ghrelin expression in hyperplastic and neoplastic proliferations of the enterochromaffin-like (ecl)...

Ghrelin in ECL Cell Proliferation 47Clinical Research

47

Department of Pathology,Tufts-New England MedicalCenter, Boston, MA.

Address for correspondence toDr. Yogeshwar Dayal,Department of Pathology,New England Medical Center,750, Washington St, Boston,MA-02111. E-mail: [email protected]

Endocrine Pathology, vol. 15,no. 1, 47–54, Spring 2004© Copyright 2004 by HumanaPress Inc. All rights of anynature whatsoever reserved.1046–3976/04/15:47–54/$25.00

Ghrelin Expressionin Hyperplastic and Neoplastic Proliferationsof the Enterochromaffin-like (ECL) Cells

Amitabh Srivastava, MBBS, Anitha Kamath, MBBS,Shepard-Annette Barry, HT(ASCP), and Yogeshwar Dayal, MD

AbstractGhrelin, a recently discovered peptide isolated from the gastric corpus mucosa, is believedto be important in the regulation of growth hormone secretion and has been shown toincrease appetite and food intake as well. It may also have other gastrointestinal andcardiac functions. Because a cell of origin for ghrelin has not been convincingly identifiedin the gastric mucosa thus far, we studied the immunohistochemical expression of ghrelinin proliferative lesions of the enterochromaffin-like (ECL) cells—a cell that is not onlyexclusively confined to the gastric corpus mucosa but is its dominant endocrine cell typeas well.Formalin-fixed, paraffin embedded tissues from three cases of gastric ECL cell hyperplasiaand five ECL carcinoids (three with coexisting foci of diffuse, linear, and micronodular hyper-plasia) were immunohistochemically stained for ghrelin, using a commercially available anti-body. The Sevier–Munger stain for ECL cells and immunohistochemical stains for chromogranin,gastrin, serotonin, somatostatin, and vesicular monoamine transporter-2 (VMAT-2) wereperformed on parallel sections for correlation with the ghrelin staining results.All ECL cell carcinoids and hyperplastic lesions were positive for both the Sevier–Mungerand the immunohistochemical stains for chromogranin and VMAT-2. Immunoreactivityfor ghrelin was seen in 4/5 ECL carcinoids, all cases of ECL cell hyperplasia, as well as in allareas with linear and micronodular hyperplasia adjacent to the ECL cell carcinoids.In each instance, such staining was confined to the Sevier–Munger, and VMAT-2 positivecells only.Our findings indicate that the ECL cells are either the ghrelin-producing cells of the gastricmucosa or acquire the capability to synthesize ghrelin during proliferative states encom-passing the entire hyperplasia to neoplasia spectrum. In view of the orexigenic and otherknown actions of ghrelin, the functional and/or biologic significance of ghrelin produc-tion in such ECL cell proliferations needs to be investigated further.Key Words: Ghrelin; enterochromaffin-like cells; VMAT-2; ECL cell hyperplasia; gastriccarcinoid; ECL cell carcinoid.

hormone (GHRH) that stimulates itsrelease from the anterior pituitary andsomatostatin that inhibits it. Both theseneuropeptides reach their respectivereceptors (GHRH-R and SS-R) throughthe portal circulation. GH release can also

Introduction

Growth hormone (GH) secretion by thepituitary somatotrophs is normally regu-lated by the antagonistic actions of twohormones—growth hormone–releasing

48 Endocrine Pathology Volume 15, Number 1 Spring 2004

be stimulated by motilin, met-enkephalin,and a number of their synthetic analogscollectively referred to as growth-hormonesecretagogues (GHS). However, these GHSstimulate GH release by acting through aspecific receptor (GHS-R) that is distinctfrom GHRH-R. The GHS-R was untilrecently referred to as an “orphan recep-tor” as no naturally occurring ligand for ithad been identified. Recent work on theneuroendocrinologic mechanisms for theregulation of appetite, body weight, energybalance, and so forth has identified a novel28-amino-acid linear peptide as the natu-ral endogenous ligand for GHS-R [1].Chiefly secreted by the gastric oxynticmucosa, this peptide has been namedghrelin (after the proto-Indo-Europeanroot of “to grow”). In addition to being apotent stimulus for GH release, it has alsobeen shown to have an important orexi-genic action in that it increases appetite andfood intake [2,3]. Because circulating lev-els of ghrelin rise before meals and fallshortly thereafter, it is also believed to playa significant role in the natural impulse toinitiate a meal [4]. Plasma ghrelin levelsare elevated during starvation and periodsof diet-induced weight loss [5] and areoften markedly so in patients with anor-exia nervosa [6]. In these latter patients,the levels revert to normalcy upon weightgain [6]. More recently, ghrelin has alsobeen implicated as an important factor inthe pathogenesis of morbid obesity [7,8].

