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Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

Gut, 1989, 30, 1630-1640 Occasional report Growth and transformation of the small intestinal mucosa - importance of connective tissue, gut associated lymphoid tissue and gastrointestinal regulatory

E 0 RIECKEN, A STALLMACH, M ZEITZ, J D SCHULZKE, H MENGE, AND M GREGOR

(This article is one ofa series linked with the Festschriftfor Christopher Booth. See Gut Festschrift 1989; 30.) Physiologically, the architecture of the small rather to focus on areas of our own interest in the intestinal mucosa is maintained by a delicate balance subject. between cell production in the crypt compartment, One of the prerequisites of the striking small migration along the villi, and extrusion of intestinal mucosal potential to change its structure mature epithelial cells from the tip of the villi into the and function is the fact that each villus possesses .' Migration and maturation of the lining cells numerous crypts with an enormous proliferative are affected in response to various types of stress and potential. The average number of crypts per villus in under conditions of mucosal damage and repair. This upper amounts to 8&3 per villus.' In is also true for the tissue components beneath the man, 23 of 33 cell positions of the lower part of the lining cells. On the other hand, although the equi- crypt cell column belong to the proliferative compart- librium of the lining cells may be disturbed by a great ment, whereas the upper third represents the matura- variety of various types of stress, the response of the tion area in which the crypt cells obtain their mucosa is rather uniform and restricted to three functional competence.7 The crypt is able to enlarge http://gut.bmj.com/ distinct mucosal patterns only - namely, hyper- and to decrease its size in diameter and depth as a trophy, hypotrophy, and mucosal transformation of consequence of changes in its proliferative capacity. the hyperregenerative type (Fig. 1).' In these In this respect, it is of interest that the crypts of mucosal patterns alterations of the absorptive cells and the zonation of villi and crypts have been studied extensively. The regulation of cell proliferation and differentiation in this system is, however, still not well understood although the importance of some on September 24, 2021 by guest. Protected copyright. factors such as luminal nutrition and the ornithine decarboxylase system have been recognised.'4 Furthermore, the role of regulatory peptides has attracted much attention, though with few concrete results, and the implications of the interaction Mucosal between the lining cells, the mesenchyme, and the hypertrophy gut associated lymphoid tissue with respect to cell proliferation and maturation have only recently Mucosal Normal gained interest.' atroph ie mucosa It is the purpose of this contribution to briefly describe the three principal mucosal responses to various types of stress and also to discuss some aspects of the connective tissue, the gut associated tissue and the in the lymphoid regulatory peptides Mucosal control of intestinal mucosal growth and their transformation of the perspectives in future research as far as the regulation hyperregenerative type of mucosal growth is concerned. It is not intended, Fig. 1 Principalforms into which the small intestinal however, to be complete within the frame given, but mucosa may be transformed. 1630 Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

Growth and transformation ofthe small intestinal mucosa 1631

Functional c compartment 0 4._ cc c ._.a1)

L- c 0)0 4._ Proliferative CO compartment

Fig. 2 Sclheme ofmigration and differentiation ofepithielial cells and subepithlelial myofibroblasts along tlie crypt-lillus axis in tle adult . Left panel sliows postulated interrelationslip between stem cells and connective tissues into differ-ent surface cells. the small intestine are surrounded by a prominent diameter, whereby the number of crypts per mucosal sheath of fibroblasts within the of the area is reduced. Concomitantly, there is a propor- http://gut.bmj.com/ bowel Fig. 2. It has been assumed that in the adult tional increase in villus size, which again leads to a small intestine, this specialised mesenchymal cell reduction in the number of villi per unit area. Thus, type, the 'pericryptal myofibroblast' undergoes a there is a largely proportional enlargement of villi rapid turnover similar to that of the epithelial cell.' and crypts which contain an increased number of Synchronous division and parallel migration of lining cells, which may undergo distinct changes in epithelial and subepithelial mesenchymal cells may size and function."1' Thus, the number of cells per

