Gut 1996; 39: 262-266

The epidermal growth factor receptor (EGF-R) ispresent on the basolateral, but not the apical,surface of enterocytes in the humangastrointestinal tract

R J Playford, A M Hanby, S Gschmeissner, L P Peiffer, N A Wright, T McGarrity

AbstractBackground-While it is clear that lumi-nal epidermal growth factor (EGF) stimu-lates repair of the damaged bowel, itssignificance in maintaining normal gutgrowth remains uncertain. If EGF isimportant in maintaining normal gutgrowth, the EGF receptor (EGF-R) shouldbe present on the apical (luminal) surfacein addition to the basolateral surface.Aims/Subjects/Methods-This study exam-ined the distribution of the EGF-R in theepithelium throughout the human gastro-intestinal tract using immunohisto-chemistry, electron microscopy, andwestern blotting ofbrush border prepara-tions.Results-Immunostaining ofthe oesopha-gus showed circumferential EGF-R posi-tivity in the cells of the basal portions ofthe stratified squamous epithelium butsurface cells were EGF-R negative. In thenormal stomach, small intestine, andcolon, immunostaining localised thereceptor to the basolateral surface withthe apical membranes being consistentlynegative. EGF-R positivity within thesmall intestine appeared to be almostentirely restricted to the proliferative(crypt) region. Western blotting demon-strated a 170 kDa protein in whole tissuehomogenates but not in the brush bordervesicle preparations.Conclusions-As the EGF-R is locatedonly on the basolateral surfaces in thenormal adult gastrointestinal tract, themajor role of luminal EGF is probably tostimulate repair rather than to maintainnormal gut growth.(Gut 1996; 39: 262-266)

Keywords: cell proliferation, gut growth, localisation,immunohistochemistry, electron microscopy, growthfactors.

Epidermal growth factor (EGF) is a potentmitogen, which is continuously secreted into thegut lumen by the salivary glands and theBrunner's glands of the duodenum.' Manystudies have shown that EGF stimulates prolif-eration ofvarious cell lines in vitro and also stim-ulates gastrointestinal growth in whole animalswhen given via the systemic circulation.2 It isalso generally accepted that luminal EGF can

stimulate repair ofthe damaged bowel.3 There iscontinuing controversy, however, regarding therole of luminal EGF in maintaining normal gutgrowth in the adult human intestine. If EGF isimportant in maintaining gut growth, one wouldexpect the EGF receptors to be present on theapical (luminal) surfaces of the gastrointestinalepithelium. We therefore examined the distribu-tion of the EGF receptor (EGF-R) in thenormal human adult gastrointestinal tract usinga combination of immunohistochemistry, elec-tron microscopy, and western blotting of brushborder preparations.

Methods

Ethical approvalThis project had local ethical approval and allsubjects gave informed written consent. Allbiopsy specimens were obtained duringroutine endoscopy performed for clinicalreasons. Immunohistochemical and electronmicroscopy analyses were performed at theImperial Cancer Research Fund, London andthe western blot analyses performed at ThePennsylvania State University, USA.

Patients and collection of samplesFresh endoscopic biopsy specimens wereobtained from the oesophagus, stomach, smallintestine, and colon of 20 histologically normalsubjects. Patients donating upper gastrointesti-nal specimens all had a final diagnosis of non-ulcer dyspepsia and patients donating colonicspecimens all had a final diagnosis of the irrita-ble bowel syndrome. Biopsy forceps FB 24Qwere used to collect oesophagus, gastric, andduodenal specimens and FG 13U forceps usedto collect colonic and ileal specimens (bothforceps obtained from Olympus Corporation,Lake Success, NY). Ileal specimens wereobtained during colonoscopy by cannulation ofthe ileocaecal valve. The average weight ofupper intestinal specimens was 9 + 1 mg and ofcolonic specimens was 11 ± 1 mg. Samples forimmunohistochemistry were fixed in formalsaline, processed routinely, and embedded inparaffin wax in the conventional manner.Fresh sections were cut and stained withhaematoxylin and eosin to establish that nohistological abnormality was present. Samplesfor western blotting were snap frozen in liquidnitrogen and stored at -70°C until assay.

