Journal of Cell Science 102, 427-436 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

427

Differential expression of the VLA family of integrins along the crypt-villus

axis in the human small intestine

JEAN-FRANgOIS BEAULIEU

Croupe de recherche en biologie du developpement, Departement d'anatomie et de biologie cellulaire, Faculte de medecine, Universite deSherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

Summary

Regulation of epithelial cell proliferation, migration anddifferentiation under physiological conditions remainspoorly understood. Interaction of the cells with theirunderlying basement membrane through integrins, aspecific subset of cell surface binding proteins, is onepotential mechanism. In the present work, I examinedthis hypothesis by investigating the distribution of avariety of epithelial basement membrane proteins andthe expression of the members of the VLA family ofintegrins in the adult intestinal epithelium. Indeed, thisrapidly renewing simple epithelium contains within itsfunctional unit, the crypt-villus axis, essentially twodistinct cell populations: the proliferative and undiffer-entiated crypt cells and the mature enterocytes on thevillas. Although immunolocalization of basement mem-brane molecules revealed that laminin, type IV collagenand heparan sulfate proteoglycan are distributed homo-geneously all along the crypt-villus axis, other non-exclusive basement membrane components were founddifferentially expressed. Tenascin was concentrated atthe base of both villus and lower crypt cells while cellularfibronectin was mostly detected in association with thecrypt cells. Moreover, VLA /?i as well as 5 of the 6 VLAa subunits tested were expressed by intestinal epithelial

cells under specific patterns of staining. The Pi and a(,subunits were strongly detected at the base of allenterocytes while as, also detected all along the crypt-villus axis, was weaker and consistently appeared with apunctated/interrupted pattern. On the other hand, theVLA of], a2 and a3 were expressed at the basolateraldomains of enterocytes under distinctive crypt-villusgradients. The ax subunit was detected at the base of allepithelial cells but lateral staining was only observed indifferentiating cells (middle and upper crypt). Finally, inmost specimens, a2 and a3 displayed strictly comp-lementary staining patterns for the lower crypt region(a2 + , a3—) and the upper crypt-to-villus region (or2-,a}+). Taken together, these data emphasize thatproliferation, migration and differentiation in thenormal state are susceptible to various influencesincluding compositional changes in the basement mem-brane and differential expression of receptors for thesecomponents.

Key words: integrins, VLA, intestinal cell, differentiation,migration, extracellular matrix, basement membrane,human.

Introduction

Cell adhesion and migration are fundamental processesfor the organization and the maintenance of tissueintegrity (reviewed by Edelman, 1985,1992; Rodriguez-Boulan and Nelson, 1989). During the past few years,major advances in our understanding of the molecularmechanisms of these cellular processes have occurred,with the identification and the characterization ofnumerous extracellular matrix (ECM) molecules(reviewed by Timpl, 1989; Yurchenko and Schittny,1990) as well as several of their cell-matrix adhesionmolecules (reviewed by Hynes, 1987; Juliano, 1987;Ruoslahti, 1988; Akiyama et al., 1990; Hemler et al.,1990).

The VLA family of integrins is an important class ofcell surface receptors involved in cell-matrix interac-

tions (Buck and Horwitz, 1987). As most othermembers of the superfamily of integrins, VLAs (for"very late activation" antigen) are transmembrane a-/5non-covalently linked heterodimers (Hynes, 1987). Sofar, at least 11 a subunits and seven /? subunits havebeen identified, which are associated to form 15 distinctheterodimers (Hemler et al., 1990; Ruoslahti, 1991). Atleast six of them belong to the VLA family (VLA 1 to 6)and are characterized by having distinct a subunits(a-j.6) that share a common /3j subunit. Ligands forVLAs have been identified as ECM molecules; namely,laminin (VLA 1,2,3 and 6), collagen I and/or IV (VLA1, 2 and 3), and fibronectin (VLA 4 and 5) (recentlyreviewed by Humphries, 1990; Ruoslahti, 1991). Thelist is however still incomplete. For example, theintegrin that binds to tenascin has not been yetidentified (Friedlander et al., 1988) while the ligands

428 J.-F. Beaulieu

for other newly identified VLAs remain to be deter-mined (Kajiji et al., 1989; Holzmann and Weissman,1989). The molecular basis for integrin-mediated cellu-lar events is not yet clearly understood but, as identifiedfrom in vitro studies, it probably involves direct linksbetween the ECM and the cytoskeleton (Burridge etal., 1990). Moreover, the functional significance of acytoplasmic /Jt tyrosine phosphorylation site remains tobe established (Tamkun et al., 1986; Hayashi et al.,1990).