form a 'unit' of both cell populations. Zajicek has unit length may increase,' and microvilli of absorp- on September 24, 2021 by guest. Protected copyright. proposed the concept of an 'intestinal proliferon', tive cells after intestinal resection are shortened.'3 In consisting of epithelial, mesenchymal, neural, and addition, specific brush border activities vascular elements, in which a turnover of the differ- have been shown to be decreased and in v,itro ent cell populations is closely coordinated.' Other absorption of non- in the hyperplastic findings, however, indicate that the division rate of mucosa is reduced. These findings have led to pericryptal myofibroblasts is lower than the division the concept that there are functionally immature rate of epithelial cells, some 3H-thymidine labelled after intestinal resection, which is in myofibroblasts remain at the basis of the crypt for a accordance with the fact that cell migration time after long time and also become polyploid."' intestinal resection is increased and the cell life span of the enterocytes is shortened. None the less, Mucosal hypertrophy Menge et al could show that in the hypertrophic mucosa brush border vesicle transport was not Mucosal hypertrophy with hyperplasia of the cells is altered after proximal resection, as judged by studies one condition in which the crypt cell compartment of the accumulation of D-.'4 Furthermore, has altered its geometry. The model in which this when using more sophisticated enzymatic in situ mucosal pattern can be studied best is the ileal studies, a differential enzyme response has been remnant after proximal small intestinal resection in observed at the cellular level'3: a significant decrease the animal." The mitotic activity increases first and is in both Vmax and Km values of neutral a-glucosidase followed by an increase in crypt depth and in crypt were already expressed four days after resection and Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

1632 Riecken, Stallmach, Zeitz, Schulzke, Menge, and Gregor confined to the apical villus while no changes in the accumulation studies also remains unaltered.'4 Thus, kinetics of the /4-glucosidase occurred. in mucosal atrophy as in mucosal hypertrophy, the Similarly, Chavez et al showed selective increments initial step of non- absorption is not in enzyme expression in ileal enterocytes after dependent on the proliferative state of the mucosa, proximal small .'` Therefore, a thereby signalling that the absorptive function of the complex pattern of cellular enzymatic and morpho- individual enterocyte remains unaltered. logical adaptation to proximal resection ensues in Similar changes of the mucosa as in the bypassed the otherwise rather uniform pattern of hyperplastic intestine do occur in longterm parenteral nutrition" ileal mucosa with increased zonation of villi and and in the starved animal in which diameter and crypts.`' 7 depth of crypts decrease, as does eventually the Because in the hypertrophic mucosa the absorptive number of villi and crypts. Mucosal atrophy may be capacity is mainly restricted to the upper third of the altered further in a number of conditions such as villi as in the normal intestine the main increase in cytostatic treatment and irradiation, the damage function per centimetre intestine is achieved by an thereby depending on dose, time course, and action increase in diameter of the small intestine.'8 This has of the drug.' 7 been experimentally proven: a 45% proximal small intestinal resection leads to an increase in villus Mucosal transformation height, mucosal circumference and glucose absorp- tion in vivo when the substrate disappearance rate is The third mucosal pattern which represents the most expressed in terms of unit serosal length. A 70% striking alteration of the mucosal geometry is resection induces a further increase in mucosal .mucosal transformation of the hyper-regenerative circumference and glucose absorption, while villus type'. It is characterised by hyperproliferation height remains unchanged.'` and mucosal growth as is the case in mucosal Intestinal mucosal hyperplasia in man largely hypertrophy. It differs, however, from mucosal corresponds to the findings in the experimental hypertrophy in that it is characterised by the trans- animal, although it has been investigated less syste- formation of the normal zonation with reduced villi matically and less completely. Booth and coworkers and enlarged crypts, the reason why we have chosen had already shown in 1961 that the functional reserve this term.'8 In addition, it is associated with damage after proximal intestinal resection is so effective that to the surface cells and the underlying connective