Immunohistochemical studies haveidentified ghrelin-positive cells in thegastric and intestinal mucosa [9], pituitarygland [10], hypothalamus [1], kidney [11],placenta [12], and the fetal lung [13].Among these various sites, their highestconcentration appears to be in the gastricoxyntic mucosa, which is regarded as themajor source of circulating plasma ghrelin.Thus on account of its preferential distri-

bution in the gastric oxyntic mucosa, andits orexigenic and other functions, ghrelinis now regarded as a gastrointestinal hor-mone with both gastrointestinal andextraintestinal functions [14,15].

The oxyntic portion of the humangastric mucosa contains at least six distinctendocrine cell types: the serotonin produc-ing EC cells, the histamine producing ECLcells, the somatostatin producing D cells,the A-like/X cells that resemble glucagonproducing A cells in the pancreas, as wellas the D

1 and P cells [16]. The secretory

product(s) of the latter cell types (X, D1,

and P) have yet to be definitively charac-terized; however, ghrelin has been local-ized to both the X/A-like cells [9,17] andthe D

1/P cells in the gastric mucosa of rats

and humans [18]. Given that ghrelin ischiefly produced in the oxyntic mucosawhere the histamine-producing ECL cellsare the dominant endocrine population,we wished to explore the possibility thatin humans ghrelin might be an ECL cellproduct. We therefore studied the immuno-histochemical expression of ghrelin inhyperplastic and neoplastic proliferationsof ECL cells to see if it might point us inthat direction.

Material and Methods

The archival files of the Department ofPathology, New England Medical CenterHospital, were searched for all cases of ECLcell hyperplasia and ECL cell carcinoidsdiagnosed between 1988 and 2000. All his-tology slides including special stains(Sevier–Munger stain for argyrophilia andimmunohistochemical stains for serotonin,gastrin, and somatostatin) were reviewedto confirm the diagnosis, and cases withsufficient tissue in the paraffin block toyield at least 10 unstained sections eachwere selected. Three biopsied cases of ECL

Ghrelin in ECL Cell Proliferation 49

cell hyperplasia and five resected specimensof ECL carcinoids were thus identified forinclusion in the study. Of the five resectedECL carcinoids, three cases also had suffi-cient tissue from the adjacent gastricmucosa showing coexistent foci of diffuse,linear, micronodular, and dysplastic ECLcell proliferations.

Serial 5-µm-thick tissue sections cutfrom selected paraffin blocks were indi-vidually mounted onto charged glassslides for immunohistochemical staining.Deparaffinized and rehydrated tissue sec-tions were subjected to antigen retrieval incitrate buffer (0.01 mol/L, pH 6.0) forthree cycles of 5 min each in a microwaveoven, allowed to cool to room temperature,and rinsed in PBS. Nonspecific reactivitywas appropriately blocked by incubationwith normal guinea pig or horse serum, andendogenous peroxidase activity wasblocked with a 0.3% solution of hydrogenperoxide in methanol. Immunohisto-chemical staining for chromogranin, sero-tonin, gastrin, somatostatin, ghrelin, andVMAT-2 were performed using commer-cially available monoclonal or polyclonalantibodies (Table 1). Formalin-fixed, par-affin-embedded sections of gastric bodyand antrum, small intestine, and pancreasserved as positive controls for VMAT-2,chromogranin, gastrin, serotonin, andsomatostatin, respectively. Sections ofhypothalamus and the pituitary glandserved as positive controls for ghrelin.

Negative controls included those in whichthe primary antibody was omitted andreplaced with PBS. Negative controls forghrelin and VMAT-2 additionally consistedof incubation with normal rabbit serum ata dilution similar to that of the primaryantibody. All immunohistochemical stainswere performed by the ABC techniqueusing diaminobenzidine (DAB) as thechromogen. For a specific identification ofnormal, hyperplastic, and neoplastic ECLcells, parallel sections were stained by theSevier–Munger (S-M) technique forargyrophilia and immunohistochemicallywith VMAT-2.

