnesenteric lymph nodes or other sites of mucosa-associated lymphoid tissue. Although it has not yet been demonstrated, it is also possible that the intraepithelial lymphocytes migrate into and out of the lamina p r ~ p r i a . ~ , ~ The predominant immunoglobulin produced in the intestinal mucosa is IgA. Interaction between plasma cell secreted IgA (and IgM) and epithelial cells (via secretory component) is essential for transport into the lumen.6 Epithelial cells as appear to be capable of presenting antigens to intraepithelial suggested by expression of MHC class I1 molecules by normal villus epithelial cells'O and by colonic epithelial cells in association with mucosal inflammation. ' I These observations underscore potentially important functional interactions between epithelial and immune cell populations. In addition to interactions with intraepithelial lymphocytes, close association between epithelial cells and macrophages may occur in the dome region of Peyer's patches where antigens are transported through M cells,'* and can be taken up by macrophages located just below.'3 Whether the M cell is in fact a specialized epithelial cell itself is uncertain, though that appears likely. Although direct interactions between epithelial cells and lymphocyte and macrophage populations are probably important, extensive interactions between epithelial cells and cells of the lamina propria may be mediated by soluble peptide factors. Macrophages are recognized to be capable of secreting many regulatory peptidesI4 and together with mucosal lymphocytes (including intraepithelial lymphocytes) may produce a wide range of peptides which could modulate both their own as well as epithelial cell functional properties. These factors include transforming growth factor p, interleukins I , 2, 3, 6, 8 and 10, tumor necrosis factor a, interferon-y, colony stimulating factors and macrophage inflammatory proteins 1 and 2.
Peptide Factors and Intestinal Epithelial Cells Despite continued loss of mature epithelial cells from villus tips and their replacement form the crypts, the integrity of the mucosal barrier and other functions are preserved. It is evident that these processes require precise regulation of proliferation and differentiation. As noted, in addition to peptide factors, components of the extracellular matrix and mesenchyme are also likely to be important. Nontransformed cell lines established from rat small intestinal epithelium and designated IEC-6, IEC-17 and IEC-18 have been useful models to study the contri-
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bution of extracellular matrix and peptide factors in intestinal epithelial cell proliferation and differentiation. These cell lines retain many of the characteristics of crypt ~el1s.l~ It has been reported that the cells can be induced to differentiate into mature epithelial cell types (enterocytes, goblet, endocrine and Paneth cells) by association with fetal rat gut mesenchyme.2In addition, enterocyte differentiation has been reported when IEC-6 cells were grown on a complex extracellular matrix without additional cellular elements. Epidermal growth factor (EGF) a homologue of transforming growth factor a (TGFa) stimulates proliferation of IEC cell lines. In contrast, transforming growth factor p (TGFp; a family of molecules produced by many different cell types which mediates a wide range of biological effects) is a very potent inhibitor of proliferation.16In addition to inhibition of proliferation, TGFpl also induces some features of the differentiated phenotype in IEC cells. Both TGFa and TGFp are produced by the IEC cell lines and their expression appears to be controlled by autocrine mechanisms." Addition of EGF (as a surrogate for TGFa) to subconfluent cells resulted in enhanced expression of TGFa mRNA which peaked at 3-6 hours. Subsequently, TGFa expression declined in parallel with increasing level of expression of TGF& in the IEC cells. In contrast, addition of TGFP to subconfluent IEC cells resulted in both autocrine induction of its own expression and suppression of TGFa expression. These observations suggest that an initial proliferative stimulus is reinforced by autocrine induction of TGFa but is ultimately downregulated through parallel induction of TGFp expression. The physiological relevance of these studies (on the IEC cells) is underscored by the observed expression of TGFa and TGFp mRNA and protein in primary rat intestinal epithelial cells.I8 Expression of EGF could not be demonstrated, suggesting that TGFa may be the physiological ligand for previously described EGF receptors on the basolateral surface of normal intestinal epithelial cells. Interestingly,a gradient of TGFa expression was observed among isolated primary intestinal epithelial cells. Paradoxically, preferential expression was seen in villus cells with progressively lower amounts in the crypts. In contrast, highest levels of TGFP expression were found in the crypts with progressively lower levels in villus cell populations. These gradients of expression may suggest mechanisms for regulation of proliferation but other functions of the peptides cannot be excluded. EGF, and by implication TGFa, can modulate nutrient and electrolyte transport as well as disaccharidase expression in enterocyte~.'~-~' The predominance of TGFP in crypt populations may imply the need for a constitutive brake on proliferation. Recent findings suggest that the insulin family of growth factors which have been shown to be important in mediatinggrowth and differentiation in many tissues may also have a role in the intestinal epithelium. Insulin-like growth factor type I1 (IGF-11) has been previously found to be expressed in late fetal development of the i n t e ~ t i n e .Recent ~ ~ . ~ ~studies in our laboratory, using isolated intestinal epithelial cells and the IEC-6 cell lines suggest that this factor is also present in the mature intestinal epithelium. Studies in the human colon cancer derived cell line CaCo2 showed low level of expression of two IGF-11 transcripts at subconfluence. This expression was enhanced upon conversion to the phenotype of differentiated enterocytes. This supports the concept of increasing expression of IGF-I1 during differentiation of epithelial cells. There is increasing evidence that other molecules in addition to the factors described above may be important in regulating epithelial cell proliferation and preservation of mucosal surface continuity. These include a recently identified family of peptides, designated trefoil proteins, with a distinct pattern of disullide