is not a problem even if 250 cm of tissue. A suitable model to study this form of mucosal http://gut.bmj.com/ intestine have been removed." In this context two response to stress is the self-filling small intestinal additional phenomena have been recognised to be blind loop in the experimental animal in which stasis, important in man. First, the intestinal remnant may bacterial overgrowth and increased concentrations of also grow in length after resection and thereby toxic deconjugated acids are the stressors to the enforce function of mucosal hyperplasia.' Second, surface . In response to these damaging the full adaptive response may require a long period agents increased exfoliation of damaged surface cells to develop, according to studies in occurs which is followed by hyperproliferation of up to 12 months.22 crypt cells in the germinative zone.'7 In longterm on September 24, 2021 by guest. Protected copyright. lactic acid perfusion studies we have shown that the Mucosal atrophy initial step of this mucosal transformation corres- ponds largely to mucosal hypertrophy in which there Mucosal atrophy is the second pattern of the small is a proportional enlargement of villi and crypts with intestine in which the geometry of the mucosa is increased mitotic activity.'8 As damage continues and distinctly altered and which has been thoroughly cell exfoliation surmounts cell proliferation, mucosal explored in Thiry-Vella fistulae to bypass the passage transformation is taking place. The villi become of ingesta and in selfemptying intestinal blind shorter and the crypts become longer. In this process loops.-3'2 Its main features are a reduction of villus the totally flattened surface with complete loss of height, crypt depth, mitotic activity, and diameter of villi, maximal elongation of crypts and high numbers the small intestine while the single enterocyte does of mitotic cells in the germinative compartment not usually exhibit features of regression. Hence, the represents an endpoint of mucosal transformation. fine structure of the absorptive cells is not altered, In contrast with mucosal hypertrophy and hypo- brush border enzyme activities are not reduced, and trophy, the surface cells in the severely transformed reduced absorptive function correlates roughly with mucosa exhibit clear cut features of damage: they are the reduction in absorptive surface. Brush border flattened, the fine structure is severely altered with vesicle transport of absorptive cells in the atrophic irregular and sparse microvilli, increased lysosomal intestine as judged by dependent D-glucose bodies and activated endoplasmatic reticulum. Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

Growth and transformation ofthe small intestinal mucosa 1633

Functionally, there is a reduction of brush border differentiation of intestinal epithelial cells. Thus, enzyme activities and sodium dependent D-glucose electronmicroscopical analysis of the developing gut accumulation in brush border vesicles is distinctly in the rat documents that the process of maturation of decreased." According to kinetic analysis in short the epithelium starts after the development of close term incubation experiments, this decrease is the cell-cell contacts between surface and subepithelial result of a reduction in the VM,,x indicating that the cells of the mesenchyme.'4 In addition, experiments number of transport in the brush border with recombined tissues grafted under the kidney of absorptive cells in the transformed mucosa is capsule of adult rats have confirmed the existence reduced. of strong inductive influences of the intestinal Electrical measurements in the experimental blind mesenchyme on the epithelium of the and loop of rat jejunum provided an increased epithelial , which lead to a differentiated intestinal resistance, which was paralleled on freeze fracture epithelium with regular microvilli and typical electron microscopy by an increase in total tight intestinal brush border .3' Furtheron, junctional depth (more pronounced in cells of the myofibroblasts of the duodenal mucosa of sucklings crypt than in the villus), and by an increase in tight rats can induce 'intestinalisation' of undifferenti- junctional strand counts in one of four regions along ated gastric epithelial cells.' These results are of the villus axis, which showed the most leaky junction, particular interest in view of the known instability of namely in the villus tip.` These changes have been the gastric epithelial phenotype in intestinal meta- interpreted as an adaptive response to reduce back plasia.3s Previous in tvitro results from our own leakage of already absorbed electrolytes across laboratory also suggested a strong specific the tight junction. In from patients with potential of the fetal intestinal mesenchyme in untreated coeliac sprue, lateral aberrant strands inducing intestinal epithelial cytodifferentiaton.' appeared in surface cells, a change which resembles In the intestinal tract itself the mesenchyme exerts that seen in the experimental .?' a strong morphogenetic induction on intestinal In addition, strand discontinuities and a more leaky epithelial cytodifferentiation, including cell lineage tight junction (decreased strand counts) were into absorptive, goblet, endocrine, and Paneth cells. observed in coeliac sprue.`' Both alterations, how- Using mouse aggregation chimaeras as an experi- ever, may be attributed to the higher degree of hyper- mental model, Ponder et al demonstrated immuno- regenerative transformation, as blind loops showed histologically that the epithelium of individual a in crypts in the small intestine is derived from a single only small reduction villus height, while coeliac http://gut.bmj.com/ sprue patients display flat mucosa. In spite of this progenitor cell. This single progenitor cell may be structural and functional impairment of the absorp- divided into several stem cells responsible for cell tive cells glucose absorption correlates closely with renewal and differentiation .`" Intestinal epithelial the decrease in mucosal surface when measured by cell lines, established from fetal rat intestine and segmental perfusion studies in man using the triple resembling in some features progenitor crypt cells, lumen tube technique.7 do not show self differentiation in vitro. Neither The clinical significance of mucosal transformation , cytokines, regulatory peptides nor of the hyper-regenerative type is considerable, and luminal factors induce features of differentiation of on September 24, 2021 by guest. Protected copyright. there are many conditions in which it is seen, its most these cells. When combined with fetal intestinal pronounced example being coeliac sprue in which mesenchyme, however, these primitive cells will most of the studies in man have been performed. actually form villous structures as well as , Wright et all' have calculated from a stathmokinetic endocrine, and Paneth cells.4' model that the cell production rate in the flat jejunum To gain further insight into the role of the intestinal of coeliacs is increased up to 155 cells per crypt subepithelial mesenchyme in the instruction of and hour, which is six times higher than normal while intestinal progenitor cells by distinct mesenchymal duration of mitosis remains constant and cell cycle cell populations, coculture studies with purified and time is reduced from 50 to 20 hours. These figures well characterised mesenchymal cells, that is, myo- show the enormous proliferative potential of the fibroblasts, neural and endocrine cells, vascular cells, mucosa and also the importance of its regulation. cells of the mucosal , and primary fetal intestinal epithelial cells are necessary. Regulation of mucosal growth in relation to In addition to diffusable molecules and the trans- connective tissue mission of signals through cell-cell contacts the possible role of the extracellular matrix in the Although the connective tissue has not attracted epithelial-mesenchymal interactions has been much attention in this respect there is increasing stressed by Grobstein.4' The epithelial mesenchymal evidence that it is important for proliferation and interface in the intestinal mucosa implies the Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