Department ofMedicine andTherapeutics,University ofLeicester, LeicesterR J Playford

Departments ofHistopathologyAM HanbyN A Wright

and ElectronMicroscopyS Gschmeissner

Imperial CancerResearch Fund,London

Division ofGastroenterology,Hershey MedicalCenter, Hershey, USAL P PeifferT McGarrityCorrespondence to:Professor R Playford,Department ofGastroenterology, LeicesterGeneral Hospital NHSTrust, Gwendolen Road,Leicester LE5 4PW.

Accepted for publication13 February 1996

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Immunohistochemistry for EGF-RMouse antihuman EGF-R monoclonal anti-

body was raised using purified human EGF-R,derived from A-431 cells, as the antigen(Triton Diagnostics, Alameda, CA, USA).Freshly cut sections were dewaxed and incu-bated in 100% alcohol. Endogenous peroxi-dase activity was blocked using a 10 minuteincubation period in methanol containing0.02% hydrogen peroxide (vol/vol). Sectionswere washed in phosphate buffered saline(PBS) and masked antigenic sites revealed byincubating in PBS solution containing 0.04%Nagarase (Cat no P-8038, Sigma UK) for 30minutes. Sections were incubated in primarymouse antihuman EGF-R monoclonal anti-body at a final dilution of 1:25 for 35 minutes,washed in PBS, and incubated in biotinylatedrabbit antimouse antibody (Dako E354) at a

final dilution of 1:500 to which 1:25 normalhuman serum had been added. Sections were

again washed in PBS followed by incubation instreptavidin-peroxidase at 1:500 for 30minutes. A brown reaction product was

obtained using a peroxidase substrate(diaminobenzidine, PBS, in addition to 0.3%hydrogen peroxide). As a negative control, theprimary antibody was omitted from the proce-

dure. In addition, the specificity of the stainingwas confirmed by taking parallel sections andincubating in the presence of excess recombi-nant EGF-R.

Rabbit antihuman EGF-R polyclonal antibody12E was produced by Gullick and coworkersusing a short synthetic peptide sequencecorresponding to residues 1164-1176 of theintracytoplasmic region of the EGF-R as theantigen.4 Sections were prepared as describedabove and incubated with the primary antibodyat a final concentration of 1/40. The secondantibody reaction consisted of incubatingsections in biotinylated swine-antirabbit anti-body at a final concentration of 1:500 to which1:25 normal human serum had been added. Asa negative control, the primary antibody was

omitted from the procedure. In addition, thespecificity of the staining was confirmed bytaking parallel sections and incubating in thepresence of excess recombinant EGF-R.

Electron microscopyFresh tissues were fixed with 4% para-formaldehyde in 0.1 M Sorensens phosphatebuffer (pH 7.4) for eight hours at 4°C. After fix-ation, the tissues were treated with 0.5 MNH4Cl in phosphate buffer for four hours andwashed in buffer overnight. The tissues were

dehydrated in a graded series of methanol con-

centrations (50, 70, 80, 90% and absolutemethanol, each for 10 minutes) at progressivelylower temperatures and infiltrated with LowicrylHM20 at -35°C. The resin was polymerised byultraviolet light for 48 hours at - 50°C.

For immunogold studies, ultrathin sectionswere mounted on carbon coated grids andlabelled as follows. After five minutes on a dropof PBS and one hour preincubation on 10%normal goat serum, 5% bovine serum albumin(BSA), and 5% ovalbumin in PBS, the grids

were transferred onto drops of rabbit anti-human EGF-R antiserum, 1 2E (1:40 dilution inPBS containing 0.5% BSA, 050/o ovalbumin,and 1% normal goat serum) for eight hours at4°C. Sections were washed three times over a 15minute period with PBS containing 0.5% BSA,0.5% ovalbumin, and 1% normal goat serumfollowed by incubation with immunogold con-jugates (1:100 goat antirabbit IgG, 5 nm gold)for two hours. After rinsing in PBS (3Xfiveminutes) and distilled water (3Xfive minutes),the sections were silver enhanced for fourminutes at room temperature using an IntenseM Silver Enhancement Kit (Amersham, UK)before contrasting with uranyl acetate and leadcitrate. Sections were then examined using aZeiss 10 C electron microscope. As a negativecontrol, the primary antibody was omitted fromthe procedure.

