72horax 1993;48: 1252-1256

Endocrine cells in diffuse pulmonary fibrosis

N J E Wilson, J R Gosney, F Mayall

Department ofPathology, Universityof liverpool,Liverpool L69 3BXN J E WilsonJ R GosneyDepartment ofPathology, LlaaidoughHospital, Penarth,South GlamorganCF6 lXXF MayallReprint requests to:Dr J R Gosney, UniversityDepartnent of Pathology,Duncan Building, RoyalLiverpool UniversityHospital, LiverpoolL7 8XWReceived 25 January 1993Retumed to authors6 April 1993Revised version received6 July 1993Accepted 14 September 1993

AbstractBackground-There is evidence to sug-gest, particularly from studies in ani-mals, that the products of pulmonaryendocrine cells, especially gastrin releas-ing peptide, may have a role in thepathogenesis of fibrosis in the lung. Thisstudy was carried out to examine themorphology, number, distribution, andcontent of pulmonary endocrine cells intissue from 49 patients with diffuse pul-monary fibrosis.Methods-Twenty patients with intersti-tial pneumonitis, 17 with early fibrosis,and 12 with frank honeycombing werestudied, together with five age matchedcontrols without pulmonary disease.Endocrine cells were immunolabelled bythe avidin-biotin complex method fortwo general markers (protein gene prod-uct 9*5 and neuron specific enolase) anda range ofnormal and aberrant secretoryproducts.Results-In the early stages, charac-terised by vigorous pneumonitis,endocrine cells were normal in appear-ance and distribution but very few innumber. They contained only thosesecretory products normally found insuch cells in health; inappropriate sub-stances were not seen. By the time ofearly fibrosis endocrine cells were evenfewer. None were identifiable in the lungsaffected by honeycombing, despite thefact that all contained intact, well pre-served epithelium.Conclusions-It seems unlikely that theproducts of pulmonary endocrine cellscan have any role in the pathogenesis ofdiffuse pulmonary fibrosis in man, thediminution in their number with advanc-ing fibrosis probably reflecting their losssimply as a consequence of generalisedepithelial damage.

(Thorax 1993;48:1252-1256)

The roles of pulmonary endocrine cells inhealthy and diseased lungs remain unclear,but they are almost certainly involved ineither the pathogenesis of, or response to, arange of pulmonary diseases.' One conditionin which they may perform an importantfunction is pulmonary fibrosis, which occurscommonly in human lungs affected byvarious diseases.2

Increased numbers of pulmonaryendocrine cells are found in rats when pul-monary fibrosis is induced by asbestos,34 andthe same phenomenon has been described inman around areas of non-specific localisedfibrosis in which aberrant substances mayappear within them.57 In addition, pul-monary endocrine cell proliferation has beenobserved in conditions such as infantilebronchopulmonary dysplasia8 and pulmonaryeosinophilic granuloma9 in which fibrosis is afeature of the disease process. One of theproducts of these cells, gastrin releasingpeptide, is a powerful mitogen for fibroblastsin vitro.'0 It has been suggested that release ofgastrin releasing peptide from proliferatingendocrine cells in situations such as thesemay be important in stimulating fibro-genesis.8 We have investigated the morphol-ogy, number, distribution, and content ofpulmonary endocrine cells in human lungs inthe various stages of development of diffusepulmonary fibrosis.

MethodsFifty four pairs of lungs were studied, allobtained at necropsy. Five were control lungsunaffected by pulmonary disease, and 20were lungs affected by pneumonitis of inde-terminate cause in which the pulmonaryinterstitium was infiltrated by chronic inflam-matory cells but showed no evidence of fibro-sis. A further 17 were affected by grade 2 andgrade 3 interstitial fibrosis in which the inter-stitium was thickened by the deposition ofcollagen. These were also of indeterminatecause. A final group of 12 patients had grade4 fibrosis with frank honeycombing. All 12patients with honeycomb lung died of theirdisease but, in the remaining 37 subjects witheither pneumonitis or early fibrosis, thecauses of death were wide ranging and unre-lated to the pulmonary disease; the cases inquestion were taken from an archive stretch-ing back many years. It was access to thisarchive that allowed us to obtain relativelylarge amounts of tissue from subjects withpneumonitis and early fibrosis; usually thiscomes to the pathologist only as pulmonarybiopsy tissue. In four pairs of honeycomblungs resulting from asbestosis, asbestosfibres were numerous in all sections studiedbut were not seen in the tissue from any ofthe other lungs.Ten blocks of tissue were taken from each