1634 Riecken, Stallmach, Zeitz, Schiulzke, Menge, and Gregor epithelium, the subepithelial basement membrane, the crypt-villus axis. This includes also the analysis and adjacent reticular lamina propria. Basement of their receptor in the normal membranes are ubiquitous extracellular matrices small intestine and in the different forms of formed from a unique set of macromolecules, mucosal transformation in response to stress and including laminin, collagen type IV, nidogen, damage. heparan sulfate proteoglycan, and fibronectin.4 During intestinal morphogenesis, the deposition of Mucosal growth and its relation to the gut associated specific extracellular matrix components at the lymphoid system epithelial-mesenchymal interface and the formation of the intact basement membrane have been found to The possible role of the gut associated lymphoid be associated with the cytodifferentiation of the tissue in the regulation of mucosal growth is derived .44 It has recently been docu- mainly from two pieces of evidence. First, several mented that the individual basement membrane immune mediated diseases of the gastrointestinal components have different modifying effects on tract are accompanied by changes in the architecture intestinal epithelial cell , cell proliferation, of the intestinal mucosa (reviewed in 46). It is and cytodifferentiation. Laminin and nidogen especi- therefore likely that local immune reactions might ally promoted cytodifferentiation of fetal intestinal directly influence intestinal epithelial growth hereby epithelial cells, whereas cell adhesion of intestinal leading to mucosal transformation. Second, recent epithelial cells was strongly promoted by fibronectin observations suggest that cytokines derived from (own results, submitted for publication). lymphoid cells are also important regulatory factors In addition to results on the importance of interac- for the growth and differentiation of other cells of tions between the mesenchyme, the extracellular either epithelial or mesenchymal origin (reviewed in matrix and the intestinal epithelium in epithelial cell 47). These factors therefore may be of importance in differentiation during organogenesis, some data the interaction between lymphoid cells and other cell indicate that the extracellular matrix plays a crucial types in the mucosa. With respect to these observa- role in intestinal disease. The restitution of epithelial tions we will first consider some characteristics of gut defects during healing of erosions and ulcers in the associated which may be of importance is bound to an intact basement for their interaction with intestinal epithelial cells. membrane on which non-injured, viable epithelial Second, recent studies will be discussed which cells proliferate and migrate from the border of the indicate that mucosal transformation is at least in part lesions.' mediated by immune mechanisms. http://gut.bmj.com/ Proliferation and adhesion of malignant intestinal epithelial cells may be influenced by cell-surface CHARACTI ERISTIICS OF MUCOSAL LYMPHOCYTIES receptors to basement membrane components (own T cells in the lamina propria, the effector com- results, submitted for publication). These cell- partment of the intestinal mucosa, have certain surface receptors mediate specific interactions of characteristics, which distinguish them from other neoplastic cells with the extracellular matrix and may populations in the body.4` Lamina

