Cell death in granulomata: the role of apoptosis · As apoptosis occurs over a period ofabout 12-18 hours, the number of apoptotic bodies we observed indicated considerable cell loss

J Clin Pathol 1987;40:1314-1319

Cell death in granulomata: the role of apoptosis

I A CREE, S NURBHAI, G MILNE, J SWANSON BECK

From the Department of Pathology, Ninewells Hospital and Medical School, Dundee, Scotland

SUMMARY Unequivocal apoptoses were seen by light microscopy in examples of leprosy, sarcoid-osis, tuberculosis, Crohn's disease and foreign body granulomata. A limited electron microscopicinvestigation showed typical apoptotic bodies in both sarcoid and leprosy granulomata. The num-ber of apoptoses and mitoses in granulomata were counted and their densities calculated. The widevariation in the results between individual lesions may reflect differences in disease activity.

Current understanding of cell turnover in granu-lomata is based largely on studies of experimentallyinduced granulomata in animals by Dannenberg,Spector, and others.' These experiments establishedthat mononuclear phagocytes in granulomata have afinite life span and that this varies with the elicitingagent. The eventual fate of mononuclear cells ingranulomata is not clear, although evidence fromsome studies suggests that cell death has a majorrole.2 Cell necrosis is not readily apparent in non-caseating granulomata, however, and hence re-gression of differentiated mononuclear phagocytes toless mature forms with subsequent emigration fromthe lesion has been proposed as an alternative expla-nation.3

Another possible mechanism for the loss of mono-nuclear phagocytes from granulomata is apoptosis, amorphologically distinctive form of cell death whichaffects isolated cells in living tissue.4 Apoptosis occursin a wide range of physiological and pathological cir-cumstances, including involution of the adrenal cor-tex,5 secretory endometrium,6 tadpole tails,7 andbasal cell carcinoma.8 Cells undergoing apoptosisshow condensation and fragmentation into a numberof membrane bound eosinophilic fragments which arethen endocytosed by surrounding cells, even if theseare not normally phagocytic.9 In contrast with truenecrosis, cells undergoing apoptosis do not provokean inflammatory response. As the process lasts amatter of hours, detection of even small numbers ofapoptotic bodies within tissues indicates appreciablecell loss.

During a recent study of the histology of leprosylesions, we observed apoptoses within both tuber-culoid and lepromatous lesions.10 We now have ex-tended these observations to include several other

Accepted for publication 28 May 1987

granulomatous conditions in man. In some of these itproved possible to count the apoptotic bodies andcalculate their density within tissues to estimate cellloss by this mechanism. As the size of the granulomareflects the balance of cell gain and cell loss mitoseswere also counted to give an estimate of gain fromthis source. It was not possible to measure cellularimmigration in fixed preparations.

Material and methods

Five granulomatous conditions were studied: leprosy,tuberculosis, sarcoidosis, Crohn's disease, and for-eign body granuloma. Biopsy specimens from theedge of leprosy lesions were obtained using a 4 mmsterile disposable skin punch (Stiefel Laboratories,Slough, England) from 29 untreated and seventreated Bangladeshi patients with leprosy. Infonnedconsent was obtained verbally in all cases and histo-pathology reports were sent back to the relevant med-ical officers. After transportation to Britain in 4%buffered formaldehyde the biopsy specimens were bi-sected, embedded in paraffin wax, cut at 5 pm, andstained with haematoxylin and eosin.

Similarly processed sections of the other conditionsobtained from the diagnostic histopathology fileswere stored at Ninewells Hospital. These wereidentified using the SNOP classification from biopsyspecimens received over 18 months (July 1984 to De-cember 1985). Cases were included in this study pro-vided that: there was no doubt about the diagnosis;necrosis was absent; and well formed granulomatawere present (table 1). A proportion of the cases ex-amined were unsuitable for counting of apoptosesdue to the presence of polymorphonuclear leucocytedebris, which it can sometimes be difficult to dis-tinguish from apoptosis. Polymorphonuclear leuco-cyte infiltration was particularly prominent in the

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Table 1 No ofcases and diagnosis

Diagnosis Total No examined No counted Granuloma area ( > 0 3 mm2)

Foreign body granuloma 13 13 13Leprosy 56 56 36Sarcoidosis 20 11 11Tuberculosis 11 4 4Crohn's disease 25 0 0Total 127 86 64

examples of Crohn's disease which were examinedand none was suitable for quantitation of apoptoses.