pair of lungs, two from the mid zone of each

1252

Endocrine cells in diffuse pulmonaty fibrosis

lobe, and fixed in 10% neutral buffered for-malin and embedded in paraffin wax.Sections were stained with haematoxylin andeosin and examined by light microscopy toascertain the stage of fibrosis. Closelyadjacent sections were immunochemicallylabelled by the avidin-biotin complex tech-nique" for neuron-specific enolase and pro-tein gene product 9 5-general markers forcells of the diffuse endocrine system'2 "-andfor a range of their secretory products. Theseincluded substances established as productsof normal human pulmonary endocrine cells(gastrin releasing peptide, calcitonin, calci-tonin gene-related peptide, and serotonin),together with several products which havebeen described in pulmonary endocrine cellsin diseased lungs.5 7 The latter includedadrenocorticotrophin, somatostatin, leucineenkephalin, growth hormone and argininevasopressin.

Following treatment with hydrogen per-oxide in methanol to inactivate endogenousperoxidase and with normal swine serumto prevent non-specific binding of antibody,sections were incubated with the primaryantiserum for a variable period of time,dependent upon the nature and concentra-tion of the antiserum being used. Thiswas then linked to avidin-biotin complexby biotin-conjugated donkey antirabbitimmunoglobulin. The chromogen used was3',3'-diaminobenzidine tetrahydrochloride.Appropriate positive and negative tissue con-trols were employed in all cases. The source,working dilution, incubation period, andnature of the control tissues for each of theprimary antisera are shown in the table.

Immunolabelled sections were examinedby light microscopy and the morphology, dis-tribution, and content of endocrine cells werenoted.

There are various ways in which pul-monary endocrine cells can be quantitated.'The most accurate is to express their number

in terms of the total epithelial population(endocrine cells/10 000 epithelial cells) orunit length of epithelium (endocrine cells/cmepithelium) but, providing sampling is consis-tent and the lungs being studied are all col-lapsed or distended to the same extent,expressing cells per cm2 tissue section is quiteappropriate. All blocks used in this studywere taken from the mid zones of the pul-monary lobes so that, apart from those inwhich honeycombing had destroyed the pul-monary architecture, all contained sections ofintrapulmonary bronchi and terminal and res-piratory bronchioles in generally equivalentproportions, together with a considerableamount of parenchyma. In addition, our pre-vious studies of endocrine cells in diseasedlungs have shown that, when they increase innumber, they appear with some frequency inalveolar ducts and alveoli, whereas in normallungs they are usually absent.' In this locationthey are impossible to quantitate in terms ofepithelial cells or epithelial length, and theonly appropriate method of assessing theirnumber is to express them per cm2. For thesereasons it was considered appropriate toexpress endocrine cells per cm2 of tissue section.

ResultsImmunolabelling for neuron-specific enolaseand protein gene product 9-5 revealedendocrine cells in the control lungs to be reg-ularly but sparsely distributed throughout thepulmonary tree, although they were mostprevalent in small intrapulmonary bronchiand none were identified in parenchyma.Most were solitary cells; only three clusterswere seen in all 10 lungs examined. Thenumber of cells in these five pairs of lungsranged from 7 to 15/cm2 tissue section with amean of 10. About 60% of them containedgastrin releasing peptide and almost all of therest calcitonin. No aberrant peptides wereidentified. This picture is typical of that seenin healthy adult human lungs.'