facilitate tumour invasion and metastasis. propria T lymphocytes are predominantly of the on September 24, 2021 by guest. Protected copyright. Preliminary results on the immunohistological helper phenotype and lack the CD45R antigen on characterisation of the basement membrane in their cell surface and therefore have the phenotype of patients with untreated with mucosal memory T cells.4 They express certain activation transformation of the hyper-regenerative type did antigens and are able to synthesise high amounts of not reveal, however, a pathological distribution and interleukin 2.'" After stimulation with antigen, these organisation of basement membrane components as T cells do not proliferate, but provide helper function compared with the mucosa of normal intestine (U for B cells.>' These T cells therefore can be character- Hahn, personal communication). ised as differentiated effector cells which respond to The process of intestinal epithelial proliferation antigen stimulation with the production of certain and cell migration along the crypt-villus axis which cytokines. Consequently, this population of T cells includes maturation and differentiation of the may be of special importance in the interaction with epithelium has been shown to follow distinct regula- epithelial cells and therefore in the maintenance of tions and we are only beginning to understand some the mucosal structure. of its molecular aspects. Of importance in this respect are detailed investigations to identify extracellular IMMUNIE ME,DIAIID MUCOSAI, matrix producing cells, to analyse their TIRANSFORMATION matrix protein metabolism and to characterise matrix Prototypes of experimental models, in which protein receptors on intestinal epithelial cells along mucosal transformation is thought to be primarily Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

Growth and transformation ofthe small intestinal muicosa 1635 mediated by immunological mechanisms, are small unless tissue is pre-exposed to gliadin in vi'ho. The intestinal allograft rejection and graft-versus-host activation of an endogeneous mechanism of toxicity reaction (GvHR) of the small bowel."'4 The thus has to be postulated before gliadin may exert its rejection of grafts of fetal intestine transplanted damaging effect. This endogeneous mechanism most heterotopically under the kidney capsule of mice is likely consists of an immunological reaction. Coeliac characterised first by infiltration of the mucosa by disease therefore most likely is another example of lymphocytes and increased mitotic activity in the immune mediated mucosal transformation of the epithelial cells in the crypts, later by flattening of the hyper-regenerative type. villi and exfoliation of surface enterocytes, and finally Of importance in this context is the finding that by destruction of the mucosa.' Several experiments patients with coeliac disease may become refractory have shown that intestinal allograft rejection to a free diet after developing an intestinal represents a local cell mediated immune reaction.5' Iymphoma. Intestinal lymphomas in coeliac disease By using a GvHR model in mice Ferguson and have recently been shown to be T cell lymphomas coworkers further investigated the hypothesis that most likely arising from IEL.`' ' In addition, these mucosal transformation occurs by direct effect of T lymphomas express certain activation antigens. The cells on the crypts rather than through damage of the activated lymphoma T cells therefore may mediate villi. The results of these experiments showed that the toxic effect on the intestinal epithelium by crypt length and crypt cell production rate increased producing certain cytokins leading to a refractory parallel to the development of GvHR; villus length sprue syndrome. was not reduced, and the number of IEL increased More direct evidence that activated mucosal T cells significantly.`'' Additional investigations by this are able to induce mucosal transformation of the group` and by Elson and coworkers'" provided sprue type is given in a recent study by MacDonald indirect evidence that the effects on the crypts in and Spencer.`3 These authors used explants from GvHR were caused by soluble T cell products rather human fetal intestine in organ culture. These than by direct T cell cytotoxicity. intestinal explants were then cultured with certain The characteristic mucosal collagen deposition in substances (pokeweed mitogen and anti-CD3 mono- chronic GvH disease of the intestine may also be clonal antibody) to activate mucosal T cells. Parallel mediated by T cell derived cytokines: supernatants to the appearance of activated T cells in the lamina from clones of T cells from mice with acute GvHR propria (measured by the expression of interleukin-2 caused fibroblasts to proliferate in vitro, and super- receptors) an increase in proliferating crypt natant of chronic GvH disease clones stimulated an epithelial cells and a reduction in villus height http://gut.bmj.com/ increase in collagen production per fibroblast.7"" occurred. Addition of cyclosporin A, a potent These experiments strengthen the hypothesis that inhibitor of T cell activation, abrogated the effects on T cell derived cytokines play a central role in mucosal transformation. These experiments show the interaction between the different cellular that activation of lamina propria T cells induces components in mucosal transformation. hyper-regenerative transformation of the small The mucosal transformation seen in allograft intestinal mucosa. rejection and intestinal GvHR resembles hyper- In the acquired immunodeficiency syndrome on September 24, 2021 by guest. Protected copyright. regenerative transformation. As discussed above (AIDS) gastrointestinal manifestations are very fre- mucosal transformation of the hyper-regenerative quent and clinical studies suggest the occurrence of type is a hallmark of coeliac disease and the question small intestinal dysfunction.' As mentioned above, if coeliac disease is caused by direct damage of intestinal lamina propria T cells are predominantly of the mucosal epithelium by gliadin or by immune the helper phenotype and are more activated than T mediated mechanisms is not yet clarified. Strong cells in other sites. These characteristics render these support for this hypothesis is provided by organ cells highly susceptible to HIV-. In fact, culture studies of intestinal biopsies from coeliac HIV-infected lamina propria mononuclear cells disease patients: biopsies from patients with active have been shown. In addition, there are indications disease under a gliadin containing diet and biopsies of small intestinal atrophy with reduced mitotic rate from patients with 'inactive' disease - that is, normal in the crypts in patients with AIDS, especially in appearing mucosa - under a gluten free diet were patients with mucosal HIV-infected mononuclear cultured in vitro with and without gliadin. A toxic cells (own results, submitted for publication). It is effect of gliadin on mucosal morphology and brush possible that infection of regulatory mucosal T cells border enzyme activity was only observed on biopsies by HIV with concurrent lymphocyte dysfunction is of patients with active disease." These studies responsible for the atrophic mucosal changes. indicate that tissue of coeliac disease patients does Mucosal HIV-infection might therefore be an not manifest gliadin induced tissue injury in vitro example for an immune mediated alteration which Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