LIGHT MICROSCOPYGranulomata were identified by scanning the sectionat low power (at a magnification of 40). In some caseswith large granulomata the areas for counting weredelineated by marking the coverslip with a black felt-tip pen; in others the whole section or granuloma wasexamined. Apoptotic and mitotic bodies were coun-ted by scanning the selected area at a magnification of400 using a 25 square eyepiece grid (Leitz) to ensurethat non-overlapping fields were examined. Struc-tures were identified as apoptotic bodies if theysatisfied three of the following four criteria: (i) ovoidor spherical shaped body; (ii) eosinophilia; (iii) sepa-ration from neighbouring cells; (iv) condensation orfragmentation, or both of nuclear material.The area of the section occupied by granuloma was

determined using an Imagan planimeter (GraphicInformation Systems, Blairgowrie, Scotland) aspreviously described.11 Statistical analysis was per-formed using Statgraphics (STSC, California, USA)on an IBM PC-AT microcomputer. Differences inapoptotic and mitotic density between the conditionsstudied were examined using the Kruskal-Wallis testfor one way analysis of variance by ranks.

ELECTRON MICROSCOPYMaterial for electron microscopy was obtained fromone case of sarcoidosis of the skin and from five casesof leprosy, all of which were classified as borderlinetuberculoid or borderline lepromatous on the Ridley-Jopling scale.12 Large granulomata were identified onthe cut surface of the fixed biopsy specimen using astereoscopic microscope and dissected out. After fur-

Table 2 No ofsections examined which containedapoptoses and mitoses

Cases Caseswith with

Diagnosis apoptosis mitosis Total

Foreign body granuloma 5 5 13Leprosy 33 19 56Sarcoidosis 14 9 20Tuberculosis 9 3 11Crohn's disease 13 15 25

Table 3 No of cases with apoptoses and mitoses in sectionswith granuloma size of > 0 3 mm2

Apoptosis Mitosis Total NoDiagnosis present present counted

Foreign body granuloma 5 5 13Leprosy 27 19 36Sarcoidosis 11 9 11Tuberculosis 4 3 4Total 47 36 64

ther fixation in osmium tetroxide the blocks were em-bedded in araldite, ultrathin sections were cut, andthen stained with uranyl acetate and lead citrate. Thesections were viewed using a JEOL IOOCX trans-mission electron microscope.

Results

Unequivocal apoptoses were seen in all of the types ofgranuloma studied, including Crohn's disease. Exam-ples of the histological appearance of these are shownin fig 1. Although prolonged searching was necessary,the electron micrographs show typical apoptoticbodies at various stages of progression (fig 2), lyingamong epithelioid cells in one case of sarcoidosis ofthe skin and one case of borderline lepromatousleprosy.Most of the apoptoses were found in areas consis-

ting mainly of epithelioid cells in all of the granu-lomata studied, with the exception of lepromatousleprosy where they were scattered among thehistiocytes. There was no microanatomical associ-ation between apoptoses and mitoses, which werecommoner at the edge of the granulomata. Table 2shows the number of cases with apoptoses and mito-ses. Foreign body granulomata (FBG) were leastlikely to contain apoptoses, followed in order of in-creasing frequency by Crohn's disease, leprosy, sar-coidosis, and tuberculosis. Mitoses were leastcommon in tuberculosis and commonest in Crohn'sdisease. Similar numbers of foreign body granuloma,leprosy, and sarcoidosis biopsy specimens containedmitoses, but these figures take no account of the areaof the granuloma examined, which has to be of a cer-tain size before rare cellular events are likely to beobserved.Measurement of the density of apoptosis in those

Apoptosis in cell death in granulomata 1315



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cases with granulomata of 0 3 mm2 area or greater There were considerable differences between(table 3, fig 3) confirm that even those foreign body different patients with the same disease, particularlygranulomata with apoptoses have a lower apoptotic in leprosy. When the results from the leprosy biopsydensity than the other types of specimen examined. In specimens were examined against the clinico-all of the pathological specimens studied the density pathological classification of the patient on theof apoptosis was much higher than mitosis, sug- Ridley-Jopling scale,'2certain patients in the border-gesting that apoptosis may be more common than line tuberculoid (BT), borderline lepromatous (BL),mitosis in granulomata in man. and lepromatous group showed high densities of apo-

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Fig 2 Electron micrographs ofapoptotic bodies in (a) sarcoidosis and (b) borderline lepromatous leprosy.




