immunohistochemical studies on egf family growth factors in normal and ulcerated human gastric...

Immunohistochemical Studies on EGF

Family Growth Factors in Normal and

Ulcerated Human Gastric Mucosa

SHINYA ABE, MD, HIRONOBU SASANO, MD, KATSUAKI KATOH, MD, SHUICHI OHARA, MD,

TADASHI ARIKAWA, MD, TETSUYA NOGUCHI, MD, SHIGERU ASAKI, MD,

WATARU YASUI, MD, EIICHI TAHARA, MD, HIROSHI NAGURA, MD, and

TAKAYOSHI TOYOTA, MD

Expression of members of the epidermal growth factor family, including epidermal growthfactor (EGF), transforming growth factor- a (TGF- a ), amphiregul in (AR), and Cripto, as wellas their putative receptor, epidermal growth factor receptor (EGFR), was studied immuno-histochemically in human gastric mucosa to evaluate their possible roles in cell proliferationof normal and regenerative gastric mucosa. We also examined the correlation between cellproliferation and EGFR by double immunohistochemical staining for proliferating cellnuclear antigen (PCNA) and EGFR. In normal gastric mucosa, TGF- a , Cripto, and ARimmunoreactivities were observed in the surface epithelial and parietal cells of gastric fundicglands, respectively. EGF immunoreactivity was not observed in any of normal mucosaexamined. EGFR immunoreactivity was detected on foveolar cells in proliferative zones andin parietal cells. Double immunostaining revealed that EGFR immunoreactivity was distrib-uted much more widely than PCNA immunoreactivity. PCNA positive epithelial cells adja-cent to gastric ulcer margin expressed relatively intense EGFR but did not express any of thegrowth factors examined. On the other hand, relatively intense immunoreactivity of bothTGF- a and Cripto was detected in PCNA-negative regenerative epithelium located distantfrom gastric ulcer margin. Relative immunoreactivity of AR in regenerative gastric epithe-lium associated with ulcer was not different from that in normal gastric mucosa. TGF- a , AR,and Cripto are considered to play important roles in normal gastric mucosal proliferation,and TGF- a and Cripto may be involved in ulcer healing, possibly via a paracrine mechanism.

KEY WORDS: epidermal growth factor; transforming growth factor- a ; amphiregulin; epidermal growth factorreceptor; Cripto; gastric ulcer.

Various polypeptid e growth factors have been dem-

onstrated to play important roles in regulating muco-

sal cell proliferation and differentiation, including

gastric ulcer healing. Among these growth factors,

growth factors with structural similarities to EGF or

EGF family growth factors have been detected in

normal human gastric mucosa. These growth factors

include transforming growth factor- a (TGF- a ) (1± 3),

amphiregul in (AR) (4 ± 6) and Cripto (7, 8). In addi-

tion, TGF- a as well as EGF (9 ± 14) have been shown

to play important roles in proliferation and differen-

tiation of mucosal cells of the gastrointestinal tract

including stomach (15± 17). Other potent biologic ac-

Manuscript received November 6, 1995; revised manuscript re-ceived October 24, 1996; accepted January 31, 1997.

From the Departments of Medicine (III) and Pathology (II),Tohoku University School of Medicine, Sendai, Japan; and De-partment of Pathology (I), Hiroshima University School of Medi-cine, Hiroshima, Japan.

Address for reprint requests: Dr. Shinya Abe, Department ofInternal Medicine (III), Tohoku University School of Medicine, 1-1Seiryou-machi, Aoba-ku, Sendai 980-77, Japan.

Digestive Diseases and Sciences, Vol. 42, No. 6 (June 1997), pp. 1199 ± 1209

1199Digestive Diseases and Sciences, Vol. 42, No. 6 (June 1997)

0163-2116/97/0600-1199$12.50/0 Ñ 1997 Plenum Publishing Corporation

tivities of EGF and TGF- a in gastric mucosa include

inhibition of gastric acid secretion (18, 19), protection

against various ulcerogenic factors (20), and stimula-

tion of gastric mucous production (21, 22). On the

other hand, the biologic activities of AR and Cripto in

human gastric mucosa have not been well studied.