1636 Riecken, Stallmach, Zeitz, Schli1dke, Menge, and Gregor leads to mucosal atrophy, although the proof for this after various stimuli."'"" An hypothesis is lacking. secreting tumour was associated with increased When summarising these different findings it can height of the villi and increased mucosal thickness be stated that, in spite of the lack of systematic which was reversed after tumour removal.` Subse- studies on the different patterns of intestinal mucosal quently, raised circulating concentrations of entero- transformation, several experimental models and glucagon have been reported in various clinical clinical findings indicate an influence of the local conditions and experimental animal models being lymphoid tissue on mucosal structure and function. associated with an increase in the intestinal epithelial Specialised regulatory T cells in the intestinal lamina cell turnover which all had in common that they propria may influence epithelial growth and differen- were characterised by an increased food supply to the tiation as well as connective tissue metabolism by the intestine.5 Especially the adaptive hyperplastic production of cytokines. So far the evidence for this response of ileal remnants after proximal bowel interaction is indirect, however, and to dissect the resection are paralleled by an increase in plasma and mechanisms of immune mediated mucosal transfor- tissue concentrations of enteroglucagon both show- mation, better experimental models are needed. ing a close relationship to the extent of the small Special emphasis should be given to the characterisa- intestine being resected." 94 tion of cytokines produced by intestinal T cells which In order to gain additional insight into the putative may be responsible for mucosal transformation. role gut GLI-I of acting as an enterotrophic regula- tory we investigated two different experi- Regulation of mucosal growth in relation to mental conditions which are known to induce gastrointestinal regulatory peptides intestinal hyperplasia not being associated with an increase in the nutritional load of the intestine: In vitro and in vivo studies have provided consider- (a) germ free rats were conventionalised with a able information on the possible physiological thermoduric flora and were held thereafter as open function of circulating gastrointestinal hormones as conventional animals. Four weeks after conven- well as locally acting regulatory peptides in the tionalisation the ileal mucosa exhibited a significant multifactorial control of adaptive gastrointestinal increase of mitotic activity and crypt length accom- epithelial cell proliferation and cell renewal.15-71 At panied by unchanged basal EG plasma concentra- the present time these studies suggest that tions and decreased tissue levels of EG in the blind may act as a growth promoting factor in the stomach contaminated group. (b) Jejunal self-filling http://gut.bmj.com/ and possibly also, although less likely, in the small loops were surgically created in germ free rats. Three intestine and colon.7'-77 Furthermore, it has been weeks postoperatively there was a significant shown that (EGF), mainly increase in mitotic activity, crypt length and villus produced in the salivary and Brunner's glands surface area in the jejunal self-filling blind loops of the of man and the rat, may have local when compared to unoperated germ free control and systemic proliferative effects on the intestinal rats. Again the basal plasma EG concentrations epithelium.78 Whereas bombesin and growth hor- remained unchanged (submitted for publication). mone releasing factor (somatocrinin) have been These two particular experiments show that there on September 24, 2021 by guest. Protected copyright. reported to stimulate intestinal cell proliferation, are situations in which intestinal hyperplasia does not may act as a growth inhibiting factor in correspond to an increase in the concentrations of the gastrointestinal tract.79"4 EG in plasma or intestinal mucosa. This implies that Enteroglucagon (EG) (gut glucagonlike immuno- in conditions which are not or only to a limited extent reactivity I; gut GLI-I) is a peptide of 69 amino acids accompanied by an increase in luminal nutrition which has been shown to be part of a larger pre-pro- other factors than EG have to be responsible for the glucagon molecule being produced by so-called regulation of the hyperplastic adaptation of the small L-cells located predominantly in the distal small intestinal mucosa. intestine and colon.` 8 It contains the entire In a further experiment we investigated the effects sequence of pancreatic glucagon linked by two pairs of longterm in *iiio immunoneutralisation of endo- of basic , lysin-arginin, at its N-terminus to genous circulating gut GLI-I by intravenous and a glicentin-related pancreatic peptide of 30 amino continuous intraperitoneal administration of newly acids (GRPP) and at its C-terminal end to a hexapep- developed monoclonal antibodies to gut GLI-I on the tide."78x The physiological role of gut GLI-I has not hyperplastic ileal response of the ileal remnant after a yet been fully elucidated. It has been suggested by 70% proximal small bowel resection.) A group of circumstantial evidences that gut GLI-I may act as a rats was given either antibody free plasmocytoma trophic factor on the intestinal mucosa which may ascites or undiluted hybridoma ascites for 14 days account for adaptive changes of the small intestine after the operation. The hybridoma ascites was Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