Experiments with a variety of cultured normal andtumor cells have provided evidence that the members ofthe VLA family of integrins, in concert with theirrespective ECM ligands, participate in fundamentalcellular processes such as adhesion, spreading, mi-gration, proliferation and differentiation. Studies onintact tissues and organs have also provided strongindications that integrins are involved in cell-matrixinteractions, acting as cell-specific receptors for base-ment membrane components. For example, in thehuman kidney the glomerular basement membrane isrecognized by different integrins in different cell types(Korhonen et al., 1990). Similarly, there are differencesin the integrin repertoire found on large vessels andcapillary endothelia (Albelda et al., 1989). Further-more, in the epidermis, the expression of severalintegrins appears closely related to the state of celldifferentiation, suggesting a role for these molecules inthe migration of terminally differentiating keratino-cytes from the basal epidermal layer (Kajiji et al., 1989;Tamura et al., 1990; Quaranta, 1990; Nicholson andWatt, 1991; Sonnenberg et al., 1991).

The aim of the present work was to obtain more basic-information on VLAs in epithelial-basement membraneinteractions and on their relationship to cell prolifer-ation, differentiation, maturation and, ultimately, sen-escence. The adult intestinal epithelium appears to bean attractive system for such a purpose. Its continuouscell renewal consists of spatially separated stem cells,proliferative and differentiated cell compartments,located, respectively, in the lower regions of the cryptsand on the villi (Bjerknes and Cheng, 1981; Leblond,1981). The molecular basis of the dynamic process ofcell production in the basal region of the crypts and oftheir migration along the basement membrane to the tipof the villi where they are extruded into the lumenremains to be established but, undoubtedly, requirescarefully controlled modulation of cell-matrix ad-hesion. Until very recently, much of the effort has beenconcentrated on the analysis of the distribution ofbasement membrane components; it revealed that mostof these constituents are distributed homogeneously allalong the crypt-villus axis (see Discussion). Herein, toget further information on the molecular basis underly-ing the mechanism of migration of epithelial cells fromthe crypt to the villus, the patterns of expression forVLA I to 6 were determined by indirect immunofluor-escence and analyzed in concert with the presence ofvarious basement membrane and interstitial ECMcomponents along the crypt-villus axis of the adulthuman small intestine. The data demonstrate that five

of the six VLA a subunits tested are expressed byintestinal epithelial cells under specific patterns, whichsupports the hypothesis that these molecules areinvolved in the control of migration and/or proliferationand differentiation of enterocytes.

Materials and methods

Primary antibodiesThe antibodies used for the immunolocalization of the VLAfamily of integrins were: the monoclonals mAb 13 and mAb16 directed against the fix and the 05 subunits, respectively(Akiyama et al., 1989; generously provided by Dr. Steven K.Akiyama); A-1A5, TS2/7 and B-5G10 directed against the fa(Hemler et al., 1983); the a{ and the a4 subunits (Hemler etal., 1987), respectively, kindly supplied by Dr. Martin E.Hemler; GoH3 directed against the a6 subunit (Sonnenberget al., 1988; Serotec, Cedarlane Laboratories, Hornby, Ont.);and P1E6 and P1B5 recognizing the a2 and the tx3 subunits,respectively (Wayner and Carter, 1987; obtained fromOncogene Science, Uniondale, NY). Type IV collagen,laminin, basement membrane heparan sulfate proteoglycan,tenascin and cellular fibronectin were immunolocalized withmono- and polyclonal antibodies as described previously(Beaulieu et al., 1991). The mature form of sucrase-isomaltase was detected with the monoclonal antibody HSI-5(Beaulieu et al., 1989).