EGF, TGF- a and AR all act by binding to their

target cell surface receptor, designated as the EGF

receptor (EGFR), which possesses intrinsic tyrosine

kinase activity. On the other hand, the receptor to

which Cripto binds has not been identi® ed. EGFR is

a single polypeptid e chain with an extracellular N-

terminus that traverses the cell membrane and has a

tyrosine-speci® c protein kinase activity in the intra-

cellular domain (23). Expression of EGFR has been

demonstrated in the gastric tissue of various labora-

tory animals using receptor binding assays (24) and

immunohistochemical methods (25, 26). However,

the relationship between expression of EGFR and

that of growth factors such as EGF, TGF- a , and AR

in the human stomach and in regenerative epithelium

of gastric ulcer not known.

Therefore, in this study, we performed an immu-

nohistochemical analysis of EGF, TGF- a , AR, and

Cripto expression in normal human gastric tissue and

in the regenerative epithelium of human gastric ulcer

tissue. In addition, the receptor± ligand relationship

between these growth factors and EGFR was evalu-

ated by immunohistochemical study of serial sections

of these tissues. In addition, we also performed dou-

ble immunostaining for EGFR and proliferative cell

nuclear antigen (PCNA) to evaluate the correlation

between EGFR expression and cell proliferation in

human gastric mucosa.

MATERIALS AND METHODS

Tissue Preparation. Forty-seven surgical pathology spec-imens of morphologically normal gastric mucosa obtainedfrom gastrectomy specimens were examined in this study.We selected these specimens after examining histologicalfeatures demonstrated by hematoxylin and eosin stain.These surgical pathology specimens did not contain foci ofneoplastic changes or signi ® cant atrophy, including intesti-nal metaplasia, and had minimum degrees of in¯ ammation.Surgical pathology specimens of perforated gastric ulcerwere retrieved from pathology ® les. Review of the charts ofpatients with perforated gastric ulcer revealed that gastrec-tomy was performed within two days following developmentof symptoms. Therefore, these specimens of gastric ulcerare all considered to be at the acute phase of ulcer forma-tion. We also examined the endoscopic biopsy specimens ofthe stomach from 10 patients who had been followed con-servatively with endoscopic observation. All patients under-went endoscopic examinations every two to three months

during medical treatment with antiulcerogenic drugs from

acute to scarred phase. Endoscopic biopsies were carried

out with consent from the patients at the endoscopicallyhealing and scarred phases. Specimens were obtained from

two different sites around the gastric ulcers, including the

region adjacent to ulcer margin and the region approxi-

mately 1 cm distant from ulcer margin. These endoscopicspecimens were ® xed in 10% buffered formalin for 18 hr at

4°C for immunohistochemistry.

Antibodies Against EGF, TGF- a , AR, Cripto, and EGFR.The antibody against EGF employed in this study wasmonoclonal antibody KME-10, purchased from WakunagaPharmaceutical Co., Ltd. (Hiroshima, Japan). The antibodyagainst TGF- a was monoclonal antibody Ab2, (OncogeneScience, Inc., Uniondale, New York). The antibody againstEGFR was monoclonal antibody 31G7 (Triton DiagnosticsCo., Ltd., Alameda, California), and the antibody againstPCNA was monoclonal antibody PC10, obtained from No-vocastral Lab (Newcastle, UK). A polyclonal antiserumagainst AR was obtained from a rabbit immunized withhuman AR 605 and was kindly provided by Dr. G. R.Johnson (FDA, Bethesda, Maryland) (27). A polyclonalantiserum against Cripto was produced from a rabbit im-munized with an oligopeptide corresponding to amino acids116 ± 127 of Cripto (5).