Western blottingBackground to method-to determine if the

apical membranes contained EGF-R, purifiedbrush border vesicles were prepared andprobed for the presence of EGF-R proteinusing western blotting. The quality of thebrush border preparations was determined byfollowing changes in the specific activity ofalkaline phosphatase activity, whose expressionis restricted to the apical membranes.5 Tworabbit polyclonal antibodies were used forthese studies; 12E, which had also been usedfor the immunohistochemical localisationstudies described above and a further affinitypurified rabbit-antihuman EGF-R raisedagainst a synthetic peptide corresponding toresidues 1005 to 1016 of the human EGFreceptor protein (EGFR-1005, Santa CruzBiotechnology, Santa Cruz, CA).

Preparation of brush border vesicles-brushborder preparations were made from duodenaland ileal tissue using the protocol described bySimpson,6 which is a modification of theoriginal technique of Kessler.5 In thesemethods, the microvesicle samples are purifiedfrom potentially contaminating intracellularorganelles by incorporating a centrifugationstep in which MgCl2 had been pre-added tothe diluent (which causes the organelles, butnot the brush border vesicles, to precipitate).This protocol produces apical membranepreparations that are essentially free from con-taminating organelles as judged by electronmicroscopy and marker enzymes.5 6 Aliquotsof the initial tissue homogenate and the finalmicrovesicle preparations were assayed fortotal protein7 and alkaline phosphatase activity(Sigma alkaline phosphatase assay kit, StLouis, USA).

Western blotting-this was performed usingone dimensional SDS-PAGE electrophoresis.Eighty pug of sample protein were loaded intoeach lane of a 7.5% SDS-PAGE gel. Samplesconsisted of whole tissue homogenate,microvesicle preparations, and prestainedmolecular weight markers. After electro-phoresis, the proteins were electroblotted ontonitrocellulose paper and stained with PonceauS to confirm complete transfer and loading

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efficiency. The nitrocellulose filters were thendestained prior to incubation in a blockingsolution for 30 minutes (5% non-fat dry milk,0.5 M NaCl, 0.02M TRIS pH 7.4, 0O1%TWEEN 20). Filters were then incubated withprimary antibodies (E12 or EGF-R 10050,each at 5 ,ug/ml) for 45 minutes and washedthree times with TBS (containing 005%TWEEN 20). Blots were then incubated withhorseradish peroxidase conjugated goatantirabbit antibody diluted 1:1000 in 5% non-fat dry milk for 30 minutes. Filters were thenwashed three times in TBS, 0.05% TWEEN

Figure 1: Immunohistochemical localisation of the EGF-R using rabbit antihumanpolyclonal antibody 12E. EGF-R positivity was visualised using DAB, giving a brownreaction product. In the oesophagus, weak EGF-R positivity is seen circumferentially incells of the basal region of the stratified squamous epithelium but EGF-R positivitydisappears as the cells approach the luminal surface (A, original magnification X400). Inthe stomach, EGF-R positivity was seen on the basolateral surfaces on surface, parietal,and mucus neck cells with particularly strong staining on the parietal cells (B, originalmagnification X400). In the small intestine, EGF-R positivity was restricted to thebasolateral membranes of the proliferative (crypt) region and the goblet cells of the villi,with little or no positivity on the villus enterocytes (C, original magnification X250). In thecolon, EGF-R positivity was seen on the basolateral surfaces of cells in all regions of thecrypts with relatively weak staining of the basolateral membranes of the surface colonocytes.(D, original magnification X400.)