TissuesTwelve samples of small intestine (3 jejunums and 9 ileums)were studied. Only tissues obtained rapidly (in less than 30min) from non-diseased parts of resected segments were usedin the present study. The normality of the tissue wasconfirmed by histology. Specimens were obtained from adultpatients with either inflammatory bowel disease or bowelobstruction, according to the protocol approved by theInstitutional Human Research Review Committee.

Indirect immunofluorescenceThe preparation and OCT (optimum cutting temperatureembedding compound; Miles Tek, Elkhart, IN, USA)embedding of specimens for cryosectioning was performed aspreviously described (Beaulieu et al., 1990). Sections 4 ,umthick were spread on gelatin-coated glass slides and air-driedfor 1 hour at room temperature before storage at —70°C.

Detection of the various antigens on cryosections byindirect immunofluorescence was performed as describedpreviously (Beaulieu et al., 1991). Fluorescein-conjugatedgoat anti-mouse IgG (Boehringer Mannheim Canada, Laval,Quebec), anti-rat IgG (Caltag, Cedarlane Laboratories,Hornby, Ont.) and anti-rabbit IgG (Boehringer MannheimCanada, Laval, Quebec) were used as secondary antibodies,at a working dilution of 1:25. After staining, preparationswere mounted in glycerol/PBS (9:1, v/v) containing 0.1%paraphenylene diamine and then viewed with a LeitzOrthoplan microscope equipped for epifluorescence underimmersion oil with xlO and X25 objectives. In all cases, nofluorescent staining was observed when the primary antibodyhad been omitted.

Results

Intestinal epithelial cell differentiationAlthough all specimens were histologically normal,evidence that a typical differentiation gradient was

Integrin expression in the small intestine 429

Fig. 1. Representative indirect immunofluorescence micrographs of basement membrane (A,B) and interstitial matrix (C-F)components in transverse sections of human adult ileum. Upper half portion of villi (A,C,E) and corresponding cryptregions (B,D,F). Antibodies used were anti-human type IV collagen antiserum (A,B), anti-human tenascin antiserum(C,D), and FN-3E2, a monoclonal antibody directed to human cellular fibronectin (E,F). LP, lamina propria; mm,muscularis mucosa; arrowheads in E and F denote the epithelial basement membrane; v and c in D denote the feet of thevilli and the crypts, respectively. Bar, 50 /m\.

displayed along the crypt-villus axis was obtained bydetermining the site of expression for mature sucrase-isomaltase with the monoclonal antibody HSI-5. Asshown previously (Beaulieu et al., 1989), this antibodyexclusively recognizes the form of sucrase-isomaltaseexpressed by differentiated villus cells.

ECM component expressionThe expression and distribution of various basement

membrane and interstitial ECM molecules were exam-ined in relation to epithelial cell differentiation alongthe crypt-villus axis of the small intestine. As summar-ized in Fig. 1, all the antibodies directed to basementmembrane molecules such as those to type IV collagen,laminin and heparan sulfate proteoglycan producedcontinuous fluorescent staining at the base of intestinalepithelial cells from the bottom of the crypts to the apexof the villi. Staining for type IV collagen and laminin

430 J. F. Beaulieu

was also found in fibrillar and cellular elements of thelamina propria and in the muscularis mucosa (Fig. 1A-B) while heparan sulfate proteoglycan was not found inthese locations (not shown).

Staining patterns displayed for tenascin and cellularfibronectin were also analyzed in relation to theorganization of the intestinal mucosal architecture.Tenascin expression was found in both the crypt and thevillus regions but with distinct patterns of labeling (Fig.1C-D). A bright staining for tenascin was detected inthe connective tissue surrounding the lower part of thecrypt (Fig. ID) and a clear gradient of decreasingintensity was observed toward the neck of the gland. Inthe villus, strong immunofluorescent staining wasobserved, which was mostly confined to the base ofepithelial cells from the foot to the tip of the villi (Fig.1C). Specific, but less-intense staining, was also ob-served in isolated muscle cells of the entire laminapropria as well as in the muscularis mucosa but notaround blood vessels (confirmed by double stainingwith anti-tenascin and anti-smooth-muscle a-actin anti-bodies; not shown). Immunolocalization of cellularfibronectin revealed some particularities in the distri-bution along the crypt-villus axis for another interstitialECM molecule (Fig. 1E-F). Specific antibodies to thismolecule stained all the elements of the lamina propriasurrounding the crypts including muscle cells, and theinterface between the connective tissue and the epi-thelial cells lining both the crypts and the bottom of thevilli (Fig. IF). Staining in the upper part of the villi wasfound to be restricted to a few isolated muscle cells andas a punctate labeling at the base of the differentiatedvillus cells (Fig. IE).