Immunohistochemistry. For each specimen, eight serial2.5- m m-thick sections were mounted on glass slides. Theywere stained as follows: hematoxylin± eosin for light micro-scopic examination; immunostaining for PCNA, EGFR,EGF, TGF- a , AR, and Cripto, and double immunostainingfor PCNA and EGFR. Immunohistochemistry was per-formed by the modi® ed streptavidin ± biotin± peroxidasemethod using the Histo ® ne immunostaining system(Nichirei Co. Ltd., Tokyo, Japan). Following deparaf® niza-tion through xylene and ethanol, sections were submergedin methanol containing 1.0% H2O2 for 30 min to blockendogenous peroxidase activity. Tissue sections werewashed in 0.01 M PBS three times for 5 min. A blockingprocedure to decrease nonspeci® c binding of second anti-bodies was performed in a moisture chamber for 30 min atroom temperature; sections were exposed to nonimmuneserum obtained from animals of the same species fromwhich second antibodies were prepared. Primary antibodieswere reacted with tissue sections at optimal dilution for 18hr at 4°C in a humidi® ed chamber. Optimal dilutions forprimary antibodies against EGF, TGF- a , AR, Cripto,EGFR, and PCNA were 1 to 150, 1 to 750, 1 to 25, 1 to1000, 1 to 100, and 1 to 200, respectively. Pretreatment withtrypsin and pepsin was performed for immunohistochemis-try of EGFR for antigen retrieval; sections were immersedin 0.1% (w/v) trypsin (Sigma, St. Louis, Missouri) and 1%(w/v) potassium chloride in 0.05 M Tris HCl (pH 7.4) for 15min at 37°C, followed by treatment with 4% (w/v) pepsin(Wako, Tokyo, Japan) in 0.01 N HCl for 15 min at 37°C.Reaction with primary antibodies was followed by incuba-tion with biotinylated second antibodies for 30 min at roomtemperature and with peroxidase-conjugated streptavidinfor 30 min at room temperature. Each step was followed bythree 5-min washes in PBS. Following the ® nal wash, sec-tions were treated with 0.06 mM 3,3 9 -diaminobenzidine and2 mM hydrogen peroxide in 0.05 M Tris HCl (pH 7.6).Immunostained sections were counterstained with 1%

ABE ET AL

1200 Digestive Diseases and Sciences, Vol. 42, No. 6 (June 1997)

methyl green. As a negative control for immunostaining,normal mouse IgG for EGFR, PCNA, TGF- a , and EGF ornormal rabbit IgG for Cripto and AR in 0.01 M PBS wasused instead of primary antibodies. None of these controlsections exhibited immunoreactivity.

Double Immunostaining for EGFR and PCNA. Doubleimmunostaining for both EGFR and PCNA was performedto examine the localization of PCNA and EGFR. Thedouble immunostaining procedure has been previously de-scribed in detail (28). Following DAB visualization of stain-ing with anti-PCNA as described above, the slides werewashed extensively three times in 0.1 M glycine HCl bufferat room temperature for 70 ± 90 min with constant stirring.Avidin and biotin blocking then was performed by exposingthe slides to an excess of both avidin and biotin using anavidin± biotin blocking system (Vector). EGFR immuno-staining was performed using biotinylated rabbit anti-mouse IgG and alkaline phosphatase-conjugated streptavi-din. Tissue sections were then reacted with fast-blue RKsalt and mounted with a glycerin± PBS solution.

Evaluations of Immunohistochemistry. Relative immu-nointensity of the tissue sections examined was scored asfollows: 2 , negative; 1 , weakly positive; 1 1 , strongly pos-itive, independently by two of the authors (S.A. and H.S.).Disconcordant cases were reevaluated by double-headedlight microscopy.

RESULTS

Immunohistochem istry. Results of immunohisto-

chemistry including relative immunointensity of EGF

family growth factors and EGFR are summarized in

Table 1.

EGF Family Growth Factor Expression in Normal

Gastric Mucosa. In all specimens of normal human

gastric mucosa examined, TGF- a , AR, and Cripto

immunoreactivity was observed, while EGF immuno-

reactivity was not observed in any of the cases we

examined. Relatively strong TGF- a immunoreactivity

was observed in the parietal cells of the gastric fundic

glands and in the surface epithelial cells, whereas

mucous neck cells were negative or weakly positive

for TGF- a (Figure 1A). TGF- a immunoreactivity

gradually intensi® ed from the neck region to the

surface epithelium. Cripto immunoreactivity was ob-

served in the surface epithelium, whereas the mucous

neck cells and parietal cells were negative or weakly

positive for Cripto (Figure 1B). On the other hand,

AR immunoreactivity was observed only in the pari-

etal cells (Figure 1C). Gastric chief cells were nega-

tive for all these growth factors above.