20 followed by one wash in TBS (each washfor five minutes). The presence of the EGF-Rproteins was then visualised using the ECL(chemiluminescence) system (Amersham,UK) by incubating in the ECL reagents for oneminute as recommended by the manufacturer.All assays were performed in quadruplicate.To determine the specificity of the reaction,

western blots were performed in which theprimary antibody had been pre-incubated inexcess recombinant EGF-R (kindly donatedbyW Gullick, ICRF).

Results

Immunohistochemistry and electron microscopyIn the oesophagus, weak membrane localisedEGF-R positivity was seen circumferentially inthe deeper portions of the stratified squamousepithelium but surface cells were negative (Fig1A). The gastric body and antrum epitheliumshowed EGF-R-like immunoreactivity in thebasolateral membranes with particularly strongstaining of the parietal cells. Surface mem-branes were consistently negative (Fig 1B).Duodenal and ileal tissue showed EGF-R-likepositivity on the basolateral surfaces of thecrypt regions (Fig 1 C) and on the goblet cellsof the villi, however, the enterocytes on the villiappeared to be EGF-R negative. Colonicmucosa showed EGF-R-like positivity in abasolateral distribution in all regions of thecrypts (Fig 1D). Distribution of EGF-R posi-tivity visualised using electron microscopy gavesimilar results (Fig 2). Therefore, theapical/surface membranes abutting the intesti-nal lumen were consistently negative through-out the gastrointestinal tract. The negativecontrol and slides in which excess recombinantEGF-R had been added showed no staining,confirming the specificity of the reaction.

Western blottingOn each occasion, brush border preparationsshowed a pronounced increase in alkalinephosphatase activity (typical specific activity ofalkaline phosphatase in ileal whole tissuehomogenate was 0O1 U/mg protein comparedwith 0.9 U/mg protein in brush borderpreparations). The major band seen in duo-denal and ileal whole tissue homogenates hadan apparent molecular weight of approximately170 kDa, with some fainter smaller bands(with apparent molecular weights of approxi-mately 150 and 100 kDa) being inconsistentlyseen in whole tissue homogenates of ilealpreparations. Brush border vesicle prepara-tions from these tissues were consistently neg-ative (Fig 3). Importantly, no bands were seenin any of the lanes in parallel western blots inwhich the primary antibody had been pre-incubated in excess recombinant EGF-R.

DiscussionUsing a variety of different techniques, we haveexamined the distribution of the EGF-R inthe human gastrointestinal tract. All results

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_:: <S .-> .............................. O ..... ............. iFigure 2: Electron microscopy ofgastric body showingEGF-R positivity (visualised using gold tagged antibody)is present on the basolateral but not the apical (luminal, L)membranes ofgastric glands. This is particularly well seenon the arrowed cell. Original magnification X6000.

suggest that, in the normal human adult gut,the apical/surface membranes abutting theintestinal lumen do not express EGF-R.We used two different antibodies (one

monoclonal and one polyclonal) to examinethe distribution of the EGF-R by immuno-histochemistry to decrease the possibility offalse EGF-R-like positivity. For all regions ofthe gastrointestinal tract, results were consist-ent between the two. The major form ofEGF-R found in tissue homogenates of duodenumand ileum had an apparent molecular weight ofabout 170 kDa, although fainter bands weresometimes seen at approximately 150 and 100kDa, probably representing partial degradationproducts of the EGF-R.EGF is a 53 amino acid peptide (molecular

weight 6400), which is secreted into thegastrointestinal lumen from the salivaryglands and Brunner's glands of the duode-num.' Gastric juice concentrations of EGFare about 500 ng/1,8 sufficient to stimulategrowth of epithelial cells in vitro. The concen-tration of luminal EGF in the small and largeintestine is less certain, but probably variesaccording to the presence or absence ofingested proteins as EGF is susceptible todigestion by pancreatic proteases.9 Thesequence and structure of the EGF-R is wellcharacterised and consists of an intracellulardomain (with tyrosine kinase activity), asingle transmembrane region, and an extra-cellular domain with a single ligand bindingsite. Although many studies have shown thatEGF is a potent mitogen for gastrointestinaltissue in vitro and when given systemically invivo,2 there is continuing debate as to its role