Growth and transformation ofthe small intestinal matcosa 1637 prepared from the clone 23.6B4 synthesising a mono- References clonal antibody directed toward the N-terminal to the central region of the glucagon molecule which repre- 1 Clarke RM. Morphological description of intestinal sents the immunoreactant common to all gut GLIs adaptation: measurements and their meaning. In: and glucagon in different species such as man, rat, Dowling RH, Riecken EO, eds. Intestinal adaptation. Stuttgart: Schattauer Verlag, 1974: 10-20. and pig." The three dimensional architecture and 2 Riecken EO. Dcrangemcnts of mucosal growth and the proliferative activity of the ileal remnant were differentiation in small-intestinal diseases. Scand J evaluated two weeks postoperatively. Despite a Gastroenterol 1988X 23 [suppl 1511: 86-93. continuous immunoneutralisation of circulating 3 Dowling RH. The influencc of luminal nutrition on endogenous gut GLI-I by monoclonal antibodies intestinal adaptation after small bowel resection and by- there was an adaptive response of the ileal remnants pass. In: Dowling RH, Riccken EO, cds. Intestinial which was of the same magnitude as in the control adaptation. Stuttgart: Schattaucr Verlag, 1974: 35-45. group but was even greater considering the increased 4 Luk GD, Baylin SB. Ornithine decarboxylase in number of mitoses per crypt. These data did not intestinal maturation, recovery and adaptation. In: Robinson JWL, Dowling RH, Riecken EO, eds. support the hypothesis of gut GLI-I being a circulat- Mechanisms ofintestinial adaptationi. Lancaster, Boston, ing enterotrophic regulatory peptide. The Hague: MTP Press, 1982: 65-78. An in vitro model was used to investigate the effect 5 Bloom SR, Polak JM. Entcroglucagon and the gut of highly purified rat G-GLI I on the proliferative profile of intestinal adaptation. In: Robinson response of primary small intestinal epithelial cells of JWL, Dowling RH, Riecken EO, eds. Mechanisms of fetal rats (submitted for publication). Gut GLI was intestinal adaptation. Lancastcr, Boston, The Hague: purified from rat small intestines by gel filtration, MTP-Prcss, 1982: 189-98. high capacity immunoaffinity chromatography using 6 Riecken EO, Sahlfeld M, Lorcnz-Meycr H. Quantita- monoclonal antibodies and reverse phase HPLC."7 tive Untcrsuchungen zur drcidimensionalen Struktur Fetal rat intestinal epithelial cells were dissected der Dunndarmschicimhaut bei Gcsunden und Patienten mit cinheimischer Sprue. Dtsch Med Wochenschr 1976; from the surrounding mesenchyma after collagenase 101: 51-3. treatment and seeded onto petri dishes previously 7 Ecknaucr R, Rommel K. Zytostatika und Dunndarm. coated with a complex basement membrane extract Klin Wochenschr 1978: 56: 579-92. (submitted for publication). After the initial attach- 8 Parker FG, Barnes KGI. The pericryptal fibroblast ment phase, the proliferation rate was assessed sheath. IV. Replication migration and differentiation of