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Cree, Nurbhai, Milne, Beck

ptosis and mitosis. The numbers of patients in eachclassification are too small for reliable conclusions tobe drawn and further studies will be necessary to es-tablish the clinical importance of a high apoptoticrate, although its presence is likely to indicate thatsuch patients have a higher rate of cell turnover.

DiscussionThe life span of mononuclear phagocytes in granu-lomata varies from a few days to several weeks. 13 - 1 5The fate of these cells has been in some doubt, but celldeath, cell division, and emigration following rever-sion to less mature forms have all been suggested.'Studies of experimental granulomata show thatmononuclear cells do not divide repeatedly after theyhave entered the granuloma.2 Both emigration ofcells from lesions16 and reversion to less matureforms have been shown to occur,3 but most authorsconsider that death of constituent cells in situ ac-counts for most of the cell loss that occurs duringgranuloma regression.' 3 Epithelioid cell death by

* necrosis is seen in tuberculosis at the edge of caseousE areas, but in leprosy and sarcoidosis caseation is a

rare occurrence and it could not account for the re-gression of the granulomata that commonly occurswith medical treatment. Apoptosis was seen in all ofthe granulomatous conditions which we studied andapoptotic bodies could be counted in the granu-lomata using the light microscope. As apoptosisaffects individual cells which may be widelyseparated, apoptotic bodies can be difficult to find byelectron microscopy. Nevertheless, we were able toconfirm their presence in both biopsy specimens frompatients with sarcoidosis and leprosy using thismethod.As apoptosis occurs over a period of about 12-18

hours, the number of apoptotic bodies we observedindicated considerable cell loss from the granu-lomata.'7 This is in keeping with the degree of cellloss found in previous kinetic studies of experi-mentally induced granulomata.' The occurrence ofboth apoptosis and caseation in tuberculosis is inter-esting, although there may be no relation between thetwo processes.

In comparison with the numbers of apoptosespresent, only small numbers of mitoses were seen ineach type of granuloma studied. There is considerablevariation of mitotic density between individuals, asthere is in apoptotic activity. The duration of mitosisis shorter than that of apoptosis, but our results sug-gest that many more cells were being lost as a result ofapoptosis than were being gained by mitosis. This is

L consistent with data obtained from experimentalgranulomata which suggest that cell influx is moreimportant in maintaining the size of the granu-loma.' 1418

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By studying the distribution and kinetics of labelledmononuclear cells in experimental granulomata, elic-ited by a variety of stimuli, Spector et al divided gran-ulomata into high and low turnover types.13` 5 18Low turnover granulomata form in response tocarrageenan and other inert particles, whereas highturnover granulomata form in response tomycobacteria, such as BCG. Histologically, epi-thelioid cells and giant cells are rare in low turnoverlesions but common in high turnover lesions. On thebasis of this correlation it has been suggested thatsarcoidosis is a high turnover granuloma.15 Ourresults show that apoptotic and mitotic activity varybetween the various types of granuloma studied, aspredicted by previous studies of experimental granu-lomata. Foreign body granulomata, which are oftenformed in response to inert materials, showed thelowest density of apoptoses and mitoses in this study.Tuberculosis and leprosy showed much higher ratesof apoptosis and mitosis, observations which are pre-dictable from their histology as well as their my-cobacterial causation. The aetiologies of sarcoidosisand Crohn's disease are still a matter of controversy.Our results confirm previous suggestions that sarcoid-osis is a high turnover granuloma, while the positionof Crohn's disease is less clear. Human granulomataseem to exhibit a spectrum of cell turnover and maynot fit neatly into high or low turnover categories.The place of individual disorders in this spectrummay correlate with the immunogenicity of the elicitingstimulus, but host factors must also be important, asshown by the variation in apoptotic and mitotic den-sity between individuals with a particular disease (fig3). The reasons for these differences are not clear fromthis study, but the possibility that they might be re-lated to disease activity is of particular interest in lep-rosy in which certain patients have a propensity forthe development of damaging immunologically medi-ated reactions, while others show no such tendency.The balance between cell gain, resulting mainly

from the influx of mononuclear cells, and cell loss aremajor determinants of the size, rate of expansion, andrate of regression of granulomata. Our results suggestthat apoptosis plays a major part in the regression ofgranulomata in man. The factors which influenceapoptotic activity and cell influx are likely to be im-portant in determining whether a particular granu-lomatous lesion progresses or regresses during thedevelopment of the disease and whether it respondsfavourably to medical intervention.