Figure 3: Western blots of tissue homogenates andmicrovillus preparations using rabbit antihuman EGF-Rantibody 12E. Eighty micrograms ofprotein were loadedper lane in the following distribution; whole tissuehomogenate (lane 1) and microvillus preparation ofduodenum (lane 2), and whole tissue homogenate (lane 3)and microvillus preparations of ileum (lane 4). Arrowsshow the position ofprotein markers of molecular weights220 and 90 kDa. Bands corresponding to full lengthEGF-R are seen in whole tissue homogenates ofduodenum and ileum, but not in the microvilluspreparations suggesting that the distribution ofEGF-R islimited to basolateral membranes. Note that ileal wholetissue homogenates (lane 3) also showed inconsistent bandsof approximately 150 and 100 kDa, probablycorresponding to a partial degradedform ofEGF-R. Nobands were seen in western blots performed in parallel inwhich the antibody had been pre-incubated withrecombinant EGF-R.

in the normal gastrointestinal tract. If luminalEGF is important in maintaining normalgastrointestinal growth (or influencing gastricacid secretion, or both), one would expect theEGF-R to be present on the apical (luminal)membranes as the tight junctions of theintestinal epithelium probably prevents para-cellular translocation of the peptide undernormal circumstances.Most ofthe previous studies in this area have

examined the distribution of the EGF-R inanimal models, particularly the rat. A paper byThompson and coworkers suggests that, inthe adult rat, the EGF-R is restricted to thebasolateral membranes.10 However, speciesdifferences may exist as Kelly and coworkersreported that EGF-Rs are present on the apicalmembranes of the pig intestine (using the tech-nique of autoradiographic localisation'1l).There have been few equivalent studies per-formed in humans and these have mainlyfocused on the distribution of the EGF-R inthe oesophagus and stomach. Using immuno-histochemical staining, Jankowski and co-workers reported EGF-R-like positivity in acircumferential distribution in the basal por-tion of the stratified squamous epithelium ofthe oesophagus,'2 in keeping with our results.However, in contrast with our findings, theyfound that the EGF-R was present on theapical and basolateral membranes of thenormal human stomach.'3 A similar apical dis-tribution has been also reported in the adultstomach and colon by Hormi and Lehy.14Reasons for these differences are unclear,although the methods used by both groupswere restricted to immunohistochemical local-isation and the antibodies used by each group(Ab-1 by Jankowski, and Ab-4 by Hormi andLehy, both monoclonal IgG's obtained fromOncogene Science, Manhaset, NY, USA) weredifferent from the two used in this study. Adifferent monoclonal antibody (528IgG) wasused by Mori and coworkers to examine the

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distribution of the EGF-R in the humanstomach using the technique of electronmicroscopy. They reported that the EGF-Rwas restricted to the basolateral membranes ofparietal cells,'5 in keeping with our findings.However, caution has to be shown in interpret-ing the results from this study as the 'appar-ently normal gastric fragments' were obtainedfrom patients having gastric resections forgastric carcinoma. It was because of theseapparently conflicting results that, in contrastto all of these other studies, we decided to useseveral different techniques and antibodies(both monoclonal and polyclonal) to examinethe distribution of the EGF-R in detail.Our confirmation that the EGF-R positivity

was restricted to basolateral membranessuggests that the role of luminal EGF in theadult gut is to act as a 'luminal surveillance pep-tide'; readily available to stimulate repair at sitesof injury (when it can reach its receptor), but ofcomparatively minor importance in maintaininggut growth.16 However, it is important to notethat the EGF-R also binds transforming growthfactor cx, amphiregulin, and heparin bindingEGF. Our findings that the EGF-R is presenton the basolateral surfaces of the parietal cells ofthe stomach and the proliferative (crypt) regionsof the small and large intestine suggests that oneor more ofthese other ligands may be importantin controlling acid secretion and proliferation ofthe gastrointestinal tract.