EGFR in Normal Gastric Mucosa. EGFR immu-

noreactivity was observed in the parietal cells and

mucous neck cells of the gastric fundic glands (Figure

2A). EGFR was localized to the basolateral cell mem-

brane, but not to the apical luminal membrane (Fig-

ure 2B). Double immunostaining for EGFR and

PCNA revealed that EGFR membrane immunoreac-

tivity was much more widely distributed than PCNA

nuclear immunoreactivity. Cells positive for both

PCNA and EGFR were predominantly observed in

the mucous neck region, which corresponds to a

morphologically identi® ed proliferative zone. On the

other hand, parietal cells expressed only EGFR (Fig-

ure 2C and D).

EGF Family Growth Factors and EGFR in Gastric

Ulceration and Ulcer Scar Tissue. Results are sum-

marized in Table 2.

In the active phase, the majority of acute phase

specimens obtained from surgical pathology ® les did

not demonstrate any histologica l evidence of epithe-

lial regeneration. However, in some of these speci-

mens, epithelial cells located around the ulcer margin

appeared immature and demonstrated PCNA posi-

tively. These PCNA-positive proliferative cells dem-

onstrated EGFR immunoreactivity at the basolateral

site of their cytoplasmic membrane, but relative im-

munointensity of EGFR was weak, compared to the

proliferative zone of normal gastric mucosa. Immu-

noreactivity for EGF, TGF- a , Cripto, and AR was not

detected in these cells. Patterns of immunolocaliza-

tion and relative immunoreactivity of TGF- a , Cripto,

and AR in the morphologically normal mucosa lo-

cated away from ulcer margins were essentially simi-

lar to those of normal gastric mucosa.

Twenty specimens obtained from 10 cases of heal-

ing-phase gastric ulcer with endoscopic biopsy dem-

onstrated various histologica l degrees of epithelial

regeneration associated with ulcer healing. Ten spec-

imens obtained from the epithelium adjacent to ulcer

margins at the healing stage demonstrated develop-

ment of immature regenerative epithelium, whereas

10 other specimens distant from the ulcer margin at

the healing stage demonstrated mature regenerative

epithelium. The maturation of regenerative epithe-

lium was characterized morphologically by increased

mucus production and formation of gastric glands,

while the immature epithelium showed neither of

these features. EGFR immunoreactivity was observed

TABLE 1. SUMMARY OF IMMUNOHISTOCHEMISTRY OF EGF FAMILY

GROWTH FACTORS AND EGFR INCLUDING RELATIVE

IMMUNOINTENSITY IN NORMAL GASTRIC MUCOSA

TGF- a Cripto AR EGF EGFR

Surface epithelial cells 1 1 * 1 2 2 2Mucous neck cells 2 2 2 2 1Parietal cells 1 2 1 2 1Chief cells 2 2 2 2 2

* 2 , negative; 1 , weakly positive; 1 1 , strongly positive.

IMMUNOHISTOCHEMICAL STUDIES ON EGF FAMILY GROWTH FACTORS


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ABE ET AL


Fig 2. Immunohistochemistry of EGFR in normal human gastric mucosa. EGFR immunoreactivity was seenin the parietal cells and mucous neck cells of gastric fundic glands (A) ( 3 150). EGFR immunoreactivity,

(higher magni ® cation of A) was localized to the basolateral cell membrane, but not to the apical luminalmembrane in parietal cells (B) ( 3 400). Double immunohistochemistry of EGFR and PCNA in normal

human gastric mucosa. Brown staining as a result of the DAB reaction represents PCNA immunoreactivity,while blue-violet staining as a result of the fast blue reaction represents EGFR immunoreactivity. Double

staining for EGFR and PCNA revealed that EGFR expression was observed extensively beyond areas withPCNA positive cells (C and D) (C: 3 150; D: 3 300). (Reduced to 77% for reproduction.)