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bonding which yields a cloverleaf-like secondary structure. Members of this family are resistant to proteolytic degradation and are expressed in the gastrointestinal tract and pancreas as well as the skin of Xenopus laevis. The peptide pS2 is expressed in the normal human stomach and porcine pancreatic spasmolytic polypeptide (PSP; as well as the mouse and human homologs mSP and hSP) in the pancreas. The functions of this family of proteins remain to be characterized. PSP has been shown to have growth factor activity and other members of the family may share the same property.24Our laboratory has recently characterized a new member of this family in the rat intestine and designated it intestinal trefoil factor (ITF).25In the gastrointestinal tract, it is expressed by goblet cells only in the small and large intestine. Other groups have demonstrated increased expression of other trefoil factors near areas of intestinal mucosal injury suggesting that they may play an important role in h e a l i ~ ~ g .However, ~ ~ . ~ ’ the full range of biological properties of this group of proteins remains to be determined. Role of TGFP in Epithelial Injury
Important components of tissue repair following injury include recruitment of macrophages and fibroblasts, production of extracellular matrix and neovascularization. Platelet derived growth factor (PDGF) and TGFP have been shown to be chemoattractants for macrophages and fibroblasts in in vivo models of wound repair. These two peptides also induce deposition of extracellular matrix by fibrob l a s t ~ . TGFP ~ ~ - ~ is~ chemotactic for endothelial cells and induces capillary tube formation.29 It is likely that fibroblast growth factor is also involved in the process of wound repair.3’ Both PDGF and TGFP have been shown to accelerate the rate as well as the strength of healing of incisional wounds in rats.28,32 TGFPl is synthesized as a 390-amino acid precursor with a C-terminal bioactive domain, an N-terminal signal sequence and a 249-amino acid pro-peptide which is g l y c ~ s y l a t e d .The ~ ~ *12.5-kD ~~ mature peptide is obtained after proteolytic cleavage from the pro-region peptide and two of these molecules are joined by disulfide bonds to form the active 25-kD TGFP molecule. TGFP, is normally secreted in a latent form which is formed by the cleaved pro-region glycopeptide remaining associated with the active dimer, most likely by noncovalent interaction^.'^*^' The latent form is biologically inactive and since receptors for TGFP are constitutively expressed by most cell types, bioactivation of the peptide may be an important regulatory step. Activation requires dissociation of the active dimer from the preregion glycopeptide. Latent TGFP can be activated in vitro by agents such as acid and urea. The mechanism of activation in vivo remains to be determined, but it has been proposed that this is mediated by plasmin which cleaves the pro-region glycopeptide with the subsequent change in conformation, leading to release of the active d i ~ n e r . ~ ~ - ” Recently we have examined the role of TGFP in epithelial responses to wounding in vitro using the cell line IEC-6. Wounds were created in confluent monolayers of IEC-6 cells with a razor blade and subsequent cell proliferation and “restitution” assessed. Cell proliferation was observed within 24 hours of creation of wounds but this response was ablated if exogenous TGFP was added to the injured monolayers. However, despite inhibition of proliferation, addition of TGFP enhanced “restitution” by accelerating migration of cells into the wounds. When conditioned media from subconfluent (nonwounded) IEC-6 cells was added to wounded monolayers, bioactive TGFP appeared to be present. TGFP bioactivity in the conditioned media was greater when the IEC-6 cells were pretreated with TGFP. This autocrine
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induction of TGFP is consistent with the studies discussed above. Requirement for proteolytic cleavage of the latent molecule to produce bioactive TGFP was suggested by abolition of TGFP bioactivity by the addition of protease inhibitors during the conditioning period. These studies suggest that the healing of in uiuo epithelial wounds may initially involve TGFP-induced rapid migration of epithelial cells to cover the defect. The TGFP may be produced by the epithelial cells and/or cells within the lamina propria, especially macrophages. This formulation is consistent with in uiuo studies of healing following superficial damage to amphibian and mammalian gastric and colonic mucosa which have shown that initial re-epithelialization occurs by a process known as restitution. In this process, epithelial barrier function is re-established by rapid migration of epithelia1cells shouldering the defect. Histologically, wounds are re-epithelialized with flattened, migrating cells in a process that occurs in less than 6 hours and within 8 minutes in some s t u d i e ~ .These ~ ~ , ~processes ~ occur much more rapidly than could be accounted for by cell proliferation which would take more than 6-12 hours. In support, studies of 3Hthymidine incorporation showed that increased proliferation does not occur during the time period of restit~tion.~' Cytochalasin B (which interferes with microfilament formation) inhibits this process4' in a manner consistent with the proposed role of cell migration in restitution.