planimetrically, correlating with the mitotic index, the subepithelial fibroblasts of the crypts and villus of the http://gut.bmj.com/ before and 48 h after the addition of highly purified rabbit jcjunum. 1974; 67: 622-35. G-GLI I in various concentrations as well as EGF. 9 Zajikzek G. The intestinal proliferon. J Tlheor Biol Whereas there was a well known growth promoting 1977; 67: 515-21. action of EGF the proliferation of rat fetal intestinal 10 Neal JV, Potten CS. A critical asscssment of the epithelial cells was inhibited by the addition of intestinal proliferation hypothesis. J Tlheoir Biol 1981; purified gut GLI-I. 91: 63-75. These results indicate that gut GLI-I does not act 11 Menge H, Hopert R, Alexopoulus 1, Riecken EO. as an enterotrophic factor but provide the first direct Three-dimensional structurc and cell kinetics at on September 24, 2021 by guest. Protected copyright. evidence different sites of the early adaptivc response to consistent with an antitrophic role of gut resection. Res Exp Med (Berl) 1982; 181: 77-94. GLI-I in the small intestine. They do not, however, 12 Weser E, Hernandez MH. Studies of small bowel rule out a paracrine enterotrophic action of gut GLI-I adaptation after intestinal resection in the rat. Gastro- in iivio controlling cell proliferation by a synergistic enterology 1971; 60: 69-75. interaction among gut GLI-I and other still unknown 13 Zeitz M, Menge H, Riecken EO. Early ultrastructural growth factors. Confirmation of our studies must adaptive changes of ileal enterocytes after proximal await the isolation and purification of sufficient small bowel resection as detcrmined morphometrically. quantities of gut GLI-I for in vi'ho infusion experi- Res Exp Med 1985; 185: 259-68. ments. 14 Menge H, Murer H, Robinson JWL. Glucose transport by brush-border membrane vesicles after proximal resection or ileo-jejunal transposition in the rat. Abteilungfur Innere Medizin, J Physiol (Lond) 1978; 274: 9-16. 15 Gutschmidt S, Kaul W, Menge H, Riecken EO. The mit Schwerpunkt Gastroenterologie, adaptive response of activities at Klinikum Steglitz der Freien different sites along the villus epithelium after proximal Univ'ersitdt, intestinal resection in the rat. A microdensitometric Hindenburgdamm 30, study. Res Exp Med (Berl) 1983; 182: 203-13. D-1000 Berlin-45, 16 Chaves M, Smith MW, Williamson RCN. Incrcased Fed Rep Germany activity of digestive enzymes in ileal cnterocytes Gut: first published as 10.1136/gut.30.11.1630 on 1 November 1989. Downloaded from

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