VLA expression and localizationTo examine the possible role of integrins in the adhesiveinteraction of enterocytes with their underlying base-ment membrane, frozen sections of normal adultintestine were stained with mAb 13 and A-1A5, whichboth recognize the human integrin pt polypeptide. Asshown in Fig. 2, the integrin /Jj chain was primarilylocalized at the basolateral aspect of intestinal epithelialcells of the villus and the crypt. Staining was alsoevident in the lamina propria, at the level of cellularelements, in the muscularis mucosa, as well as in theouter muscle cell layers (longitudinal and circular layersof the muscularis propria; not shown).

To explore further the distribution of the & integrins,I then examined a set of normal small intestinalspecimens with a series of subunit-specific monoclonalanti-VLA integrin antibodies. Five out of the six asubunits tested were found to be expressed undervarious patterns by intestinal epithelial cells. Subunit a4was not detected at the basolateral aspect of theepithelium (Fig. 3A-B). However, because of a strongstaining of pericryptal cellular elements (such as thosethat express tenascin (Fig. ID) and smooth muscle aactin (not shown)) with the anti-a-4 subunit antibody,some basal expression of this molecule by the epithelialcells of the crypt can not be ruled out (Fig. 3B). TwoVLA subunits (a5 and o )̂ were found expressed along

Fig. 2. Representative indirect immunofluorescencemicrograph of transverse section of human adult ileumstained for the detection of p{ integrins with the mAb 13monoclonal antibody. Staining is evident at the basolateralaspect of epithelial cells on the villus (v) and in the crypt(c), and in the muscle cells of the lamina propria (LP) andof the muscularis mucosa (MM). Bar, 100 urn.

the entire crypt-villus axis. Antibody GoH3 directed to(% stained intensively the basolateral surface of entero-cytes on the villus and in the crypt (Fig. 3C-D) whilemAb 16, specific for a5, stained the basal aspect of cryptand villus epithelial cells according to a faint punc-tate/interrupted pattern (Fig. 3E-F). Integrin subunits<*!, a2 and a3 were distributed according to a specificgradient along the crypt-villus axis (Fig. 4). AntibodyTS2/7 to ai stained evenly and with a good intensity thebasolateral aspect of epithelial cells located in themiddle and upper parts of the crypt (Fig. 4B) while thestaining in the lower part of the crypt and on the villuswas weaker and more restricted to the basal margin ofthe epithelium (Fig. 4A-B). Subunit a2, specificallyrecognized by the antibody P1E6, was found confinedto the crypt epithelium. Basolateral aspects of the cellswere strongly stained from the bottom to the middlepart of the crypt but a clear gradient of decreasingintensity was observed from the middle to the upperparts of the crypt (Fig. 4D); the entire villus epitheliumwas negative (Fig. 4C). In contrast, staining for the a3subunit with the P1B5 antibody revealed that the

Integrin expression in the small intestine 431

Fig. 3. Representative indirect immunofiuorescence micrographs of transverse sections of human adult ileum stained for thedetection of the 0-4 (A,B), &6 (C,D) and a5 (E,F) subunits of integrins. Subunit o-4 was mainly detected in close associationwith muscle cells in the lamina propria of the villi (A) and around the crypts (B) as well as in the muscularis mucosa(mm;B). On the contrary, the o^ subunit was exclusively found at the basolateral aspect of epithelial cells lining the villus(C) and in the crypts (D). The 05 subunit was found at the basal aspect of both villus (E) and crypt (D) epithelial cellsaccording to a faint punctate/interrupted pattern and in muscle cells of the lamina propria and muscularis mucosa. Arrowsin A,C,E and arrowheads in B,D,F denote basal surfaces of villus epithelial cells and crypt cells, respectively. Bar, 50 /zm.

molecule was only expressed by enterocytes lying in theupper part of the crypt and the entire villus (Fig. 4E-F).The complementary patterns of staining for a2 and a3along the crypt-villus axis were striking.