We thank Drs D Hopwood, I H Cochrane, T Ali andMr S Halda for their assistance. We are grateful toMr G Coghill for technical assistance and to Mr RFawkes for preparation of the photographs. Thework in Bangladesh was funded by the award of the

Becton Dickinson travelling scholarship to IAC bythe Royal College of Pathologists. The laboratorywork was funded by grants from LEPRA and JamesFinlay plc.

References

1 Adams DO. The granulomatous inflammatory response. A re-view. Am J Pathol 1976;84:161-83.

2 Dannenberg AM, Ando M, Shima K. Macrophage accumu-lation, division, maturation and digestive and microbiocidalcapacities in tuberculous lesions. III. The turnover of macro-phages and its relation to their activation and antimicrobialimmunity and in primary BCG lesions and those of reinfection.J Immunol 1972;109:1 109-21.

3 Adams DO. The structure of mononuclear phagocytesdifferentiation in vivo. II: The effect of Mycobacterium tuber-culosis. Am J Pathol 1975;80:101-16.

4 Kerr JFR, Bishop CJ, Searle J. Apoptosis. Recent Adv Histo-pathol 1984;12:1-15.

5 Wyllie AH, Kerr JFR, Macaskill IAM, Currie AR. Adre-nocortical cell deletion: the role of ACTH. J Pathol1973;1 11:85-94.

6 Hopwood D, Levison DA. Atrophy and apoptosis in the cyclicalhuman endometrium. J Pathol 1976;1 19:159-66.

7 Kerr JFR, Harmon B, Searle J. An electron microscope study ofcell deletion in the anuran tadpole tail during spontaneous met-amorphosis with special reference to apoptosis of striated mus-cle fibres. J Cell Sci 1974;14:571-85.

8 Kerr JFR, Searle J. A suggested explanation for the paradoxicallyslow growth rate of basal cell carcinomas that contain numer-ous mitotic figures. J Pathol 1972;107:41-4.

9 Weedon D, Searle J, Kerr JFR. Apoptosis. Its nature and impli-cations for dermatopathology. Am J Dermatopathol1979;1:133-44.

10 Cree IA, Gardiner CA, Beck JS. Studies of cell death (apoptosis)and cell division in leprosy granulomata. International Journalof Leprosy 1986;54:607-13.

11 Cree IA, McDougall AC, Coghill G, Beck JS. Quantitation of thegranuloma fraction in leprosy skin biopsies by planimetry. In-ternational Journal of Leprosy 1985;53:582-6.

12 Ridley DS, Jopling WH. Classification of leprosy according toimmunity: a five group system. International Journal ofLeprosy1966;34:255-73.

13 Ryan GB, Spector WG. Natural selection of long-lived macro-phages in experimental granulomata. J Pathol 1969;99:139-5 1.

14 Ando M, Dannenberg AM Jnr, Shima K. Macrophage accumu-lation, division, maturation and digestive and microbiocidalcapacities in tuberculous lesions. II. Rate at which mono-nuclear cells enter and divide in primary BCG lesions and thoseof reinfection. J Immunol 1972;109:8-19.

15 Spector WG. Immunologic components of granuloma formation.Epithelioid cells, giant cells and sarcoidosis. Ann N Y Acad Sci1976;278:3-6.

16 Smith JB, McIntosh GH, Morris B. The migration of cellsthrough chronically inflamed tissues. J Pathol 1970;100:21-9.

17 Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance ofapoptosis. Int Rev Cytol 1980;68:251-306.

18 Papadimitriou JM, Spector WG. The ultrastructure of high- andlow-turnover inflammatory granulomata. J Pathol1972;106:37-43.

Requests for reprints to: Dr IA Cree, Department of Pathol-ogy, Ninewells Hospital and Medical School, PO Box 120,Dundee DDI 9SY, Scotland.



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Cell death in granulomata: the role of apoptosis · As apoptosis occurs over a period ofabout 12-18 hours, the number of apoptotic bodies we observed indicated considerable cell loss