predominantly in the immature regenerative epithe-

lium around ulcer margins, and PCNA-positive cells

were scattered throughout the EGFR-positive epithe-

lium. In ulcer margins at the healing stage, a single

regenerative epithelial layer, positive for EGFR but

negative for PCNA, extended toward the ulcer base

from proliferat ive zones (Figure 3). None of the

growth factors examined were present in the imma-

ture regenerative mucosa, including the proliferat ive

zone near the ulcer margin. On the other hand,

mature regenerative epithelium distant from the ulcer

margins demonstrated TGF- a and Cripto immunore-

activity, with their immunointensity nearly equal to

those of parietal cells of normal gastric mucosa (Fig-

ure 4). No increase of AR immunoreactivity was

observed in any of the regenerative epithelial areas in

any of the gastric ulcer cases examined. EGF immu-

noreactivity was not observed in healing stage of

gastric ulcer.

Scarred lesions were completely covered with re-

generative epithelial cells. Immunohistochemistry

showed both PCNA- and EGFR-positive cells, pre-

dominantly from the bottom to the neck of the glands.

Cells positive for TGF- a and Cripto were observed

predominantly in the upper part of the regenerative

epithelium adjacent to ulcer scar tissue. AR and EGF

immunoreactivity was not identi® ed in the regenera-

tive epithelium and glands of gastric ulcer scar tissue.

Results of immunolocalization of EGF family

growth factors and EGFR in human gastric ulcers are

summarized in Figure 5.

DISCUSSION

Several studies have demonstrated expression of

TGF- a , AR, and Cripto in normal gastric mucosa in

humans and other mammalian species. Beauchamp et

al (3) reported that in mammalian gastric mucosa,

TGF- a mRNA was detected predominantly in pari-

etal cell-enriched fractions. Immunolocalization of

TGF- a in the normal human stomach also has been

reported using the same anti-TGF- a antibody em-

ployed in the present study (29, 30). Results from

these studies are consistent with the TGF- a immuno-

localizatio n reported in this study. AR and Cripto,

which are newly identi® ed members of the EGF fam-

ily, have been detected in normal gastric mucosa at

the mRNA level by northern blot analysis (7, 8), but

expression of AR and Cripto in the human stomach

has not been analyzed immunohistochemically. In the

present study, Cripto immunoreactivity was observed

in the surface epithelium and was negative or faintly

positive in the parietal cells of the gastric mucosa,

while AR was identi® ed only in the parietal cells.

These differences in immunoloca lization between

Cripto and AR have been also reported in the human

colon. Saeki et al demonstrated that AR is expressed

in normal colon, while Cripto is not (31). These

reports indicated that TGF- a , AR, and Cripto are

produced locally in normal gastric mucosa and play a

biologic role in the human stomach. However, the

possible biological roles of AR and Cripto in gastric

mucosa are not clearly understood. Various in vitro

studies have suggested that AR has biological func-

tions different from those of EGF and TGF- a . For

instance, AR does not promote anchorage- indepen-

dent growth of normal rat kidney ® broblasts in the

presence of transforming growth factor- b (5). The

biological function mediated by Cripto has not been

well-studied in vivo, since its liganded receptor has

not been identi® ed yet. Therefore, expression of

Cripto and AR in normal gastric mucosa has been

con® rmed but further investigations are needed to

clarify the biological signi® cance. On the other hand,

immunohistochemical analysis of EGF did not dem-

onstrate immunoreactivity in any specimens of human

gastric mucosa. This ® nding is consistent with previ-

ous studies on both human (13) and rat (26) gastric

mucosa. In addition, EGF mRNA has not been de-

tected in gastric tissues from mammals, including

TABLE 2. SUMMARY OF IMMUNOHISTOCHEMISTRY OF EGF FAMILY GROWTH

FACTORS AND EGFR INCLUDING RELATIVE IMMUNOINTENSITY IN ULCER MARGINS

OF HEALING STAGE

TGF- a Cripto AR EGF EGFR PCNA

Single regenerative

epithelial layer2 * 2 2 2 1 2

Immature regenerative

epithelium2 2 2 2 1 1

Mature regenerative

epithelium1 1 1 2 2 2 2

* 2 , negative; 1 , weakly positive; 1 1 , strongly positive.

ABE ET AL


humans (32). Therefore, EGF, which exerts biologic

effects on normal human gastric mucosa may origi-

nate predominantly from other organs, including the

salivary glands and the duodenal Brunner’ s glands, in

which EGF synthesis has been reported (12, 13).