Expression of integrin subunits was also observed inclose association with muscle cells in the laminapropria, muscularis mucosa and the outer muscularispropria. As summarized in Table 1, smooth muscle cellsfrom all sites express evenly ft, a^ and a5. Staining fora4 was also found widespread but much weaker in theouter muscular layers. The <*2 and <x$ subunits were notdetected in muscle cells in any location. Finally, theexpression of the a3 subunit was found to be restricted

to only particular areas of the muscularis mucosa andmuscularis propria: as shown in Fig. 4F, muscle cells inthe muscularis mucosa were predominantly stained onthe mucosal side, the lower (submucosal) side remainedonly weakly stained; similarly, the staining for a3 in theouter muscle layers was strictly limited to the submuco-sal aspect of the circular layer (not shown).

Discussion

The intestine has been proven to be a valuable modelfor the study of cell growth and differentiation. This

432 J.-F. Beaulieu

Fig. 4. Representative indirect immunofluorescence micrographs of transverse sections of human adult ileum stained for thedetection of the ai (A,B), a2 (C,D), and a3 (E,F) subunits of integrins. Subunit a-, was detected at the base of the villusepithelium (A), at the basolateral aspect of crypt cells (B), and in muscle cells of the lamina propria and the muscularismucosa. Subunit <x2

w a s found restricted to the basolateral and the apical surfaces of the lower crypt epithelial cells (C,D),while, on the contrary, subunit a3 was only expressed at the basolateral surface of villus (E) and upper crypt epithelialcells (F) and on the mucosal side of the muscularis mucosa (mm). Arrows in A,C,E and arrowheads in B,D,F denote basalsurfaces of villus epithelial cells and crypt cells, respectively. Bar, 50 /an.

Table 1. Distribution of VLA 1 to 6 in smooth muscle cells at various locations in the human small intestine

Lamina propria ++ + + — + + —Muscularis mucosa +4- +• - +/— + ++ -Muscularis propria ++ + - +/— ± ++ -

+ + to - : intensity of staining: bright (++) , good (+), faint (±), or no (—) staining detected at the basal surface of intestinal epithelialcells.

+ / - : specific regions stained while others negative.

rapidly renewing simple epithelium contains within itsfunctional unit, the crypt-villus axis, essentially twodistinct cell populations, the proliferative or undifferen-tiated crypt cells and the mature enterocytes of thevillus. Indeed, the fully differentiated villus cell has

typical morphological and functional properties thateasily distinguish it from the crypt cell (Weiser, 1973;Altmann, 1976; Baylin et al., 1978; Leblond, 1981;Quaroni and Isselbacher, 1985; Raul and von derDecken, 1985). How cells move out of the crypt and

Integrin expression in the small intestine 433

migrate onto the villus and what the molecularmechanisms are that prompt epithelial cells to stopproliferation and undertake differentiation still remainsto be elucidated. In the present work we haveapproached these basic questions by studying thedistribution of extracellular matrix proteins and theirreceptors along the crypt-villus axis in the adult humanintestinal mucosa.