It will be of interest to extend these studies toexamine the distribution of the EGF-R in theneonatal or adult damaged bowel as animalstudies suggest that, in these conditions, lumi-nal EGF is capable of stimulating gut growthand repair. This may be because of an altereddistribution of the EGF-R, or alternatively, thatthe increased permeability of the neonatal ordamaged bowel allows luminal EGF to reachits receptor on the basolateral membranes.

We thank the Medical Research Council and the WellcomeTrust for funding.

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2 Goodlad RA, Wilson TJG, Lenton W, Gregory H, McClaghKG, Wright NA. Intravenous but not intragastric urogas-trone (EGF) is trophic to the intestine of parenterally fedrats. Gut 1987; 28: 573-82.

3 Itoh M, Imai S, Kawai T, Katsumi K, Yokochi K, TakeuchiT. Protection of gastric mucosa against ethanol injury byintragastric bolus administration of epidermal growthfactor combined with hydroxypropylcellulose. J ClinGastroenterol 1992; 14: S 127-30.

4 Gullick WJ, Hughes CM, Mellon K, Neal DE, LemoineNR. Immunohistochemical detection of the epidermalgrowth factor receptor in parrafin-embedded tissue.Jf Pathol 1991; 164: 284-9.

5 Kessler M, Acuto 0, Storelli C, Murer H, Muller M,Semenza G. A modified procedure for the rapid prepara-tion of efficiently transporting vesicles from small intestinebrush border membranes. Biochem Biophys Acta 1978;506: 136-54.

6 Simpson RJ, Peters TJ. Studies ofFe3+ transport across iso-lated intestinal brush border membrane of the mouse.Biochem Biophys Acta 1984; 772: 220-6.

7 Schacterle G, Pollack R. A simplified method for the quan-titative assay of small amounts of protein in biologicalmaterial. Anal Biochem 1973; 51: 654-5.

8 Playford RJ. Marchbank T, Calam J, Hansen FH. EGF isdigested to smaller, less active, forms in acidic gastricjuice. Gastroenterology 1995; 108: 92-101.

9 Playford RJ, Watanaba P, Woodman AC, Deprez PH,Calam J. Effect of luminal growth factor preservation onintestinal growth. Lancet 1993; 341: 843-8.

10 Thompson JF, Van Den Berg M, Stokkers PCF.Developmental regulation of epidermal growth factorreceptor kinase in rat intestine. Gastroenterology 1994; 107:1278-87.

11 Kelly D, McFadyen M, King TP, Morgan PJ.Characterisation and localisation of the epidermal growthfactor receptor in the jejunum of the neonatal and weanedpig. Reprod Fertil Dev 1992; 4: 183-9.

12 Jankowski J, Murphy S, Coghill G, Grant A, Wormsley KG,Sanders DSA, et al. Epidermal growth-factor receptors inthe oesophagus. Gut 1992; 33: 439-43.

13 Jankowski J, Al-Rawi HJ, Johnston DA, Hopwood D, FilipeMI, Coghill G, et al. Growth regulatory peptides in gastricmucosa. Clin Sci 1992; 82: 581-7.

14 Hormi K, Lehy T. Developmental expression of transform-ing growth factor a and epidermal growth factor receptorproteins in the human pancreas and digestive tract. CellTissue Res 1994; 278: 439-50.

15 Mori S, Morishita Y, Sakai K, Kurimoto S, Okamoto M,Kawamoto T, et al. Electron microscopy evidence for epi-dermal growth factor receptor (EGF-R) like immunoreac-tivity associated with the basolateral surface of gastricpariental cells. Acta PatholJ'pn 1987; 37: 1909-17.

16 Playford RJ. The role of peptides in mucosal integrity andrepair. Gut 1995; 37: 595-7.

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