The biologic effects of various growth factors are

mediated generally via cellular surface receptors.

EGF, TGF- a , and AR bind to a common cell surface

receptor, EGFR, which is a tyrosine kinase-associated

receptor. On the other hand, interactions between

Cripto and EGFR have not been well established

(33). In the present study, EGFR immunoreactivity

was identi® ed in mucous neck cells and parietal cells

of the fundic glands and was found only at the baso-

lateral cell membrane. These results are consistent

with ultrastructural immunohistochemical studies by

Fig 3. Immunohistochemistry of EGFR and PCNA in gastric mucosa obtained from areas of

ulceration in the healing stage. EGFR immunoreactivity was identi® ed mainly in the immatureregenerative epithelium around ulcer margins (A), and PCNA positive proliferative cells were

scattered among these epithelial cells (B) ( 3 200).



Mori et al (34) and radiolabeled assays by Scheving et

al (35). Therefore, growth factors such as EGF,

TGF- a , and AR must be delivered to the basolateral

aspect of target cells through endocrine, paracrine,

and/or autocrine mechanisms in order to exert their

EGFR-mediated effects. This ® nding is consistent

with the suppression of gastric acid output by paren-

teral, but not enteral, administrat ion of EGF along

with gastric mucosal protection against ulcerogenic

agents (19, 36).

Double immunostaining for EGFR and PCNA

demonstrated that EGFR-positive cells were more

extensively distributed than cells expressing both

EGFR and PCNA. Such double-po sitive cells were

observed predominantly among mucous neck cells in

the proliferative zone. In addition, PCNA-positive

Fig 4. Immunohistochemistry of TGF- a and Cripto in gastric mucosa obtained from areas of

ulceration in healing stage. TGF- a (A) and Cripto (B) immunoreactivity were observed in matureregenerative epithelium apart from the ulcer margin ( 3 200).

ABE ET AL


cells were nearly always positive for EGFR. There-

fore, the expression of EGFR is closely correlated

with the proliferative activity of the mucous neck

cells. The mucous neck cells, which were positive for

both EGFR and PCNA, did not demonstrate immu-

noreactivity for any of the growth factors examined.

This ® nding suggests a paracrine control mechanism

operating between EGFR and bound growth factors,

ie, the mucous neck cells in the proliferative zone

express EGFR, while neighboring surface epithelium

and parietal cells produce TGF- a and AR. This is

consistent with the report of Chen et al (37), who

have demonstrated paracrine control of TGF- a on

mucosal cell growth in vitro. Parietal cells expressed

EGFR but not PCNA. Therefore, EGFR expression

in the parietal cell is not associated with cell prolif-

eration, but may rather re¯ ect effects of EGF on acid

output. It is well known that parenteral administra-

tion of EGF suppresses gastric acid output (19, 36),

but the inhibitory activity of EGF on gastric acid

output requires supraphysio logic or pharmacological

amounts of parenteral EGF. Therefore further inves-

tigation is required to clarify the biologic roles of

EGFR in human gastric parietal cells.

Various polypeptid e growth factors, including EGF

family growth factors, are involved in tissue repair

following injury. Therefore, understand ing of the pos-

sible involvemen t of various growth factors in the

development of regenerative epithelia following gas-

tric ulceration becomes important . Results from sev-

eral studies have demonstrated that both EGF and

TGF- a may play important roles in the reparative

events following gastric mucosal injury. Wright et al

have reported that ulceration of human gastrointes-

tinal tract induces EGF immunoreactivity around ul-

cer margins (38). Lee et al have reported increased

levels of EGF immunoreactivity in chronically injured

rat gastric mucosa treated with acetic acid (39). Both

of these studies suggested that ulceration of the hu-

man gastrointestinal tract induces development of

novel cell lineages from stem cells that secrete EGF.