Details of antisera used in study

Antigen

Neuron-specific enolase

Protein gene product 9-5

Gastrin releasing peptide

Calcitonin

Calcitonin gene-relatedpeptide

Serotonin

Adrenocorticotrophin

Somatostatin

Leucine enkephalin

Growth hormone

Arginine vasopressin

Source of antiserum

Dako CorporationHigh Wycombe, Bucks, UKBiogenesisBournemouth, UKPeninsula LaboratoriesSt Helens, Merseyside, UKDako CorporationHigh Wycombe, Bucks, UK

Peninsula LaboratoriesSt Helens, Merseyside, UK

Peninsula LaboratoriesSt Helens, Merseyside, UKDako CorporationHigh Wycombe, Bucks, UKSeralabCrawley Down, Sussex, UKCRBNorthwich, Cheshire, UKDako CorporationHigh Wycombe, Bucks, UKICN BiochemicalsHigh Wycombe, Bucks, UK

Workingdilution

1:200

1:2000

1:4000

1:2000

ControlIncubation tissue

45 min Human pancreas

Overnight (18 h) Human pancreas

Overnight (18 h) Human fetal lung

45 min

1:4800 45 min

1:1000

1:1000

1:1600

1:800

1:800

1:2000

45 min

45 min

45 min

45 min

45 min

45 min

Medullarycarcinomaof thyroidMedullarycarcinomaof thyroidHuman ileum

HumanadenohypophysisHuman pancreas

Human adrenalmedullaHumanadenohypophysisHumanneurohypophysis

1 253

Wilson, Gosney, Mayall

20.

*.x...,;.-...... ic./.:jis,Ze,

.:....>...y-t:'....'.N :...

:.a

Figure I Typical appearances of the three groups of diseased lungs studied: (a) earlystage ofpneumonitis with, in this case, a prominent desquamative component; (b) earlfibrosis of alveolar walls; and (c) lowerpower view ofa typical example ofcoarsefibrowith honeycombing. Haematoxylin and eosin; original magnification x 700; x 550;x 50, increased to 114% during origination

Endocrine cells were identified in just fourof the 20 pairs of lungs affected by pneu-monitis (fig la). In these four cases the cellscontained gastrin releasing peptide, or calci-tonin, or both; no inappropriate secretoryproducts were identified. They were individu-ally normal (fig 2) and distributed regularly

but sparsely, a picture indistinguishable fromthat seen in the control lungs. In one casethere was a single, localised area in whichthere was a significant increase in their num-ber (fig 3), but this was associated with anarea of pneumonic consolidation. When justthe four pairs of lungs in which endocrinecells were found were considered, their num-bers ranged from 4-21/cm2 with a mean of10/cm2 tissue section. When the whole groupof 20 showing pneumonitis was consideredthe mean was two cells/cm2 tissue.

In three of the 17 pairs of lungs affected byearly interstitial fibrosis (fig lb) endocrinecells were again normal in morphology anddistribution and contained the same two pep-tides as were found in the lungs affected bypneumonitis. Other secretory products couldnot be shown. No cells were seen in the tissuefrom the remaining 14 pairs of lungs affectedby interstitial fibrosis. When just the threepairs with identifiable endocrine cells wereconsidered, they contained between six andeight endocrine cells/cm2 with a mean ofseven. When all 17 were considered, themean was just one.No pulmonary endocrine cells were identi-

fied in any of the 12 cases of "honeycomblung" (fig lc) despite the fact that all con-tained numerous strips of well preservedepithelium lining the abnormal spaces (fig 4).

DiscussionIn conditions in which diffuse pulmonaryfibrosis arises details of the underlying fibro-genic process are poorly understood,although stimulation of fibroblast activity is akey step and a number of different mecha-nisms and chemical mediators has been pro-posed.2 One possible mediator is gastrinreleasing peptide, the mammalian equivalentof bombesin, which is a powerful mitogen for3T3 fibroblasts in vitro.'0 For example, Dayet al'4 1' have shown a 2-2-5 times increase inthe levels of extractable bombesin in thelungs of rats exposed to asbestos, and studiesof pulmonary endocrine cells in animals simi-larly exposed describe changes which alsosuggest a role for their products in pulmonaryfibrosis. Thus, Johnson et al3 and Sheppard etat4 have described increased numbers of thesecells, identified by electron microscopy andimmunolabelling for neuron-specific enolaserespectively, in the fibrotic lungs of rats withexperimentally induced asbestosis.

It is tempting to associate proliferation ofpulmonary endocrine cells and elevated levelsof extractable bombesin with the develop-ment of fibrosis, but there are problems withthis hypothesis. For example, the elevation oflevels of extractable bombesin reported byDay et al'41' occurred not when fibrogenesiswas most active, but after 6-9 months ofexposure when it was well established. Inaddition, and more fundamentally, nobombesin-like peptide has yet been identifiedin the pulmonary endocrine cells of rodents,and its source in the above studies is unclear.