ECM components, namely those present at thebasement membrane, are important macromoleculesfor tissue organization and maintenance (Timpl, 1989;Yurchenko and Schittny, 1990). There is also accumu-lating evidence that these components play a pivotalrole in epithelial polarization and differentiation in thedeveloping small intestine. Such involvement of ECMmolecules was first suggested by the observation ofchanges in their deposition and distribution in develop-ing rodents (Simon-Assmann et al., 1986; Aufderheideand Ekblom, 1988) and human intestine (Beaulieu etal., 1991), and is strongly supported by in vitro studiesdemonstrating their necessity to allow cell differen-tiation (Dauca et al., 1990; Hahn et al., 1990; Simon-Assmann et al., 1990). In this study, in the maturehuman intestine, immunolocalization of basementmembrane molecules revealed that laminin, type IVcollagen and heparan sulfate proteoglycan are distrib-uted homogeneously all along the crypt-villus axis in theepithelial basement membrane. This observation,which is in accordance with previous work performed inthe rodent small intestine (Laurie et al., 1982; Abra-hamson and Caulfield, 1985; Simon-Assmann et al.,1986; Senior et al., 1988; Alho and Underhill, 1989),may suggest that basement membrane macromoleculessupport intestinal cell migration and differentiation butdo not govern them. Several arguments plead againstthe latter assumption. First, the recent finding that thelaminin A chain is preferentially located in thebasement membrane of intestinal crypts while B chainsare found all over the crypt-villus axis raises thepossibility of a specific involvement for a basementmembrane component in the process of cell growthand/or polarization (Simo et al., 1991). Second, asexemplified in the present work by the immunolocaliz-ation of tenascin and cellular fibronectin, some ECMcomponents must also be considered in the crypt-villusaxis gradient of basement membrane components.Indeed, both macromolecules were identified in thebasal lamina of intestinal epithelial cells (Laurie et al.,1982; Probstmeier et al., 1990) and therefore may play arole in intestinal cell adhesion/shedding (Quaroni et al.,1978; Probstmeier et al., 1990). It is also noteworthythat such a preferential distribution of tenascin at thebase of both villus and lower crypt cells and of cellularfibronectin in association with crypt cells is alreadyestablished by mid-gestation in the human fetal intes-tine (Beaulieu et al., 1991; Jutras and Beaulieu,unpublished). Third, some monoclonal antibodies topresumed basement membrane components are knownto exhibit distinct staining patterns along the crypt-villus axis (Cleutjens et al., 1989; Millane et al.,unpublished), suggesting that other uncharacterized

basement membrane components and/or unusual sub-units may also participate in the establishment of thegradient. Taken together, these observations suggestthat compositional changes in the basement membranealong the crypt villus-axis play a role in the control ofepithelial cell migration and/or differentiation.

As demonstrated herein by the immunolocalizationstudy of the members of the VLA family of integrins,differential expression of cell-surface receptors forbasement membrane components must also be con-sidered among the potential regulatory mechanisms ofthe dynamic process of the enterocyte in the crypt-villusunit. Indeed, five out of the six VLA a'subunits testedwere found to be expressed by intestinal epithelial cellsunder specific patterns of staining. Only the a6 subunitwas, as observed for the ft subunit, present at the baseof all enterocytes. This is in accordance with previousarticles reporting widespread expression of this specificlaminin receptor (Sonnenberg et al., 1988) in intestinaland colonic epithelial cells both in vivo (Choy et al.,1990; Koretz et al., 1991) and in vitro (Schreiner et al.,1991; Lotz et al., 1990). In contrast, the a4 subunit,which seems to be important in immunological eventssuch as T cell activation (Hemler, 1988), was notdetected in direct association with enterocytes. How-ever, a4 being strongly expressed in cellular elementslying just beneath the epithelium, a basal expression ofthis subunit by enterocytes cannot be ruled out at thistime. Although expressed only at a low level, thefibronectin receptor (05ft complex) was consistentlydetected at the base of enterocytes from both the villusand the crypt in a punctate pattern. In general, VLA 5(osft) is poorly expressed in most tissues includingepithelia (Toda et al., 1987; Wayner and Carter, 1988;Albelda et al., 1989). However, as reported recently(Guo et al., 1991), the expression of the 05ft complexat the basal surface of migratory epithelial cells may beof functional importance. Moreover, it is worth empha-sizing the close similarity in staining patterns for cellularfibronectin and the 05 subunit (compare Fig. 1E,F withFig. 3E,F) in the context of the existence of a potentialrelationship between the expression of the fibronectinreceptor and its ligand (reviewed by Akiyama et al.,1990).

Another interesting finding arising from the presentset of observations is the apparent gradient of ex-pression along the crypt-villus axis for the threemultispecific VLAs, a'i(ft), ^ ( f t ) and a3(fli), whichindeed may bind to various components present in theintestinal basement membrane such as type IV col-lagen, laminin or fibronectin (Humphries, 1990; Ruos-lahti, 1991). In three tissue samples of the smallintestine, Choy et al. (1990) reported a widespreaddistribution of a2 and a3 along the crypt-villus axissimilar to that observed for a6, while a^ was concen-trated in the crypt cells. On the other hand, Koretz etal. (1991) reported recently that the VLA-ar2 chain wasrather preferentially associated with the cryptic glands.In our study, we found that in most cases, <x2 and a'3displayed complementary staining patterns for thelower crypt (a2+, a3—) and the upper crypt-villus