Results

All the eight cases studied representedexamples of one or more stages in the ECLcell hyperplasia–neoplasia sequence, rang-ing from diffuse, linear, and micronodularhyperplasia to dysplasia and overt ECL cellcarcinoids. In each of these proliferativelesions, we were able to demonstrate astrong black, granular cytoplasmic stain-ing (argyrophilia) with the Sevier–Mungerstain and a diffuse granular cytoplasmicimmunopositivity for chromogranins andVMAT-2 within the hyperplastic and neo-plastic cells. Although the adjacent gastricmucosa showed only an occasional neu-roendocrine cell positive for serotonin andsomatostatin, the VMAT-2 positive prolif-erating cells were completely negative foreach of these hormones as well as forgastrin. The histochemical and immuno-histochemical profile of these proliferativeneuroendocrine lesions thus confirmedthem to be of ECL cell origin.

Immunohistochemical staining forghrelin showed a diffuse, brown, granularcytoplasmic staining within the proliferat-ing hyperplastic and neoplastic ECL cellsin 7/8 cases studied (3/3 ECL cell hyper-

Table 1. Primary Antibodies Used in Immunohistochemical Analysis

AntigenAntibody Vendor Detail retrieval Dilution

Chromogranin Ventana Clone: LK2H10 Yes PredilutedSerotonin Ventana Clone:5HT-H209 Yes PredilutedGastrin Ventana Rabbit Yes PredilutedSomatostatin Ventana Rabbit Yes PredilutedVMAT-2 Chemicon Rabbit Yes 1:400Ghrelin Phoenix Pharma Rabbit Yes 1:100


plasias and 4/5 ECL cell carcinoids).In each case of ECL cell hyperplasia, theghrelin immunoreactivity was seenalmost exclusively in the hyperplasticECL cells and was most easily identifiedin areas of linear and micronodular ECLcell hyperplasia (Figs. 1A,B). No suchstaining was seen in the surface or glan-dular epithelium or in the parietal orchief cells. Four of the five ECL cellcarcinoids showed a diffuse cytoplasmicstaining for ghrelin in the tumor cells(Figs. 2A,B). In three of these carcinoidswhere coexistent foci of diffuse, linear,and micronodular ECL cell hyperplasiawere present in the adjacent mucosa, a

similar cytoplasmic immunoreactivityfor ghrelin was seen confined exclusivelyto these hyperplastic lesions.

Correlation of the results of the Sevier–Munger stain for argyrophilia and theimmunohistochemical stains for chromo-granin, VMAT-2, serotonin, and soma-tostatin with those of ghrelin showed theECL cell proliferative lesions to be uni-formly Sevier–Munger, chromogranin,VMAT-2, and ghrelin positive, but nega-tive for serotonin, somatostatin, and gas-trin. The ghrelin immunoreactivity couldthus be clearly identified as being confinedto the proliferating hyperplastic and neo-plastic ECL cells.

Fig. 1. Low-power photomicrographs showing clusters of micronodular ECL cell hyperplasia stained positively with (A) VMAT-2 and(B) ghrelin.


Discussion

The identification of ghrelin from therat gastric mucosa, its characterization as a28-amino-acid peptide, and its acceptanceas a gastrointestinal regulatory peptide rep-resents the culmination of a search for theendogenous ligand for GHS-R. Ghrelinhas since been localized in a wide varietyof other tissues including pituitary [10],hypothalamus [1], placenta [12], kidney[11], fetal lung [13], and, most impor-tantly, the gastrointestinal tract [2,9,19].Although initial studies focused on itsgrowth hormone–releasing properties, sub-sequent work has shown it to have a muchwider spectrum of biologic action. It has

also been shown to stimulate lactotrophand corticotroph secretion, modulate sleepand cardiovascular functions, and to evenexert an antiproliferative effect in neoplas-tic tissues [20–24]. More significantly,however, it has now been shown to alsoplay a critical role in normal energyhomeostasis [15,25] by increasing foodintake and decreasing the utilization of fatfor caloric needs [2,3]. Additionally, it ispossibly responsible for meal initiation aswell [4]. Thus, by virtue of its significantorexigenic and adipogenic properties,ghrelin has now also been implicated in thepathogenesis of morbid obesity [7,8] andanorexia nervosa [6].

Fig. 2. Parallel sections of an ECL cell carcinoid showing (A) VMAT-2 and (B) ghrelin immunoreactivity in tumor cells.