Results of studies of naturally occurring

... w . . ........... w... =. s i .-: r ._S°. - #.' '%.-. - # r ... ::..x§.i:.::-Lis * ! .... I i 11111 - ....... iiEL. i rsiiiL; :^

I-,w .;-. w F:

ds:i ! -:>. .:'Fw-S.:. # .::.. :::f.j^' i* .o' .,;§ .# .. |.* :* .... : .::^ :. .;: . : : :;.

*Fi:' ^ .: :::^' d,d'S i9iS'¢

j..F. .wF .6.

*.i d XfR:'. e ii¢C: ! . g . . .. a/j,RrS e S; J.: .c s s*° .^ F.'^ 3w''. ... '.' .. ' .

.. S:

: .: .:

*. iR .:

.w W

... W .:._ F.,.! . oF e ... ^ 9g g ... 41K:

,,; n w.g..... o. 1gS ''7 :R. 2;f. .S:--^ a>'

F:-:. ^ ::: ^::

v :.:,o.,- vt .lm -eo ;.: tUd.ii4 beM w#.oMM;.ee ............... . W;zi: :. :.e .. E i : .... . . u.,° B :8 ! .......... :: ._E ... ..... 1N' w.:.:.:s 3F W. .......... s :

a .i

v :. s.xsju.s c.; ,.

1254

Endocrine cells in diffuse pulmonary fibrosis 15

r

original~~~~......

manfctoM

Figure 3 Intermpted row of endocrine cells in epithelium lining an inflamed airway in an

area ofpneumonia. Avidin-biotin complex for neuron-specific enolase; originalmagnification x 550, increased to 122% during origination.

Figure 4 Strip of intact epithelium lining an abnormal space in a "honeycomb" lung.Despite the prevalence of such intact epithelium in these lungs, no endocrine cells were

identifiable. Avidin-biotin complex for neuron-specific enolase; original magnificationx 550, increased to 136% during origination.

respiratory disease in man have also been

interpreted as supporting a role for gastrin

releasing peptide in pulmonary fibrogenesis.In bronchopulmonary dysplasia, for example,a condition of infancy in which fibrosis is a

prominent feature, Johnson et a18 have shown

a three fold increase in pulmonary endocrine

cells containing gastrin releasing peptide.

Aguayo et a19 found a tenfold increase in

pulmonary endocrine cells with bombesin-like immunoreactivity compared with con-trols in adults with eosinophilic granuloma.Tsutsumi et all found a proliferation of gas-trin releasing peptide-containing endocrinecells in four out of nine lungs containingareas of non-specific fibrosis.

In the light of the results of these previousinvestigations, the paucity of endocrine cellsin the lungs affected by pneumonitis andinterstitial fibrosis and their absence fromthose destroyed by end stage fibrosis in thepresent study seems initially difficult toexplain. It is possible that the methods weused to identify these cells failed to do so, butthis is highly unlikely, both protein geneproduct 9-5 and neuron-specific enolasebeing excellent labels for them.' It is possiblealso that our sampling was responsible, theairways and parenchyma contained in the sec-tions we examined being unrepresentative.However, all sections contained well pre-served lengths of epithelium, even those fromthe lungs with honeycombing, and the classesof airways included in them are relatively wellendowed with endocrine cells in normallungs, notwithstanding the well documentedvariability in their concentration at differentlevels of the respiratory tree.'

It is interesting to note that Aguayo et al19were also unable to show any significantincrease in pulmonary endocrine cells in thelungs of eight patients with idiopathic diffusepulmonary fibrosis which were included intheir study of eosinophilic granuloma.Indeed, in their investigation these were actu-ally used as controls.We believe it is probable that the pul-

monary endocrine system and the peptides itsecretes are involved, not in fibrosis, but inthe response to infection in the lung and theregeneration of damaged pulmonary tissues.In nearly all the conditions in which increasednumbers of these cells have been described inhuman lungs, either acute or chronic inflam-mation due to infection or, more often, vigor-ous regeneration, especially of epithelium,have been present. Indeed, in the presentstudy the single focus of endocrine cell prolif-eration that we identified surrounded, andwas strictly localised to, an area of infectiveconsolidation.The paucity of endocrine cells in the lungs

affected by pneumonitis and early interstitialfibrosis in the present study is probablysimply a reflection of epithelial damage. Theabsence of these cells from the epitheliumlining the abnormal air spaces in the lungsaffected by honeycombing is interesting andhas not been previously noted. Perhaps itreflects the abnormal envirornment in whichthis epithelium exists.