Interleukin 2 Response in Epithelial Cells As noted above, several peptide factors produced by macrophagesand lymphocytes (also known as cytokines) are capable of affecting epithelial cell function. Interferon-? has been shown to induce expression of MHC class I1 molecules,42 enhance expression of secretory component43and affect permeability of epithelial cells." Interleukin 145 and tumor necrosis factor a* also influence epithelial cells. It is likely that other factors, which can also be produced by lymphocytes and macrophages in the intestinal mucosa, will affect epithelial cells. Indeed, recent studies in our laboratory suggest that interleukin 2 may have potent functional effects on intestinal epithelial cells. Interleukin 2 (IL2) is a peptide produced by T cells which induces growth and differentiation of lymphocyte^.^^ Studies in lymphoid cells have shown that the IL2 receptor is present in three forms, high-, intermediate- and low-affinity M and M,respectively. These with dissociation constants of lo-'' M, binding affinities are mediated by two polypeptide chains IL2Ra (Tac antigen, p55), which binds IL2 with low affinity, and IL2RP (p70-75) with intermediate affinity. Association of both IL2Ra and IL2RP constitutes the high affinity form of the re~eptor.~' In addition to T cells, IL2 receptors are expressed by activated B cells and m o n ~ c y t e sand ~ ~ also intestinal macrophages in active inflammatory bowel disease.48 In the latter study, a sensitive alkaline-phosphatase anti-alkaline phosphatase (APAAP) technique was used but epithelial cells could not be studied because of endogenous alkaline phosphatase activity which is difficult to block and produces nonspecific staining. Our recent studies have shown that epithelial cells can specifically bind and respond to IL2. Scatchard plot analysis on IEC-6 cells indicates the existence of two distinct populations of specific receptors. Studies on proliferative responses demonstrated that the receptors to IL2 are functional as demonstrated by enhanced expression of TGFP and mac-2.
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Potential Influence of Epithelial Cell-Derived Peptide Factors on Lamina Propria Cells
As noted, a number of peptide factors produced by epithelial cells are known to have significant functional effects on types of cells present in the lamina propria. Most notable among these is TGFP, which has been shown to inhibit T and B cell p r o l i f e r a t i ~ n . In ~ ~addition, .~~ this factor can selectively suppress IgG and IgM production. It is also a potent deactivator of macrophages in picomolar concentrat i o n ~ . ~A' large proportion of intestinal macrophages have been shown to be downregulated in their capacity to produce oxygen radicals,52and it is conceivable that this effect is mediated by TGFP produced by the epithelial cells. Products of activated macrophages can be toxic to surrounding cells, and deactivation of the cells by molecules such as TGFP could be a physiological mechanism for protection against such injury. TGFP is also a potent chemotactic agent for monocytess3and polymorphonuclear leukocyte^^^ and thus is likely to play an important role in recruiting these cell populations into the intestinal mucosa following injury. The elicited monocytes would be capable of producing a variety of peptide factors, including TGFP, which can subsequently influence epithelial cell function. Since redundancy is an important feature of the network of peptide factors, it is likely that a number of molecules are involved in responses to injury and probably a different spectrum of peptides in the subsequent healing process. Interestingly, a number of studies suggest that TGFP may make a significant contribution in all the stages from onset to resolution of injury.55Although the mechanisms involved in these processes are likely to be complex, a model of epithelial lamina propria interaction which can play a role in mucosal response to injury can be outlined. Induction of lymphocyte activation and the subsequent release of IL2 may enhance synthesis of TGFP by the epithelial cells as noted above. The increased TGFP levels may facilitate repair of epithelial wounds as well as elicit monocytes and fibroblasts for more extensive wound repair. It could also provide a feedback mechanism to inhibit T cell proliferation and subsequent production of IL2. REFERENCES 1.
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