In all mammalian species studied so far,each of the specific neuroendocrine celltypes is associated with a dominant secre-tory product. Although some neuroendo-crine cell types are known to produce twoor more regulatory peptides, production ofa given peptide by two or more differentcell types has not been described. In thiscontext, the widespread distribution ofghrelin, in a variety of different organ sys-tems, none of which shares a common neu-roendocrine cell type, is not only a distinctoddity but also raises the question ofwhether: (a) ghrelin might possibly be pro-duced by an as yet unidentified neuroen-docrine cell type that is widely dispersedin the pituitary, stomach, intestines, pla-centa, kidney, and so on, or (b) that ghrelinis colocalized in a variety of different neu-roendocrine cells, some of which may bedispersed in at least some, if not all, of theseorgans. The bulk of the circulating plasmaghrelin appears to be derived from the gas-tric oxyntic mucosa, which has been shownto have the highest concentration ofghrelin-producing cells [2], even thoughthere seems to be some disagreement as totheir specific identity. Ghrelin productionhas thus far been ascribed to either theX/A-like [9,17] or the D

1/P cells [18] of

the normal gastric oxyntic mucosa, and thealpha cells in the pancreas [26]. Ghrelinexpression has also been demonstrated ina variety of neoplastic endocrine prolifera-tions, e.g., of the pituitary, thyroid, pan-creas, and GI tract [27–30], but there islittle data on the expression of ghrelin inhyperplastic and pre-neoplastic neuroen-docrine cell lesions. Because ECL cells con-stitute the dominant neuroendocrine cellpopulation of the human gastric oxynticmucosa and have a well-delineated hyper-plasia–neoplasia sequence, we decidedto study ghrelin expression in lesionscovering the entire spectrum of ECL cellproliferations.

We were able to demonstrate ghrelinimmunoreactivity in 7/8 cases encompass-ing the entire ECL cell hyperplasia–neo-plasia sequence. In each of these we foundghrelin immunoreactivity to be almostexclusively confined to foci of hyperplas-tic, dysplastic, and neoplastic ECL celllesions that were argyrophilic by theSevier–Munger stain and immunoreactivefor chromogranin and VMAT-2 but notfor gastrin, serotonin, and somatostatin.

As mentioned earlier, ghrelin has beenpostulated to be a product of A-like/X-likecells or the D

1/P cells of the gastric mucosa.

Our study clearly demonstrates its presencein proliferating ECL cells as well, and ourresults are similar to previously publishedreports of ghrelin expression in prolifera-tive ECL cell lesions [29,31]. Ghrelin-producing cells in our study showedsubstantial immunoreactivity with VMAT-2, which is now recognized as a specificmarker for ECL cells [32]. It could beargued that the ECL cells acquire thecapacity to synthesize ghrelin during neo-plastic transformation. Because hyperplas-tic neuroendocrine cell proliferationsinvariably retain their original secretoryprofile and have never been shown to beassociated with ectopic hormone produc-tion, the presence of ghrelin in ECL cellhyperplasia would indirectly suggest thatit might be an ECL cell product. How-ever, the possibility that stimuli that trig-ger the hyperplastic proliferation in ECLcells also somehow confer on these prolif-erating cells the ability to synthesize ghrelincannot be totally discounted. It would beinteresting to see if the plasma ghrelin lev-els demonstrate any correlation with theimmunohistochemical expression ofghrelin in such proliferative lesions. How-ever, no such data are currently available.Also, given the wide spectrum of physi-ologic actions of ghrelin, it would beworthwhile to explore the effects, if any,


of ghrelin overexpression on food intake,sleep patterns, or other GI and cardiovas-cular functions. The possibility thatelevated plasma ghrelin could lead to awell-defined clinicopathologic syndromeby virtue of its GHS and/or other biologiceffects also needs to be investigated further.

In summary, we have demonstratedghrelin immunoreactivity in 7/8 cases ofECL cell proliferations spanning its hyper-plasia–neoplasia sequence. Ghrelin reactiv-ity was clearly confined to proliferating cellsthat showed argyrophilia with the Sevier–Munger stain and positivity for chromo-granin and VMAT-2 but not for gastrin,serotonin, and somatostatin. Because thehistochemical and immunohistochemicalprofile of the proliferating cells conformedto that of ECL cells, we suggest that thegastric ECL cells are either the ghrelin-secreting cells in the human oxynticmucosa or that they acquire the capacityto secrete ghrelin early in their hyperpla-sia–neoplasia sequence. Whether such anexpression might lead to as yet unrecog-nized physiologic disturbances needs to beevaluated.

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ghrelin expression in hyperplastic and neoplastic proliferations of the enterochromaffin-like (ecl)...

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