434 J.-F. Beaulieu

regions (a2—, a3+). On a few occasions, staining forthe a2 subunit extended beyond the basal half of thecrypt but the a3 subunit was always poorly expressed inthis latter region. Interestingly, the cr2ft complex, butnot the 03/?!, was recently reported to be involved incolon carcinoma cell adhesion to type I collagen andlaminin (Lotz et al., 1990). The ligand for the ar3ftcomplex in the intestinal epithelium remains to bedetermined but, since colon carcinoma cell adhesion tofibronectin is very poor, even though these cells expresssignificant amounts of the a3 and ft subunits (but nota5; Lotzetal., 1990; Schreineret al., 1991), fibronectin,like laminin and type-I collagen, appears to be a poorcandidate. Although it was not in the main focus of thepresent work, it is germane that all integrin subunitsexpressed in the intestinal epithelium were alsodetected in proportional amounts in the lateral (at, a2,a3, a5, <x6 and ft) and even apical (a2 and a4) domainsof the epithelial cells (see Figs 2-4). This agrees withprevious reports (De Strooper et al., 1989; Pignatelli etal., 1990; Virtanen et al., 1990; Koretz et al., 1991),which may suggest some involvement for these mol-ecules not only in cell-matrix but also in cell-cellinteractions as suggested recently (Kaufmann et al.,1989; Larjava et al., 1990). In this regard, the pattern ofexpression for the ax subunit is intriguing, since on onehand the molecule was found widely expressed at thebase of epithelial cells all along the crypt-villus axis,while on the other hand staining of the lateral domain,which was strong in the middle and upper parts of thecrypt, appeared much weaker or absent in the upperhalf of the villi as well as in the bottom of the crypts.The significance of such a differential pattern of stainingfor this putative type IV collagen (Vandenberg et al.,1991) and/or laminin receptor (Humphries, 1990;Ruoslahti, 1991) is unknown, but could be related to thecell state, since weak lateral staining for ax wasobserved in sites where the epithelium was most fullymature (i.e. on the villus, and also at the base of thecrypt where the down-migrating Paneth cells concen-trate; Bjerknes and Cheng, 1981; Leblond, 1981).

Finally, diversity among integrin subunit expressionin a variety of tissues (see, for example, Albelda et al.,1990; Virtanen et al., 1990) is exemplified by musclecells of the intestinal wall that, although these are allderived from the embryonic intestinal mesenchyme,express various integrins according to their locationand, therefore also, to their functions. It is pertinent tonote that in the intestine, muscle cells possess abasement membrane of similar composition to that ofepithelial cells (Simon-Assmann et al., 1986; Simo etal., 1991; Beaulieu, unpublished data) but, as clearlyshown herein, express a quite different set of integrins.

In conclusion, these data taken together stress thepotential complexity of epithelial cell-matrix interac-tions in a relatively simple system such as the intestinalepithelium. It now appears clear that proliferation,migration and differentiation are processes susceptibleto various influences, including compositional changesin the basement membrane and differential expressionof receptors for these components. What controls these

changes along the crypt-villus axis? It is hoped thatfurther in vivo as well as in vitro studies will help toanswer this question, which is crucial for our under-standing of the mechanisms underlying cell differen-tiation under normal as well as pathological conditions.

The author thanks Drs. Steven K. Akiyama, Martin E.Hemler and Andrea Quaroni for their generous donations ofantibodies, and Dr. Jacques Poisson from the Department ofGeneral Surgery and the staff of the Department of Pathologyof the CHU of Sherbrooke for their cooperation in providingspecimens for this study. I thank F. Racicot and D. Morin fortechnical assistance and F. E. Herring-Gillam for reviewingthe manuscript. This work was supported by grants from theMRC of Canada (MT-11289), The Cancer Research SocietyInc. and the Canadian Foundation for Ileitis and Colitis.J.F.B. is a "Chercheur boursier du Fonds de la recherche ensante du Quebec".

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(Received 22 January 1992 - Accepted 26 March 1992)