Nowhere, to our knowledge, have changesin the human pulmonary endocrine systembeen described in lungs affected by sterileinflammation or purely by fibrosis, and stud-ies in animals are simply not comparable. Arole for the products of pulmonary endocrinecells in inducing pulmonary fibrosis in manseems unlikely.

1255

I

I.f.

.0A..

A

Wilson, Gosney, Mayall

1 Gosney JR. Pulmonary endocrine pathology: endocrine cellsand endocrine tumours of the lung. Oxford: Butterworth-Heinemann, 1992.

2 Dunnill MS. Pulmonary fibrosis. Histopathology 1990;16:321-9.

3 Johnson NF, Wagner JC, Wills HA. Endocrine cell pro-liferation in the rat lung following asbestos inhalation.Lung 1980;158:221-8.

4 Sheppard MN, Johnson NF, Cole GA, Bloom SR,Marangos PJ, Polak JM. Neuron specific enolaseimmunostaining. A useful tool for the microscopicaldetection of endocrine cell hyperplasia in adult ratsexposed to asbestos. Histochemistry 1982;74:505-13.

5 Tsutsumi Y, Osamura RY, Watanabe K, Yanaihara N.Immunohistochemical studies on gastrin-releasing pep-tide and adrenocorticotropic hormone-containing cellsin the human lung. Lab Invest 1983;43:623-32.

6 Fukayama M, Hayashi Y, Shiozawa Y, Furukawa E,Funata N, Koike M. Human chorionic gonadotropina-subunit in endocrine cells of fibrotic and neoplasticlung. Lab Invest 1990;62:444-51.

7 Gould VE, Linnoila RI, Memoli VA, Warren WH.Neuroendocrine components of the bronchopulmonarytract: Hyperplasias, dysplasias and neoplasms. LabInvest 1983;49:519-37.

8 Johnson DE, Georgieff MK. Pulmonary neuroendocrinecells. Their secretory products and their potential rolesin health and chronic lung disease in infancy. Am Rev

Respir Dis 1989;140:1807-13.9 Aguayo SM, King TE, Waldron JE, Sherritt KM, Kane

MA, Miller YE. Increased pulmonary neuroendocrinecells with bombesin-like immunoreactivity in adultpatients with eosinophilic granuloma. Clin Invest1990;86:838-44.

10 Rozengurt E, Sinnett-Smith J. Bombesin stimulation ofDNA synthesis and cell division of cultures of Swiss3T3 cells. Proc Nad Acad Sci USA 1983;80:2936-40.

11 Hsu SM, Raine L, Fanger H. Use of avidin-biotin peroxi-dase complex (ABC) in immunoperoxidase techniques:a comparison between ABC and unlabelled antibody(PAP) procedures. JHistochem Cytochem 1981;29:577-80.

12 Wharton J, Polak JM, Cole GA, Marangos PJ, PearseAGE. Neuron-specific enolase as an immunocytochemi-cal marker for the diffuse neuroendocrine system inhuman fetal lung. Y Histochem Cytochem 1981;29:1359-64.

13 Thompson RJ, Doran JF, Jackson P, Dhillon AP, Rode J.PGP 9-5, a new marker for vertebrate neurones andneuroendocrine cells. Brain Res 1983;278:224-8.

14 Day R, Lemaire I, Masse S, Lemaire S. Pulmonarybombesin in experimentally induced asbestosis in rats.Exp Lung Res 1985;8:1-13.

15 Day R, Lemaire S, Nadeau D, Keith I, Lemaire I.Changes in autacoid and neuropeptide contents of lungcells in asbestos-induced pulmonary fibrosis. Am RevRespir Dis 1987;136:906-15.

1256