On the other hand, Polk et al have observed increased

expression of TGF- a mRNA and protein in rat gastric

mucosa following acute injury induced by hydrochlo-

ride and taurocholate, whereas EGF was not in-

creased during the following acute phase of gastric

injury (32). In this study of human gastric ulceration ,

we did not ® nd EGF immunoreactivity in the regen-

erative mucosa around gastric ulcers, consistent with

the study of Polk et al (32). This discrepancy in EGF

expression from previous studies may be due to dif-

ferences in the antibody employed, tissue prepara-

tion, or stage of the gastric ulcers examined. Immu-

noreactivity of all the growth factors examined in this

study was not detected in the epithelia around ulcer

margins of acute-phase gastric ulcer. However,

EGFR expression was detected in some of these

proliferative epithelia. These ® ndings suggest that

extrinsically produced EGF family growth factors,

including EGF secreted from salivary gland and du-

Fig 5. Summary of immunolocalization of EGF family growth factors, EGFR, and PCNA in

areas of healing human gastric ulceration.



odenal Brunner’ s gland, are considered to play im-

portant roles in gastric ulcer healing. This ® nding is

also consistent with the report by Imai et al, which

demonstrated that submandibularectomy prevented

gastric ulcer healing in rats (22). In addition, in acute-

stage perforated ulcer, various growth factors includ-

ing basic ® broblast growth factor may have some

effects (40).

The immature epithelium adjacent to the ulcer

margin did not express any of the EGF family growth

factors in healing-stage gastric ulcer mucosa exam-

ined in this study. However, intense EGFR immuno-

reactivity was identi® ed in this area. In these EGFR-

positive epithelial cells, areas of PCNA-positive cells

were identi® ed and were considered to correspond to

the proliferative zone of immature epithelium. In

addition, the regenerative epithelium is histologica lly

continuous with such proliferative zones described

above toward the ulcer base and subsequently ap-

pears to cover the mucosal defect. The regenerative

epithelia of healing-stage gastric ulcers gradually dif-

ferentiated and obtained the biologic characteristics

of mature epithelium, including growth factor expres-

sion. Relative immunointensity of TGF- a and Cripto

is nearly equal to that of parietal cells of normal

gastric glands in the mature regenerative epithelia

present at sites distant from healed-phase ulcer mar-

gin. AR immunoreactivity was not detected in these

mucosas. Therefore, among EGF family growth fac-

tors, TGF- a and Cripto are considered to play impor-

tant roles in the repair of gastric ulcers. However, AR

does not appear to play a signi® cant role in human

gastric ulcer repair.

Based on these ® ndings, regenerative epithelium

can be classi® ed into three regions proceeding out-

ward from the ulcer base in terms of EGFR and EGF

family growth factor expression patterns, as shown in

Figure 5. The area nearest to the ulcer base is the

growing regenerative epithelial zone. The next is the

proliferative zone, which expresses PCNA. The last,

which is farthest from the ulcer base, has cells positive

for growth factors including TGF- a and Cripto.

These patterns of immunolocalization of EGFR and

the growth factors were consistently seen during ulcer

healing. Therefore, TGF- a and Cripto, which are

derived from the mature regenerative epithelium

around gastric ulcers, are considered to play impor-

tant roles as locally synthesized EGF family growth

factors in promoting ulcer healing in a paracrine

manner.

In summary, we have demonstrated that immuno-

reactive forms of TGF- a , AR, and Cripto are present

in the surface epithelium and parietal cells of normal

human gastric mucosa. EGFR, which is a receptor for

EGF, TGF- a , and AR, was expressed in the mucous

neck cells and was associated closely with cell prolif-

eration in the gastric mucosa. The correlation be-

tween growth factor expression and EGFR immuno-

localizatio n suggests paracrine regulation of TGF- aand AR on human gastric mucosa. During gastric

ulcer healing, the immature regenerative epithelium

around the ulcer margin expressed EGFR and

formed a proliferative zone. The mature regenerative

epithelium adjacent to the proliferative area demon-

strated increased immunoreactivity of TGF- a and

Cripto, but not of AR. Therefore TGF- a and Cripto

may play important roles in promoting gastric ulcer

healing as locally synthesized growth factors. EGF

immunoreactivity was not present in the gastric mu-

cosa around ulcers; EGF therefore appears to exert

its actions through endocrine and/or possibly exocrine

mechanisms and is delivered from distant organs such

as the salivary glands.

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