The composition of gingivalinflammatory cell infiltrates inchildren studied by enzymehistochemistry

G)l Alves Pessanha Alcoforado '̂̂Tore Krisloffersen^ Anne ChristineJohannessen^ and Rune Nilsen^'Department of Periodontology, EscolaSuperior de Medicina Dentaria, Lisboa.Portugal: ^Department of Periodontology and^Department of Oral Pathology and ForensicOdontology, Sotiool of Dentistry, University ofBergen, Bergen, Norway

Alcoforado GAP, Kristoffersen T. Johannessen AC and Nilsen R. The compositionof gingival irtflammatory eell infiltrates in children studied by enzyme histochemistry.J Clin Periodoniol 1990; 17: 335-340.

Abstract. Gingival biopsies were obtained from 23 children, aged 5-11 years(8.6+ 1.8 years). Specimens were taken from areas of the gingiva adjacent to theteeth which were to be extracted because of caries or its sequelae and whichclinically had a gingival index score of at least 1. Staining for a-naphthyl acetateesterase with unspecific esterase at pH 5.8 (ANAE) permitted identification of Tlymphocytes, monocytes/macrophages. plasma cells and non-reactive (ANAE-negative) cells. Cells which tentatively were identified as "natural killer" (NK)cells were also observed. Differential cell counting was performed for 10 specimens,selected on the basis of the presence of a well-defined inflammalory infiltrate,clear morphology throughout and good ANAE staining. Cell counts confirmedearlier studies showing that lymphocytes predominate in the inflammatory infil-trates in childrens" gingivitis. T lymphocytas dominated particularly in theperiphery of the most densely infiltrated areas. Relatively few plasma cells wereseen. It was concluded that T lymphocytes dominate in the inflaimnatory infiltratein childrens' gingivitis.

Key words: children: gingivitis; histochem-istry: infiltrate.

Accepted for publication 26 June t989

Chronic inflammatory periodontal dis-ease (CIPD) is not uncommon inchildren (Jamison 1960). Although itmay clinically produce more dramaticsuperficial changes than commonly seenin adults, it usually develops more slow-ly and does not have the same tendencyto produce irreversible destructive de-fects (Mattsan 1978), Mackler & Craw-ford (1973) and Cox et al. (1974) sug-gested that in the preschool child, gingi-vitis would develop much more slowlythan in the aduh for a given plaqueamount. These findings were confirmedby Mattson (1978). He compared thedevelopment of gingivitis in preschoolchildren and young adults and foundthat the latter group had a higher pro-pensity to develop gingivitis.

It has been suggested (Longhurst etal, 1977) that tbe histological appear-ance of the tissue of a biopsy taken froma preschool child with gingivitis re-sembles an -'early lesion" (Page &Schroeder 1976).

Established gingival inflammatory in-filtrates in adults contain a large pro-portion of plasma cells (Brandtzaeg

1966. Lindhe et al, 1980). The proportionof plasma cells appears to increase withincreasing severity of inflammation(Lindhe et al. 1980). Immunohistochem-ical studies indicate that there is a gradu-ally increasing proportion of plasmacells producing IgG with increasing se-verity of the inflammatory changes(Brandtzaeg & Tolo 1977),

Longhurst et al, (1977, 1980) andSeymour et al, (1981) have presentedstudies based on morphologicalmethods which may indicate that inchildren, the gingival inflammatory in-filtrate contains a higher proportion oflymphocytes than usually seen in CIPDin adults, Seymour et al, (1979), on thebasis of histochemical and immunofluo-rescence studies, have presented evi-dence suggesting that the change froma stable to a destructive lesion may in-volve a shift from a T cell to a B cell(plasma cell) dominated lesion. How-ever. Gillett et al. (1986) held that smallB lymphocytes dominate when the in-flammatory lesion is small and quies-cent, whilst on activation, tbe lesion in-creases in size and many B lymphocytes

are transformed into plasma cells. Thisfmding was refuted by Walsh et al.(1987) on the basis of an extensivestudy, using a panel of monoclonal anti-bodies and histochemical markers.

Thus, the gingival inflammatory reac-tion in children in some respects is simi-lar to the reaction described in adultswith gingival inflammation, but withoutmarked or active destruction of sup-porting tissues (stable lesion) (Seymouret al. 1979). Other authors (Page &Schroeder 1982) have expressed doubtsconcerning tbe validity of the hypothe-sis of Seymour et al, (1979).

Several methods which may be help-ful in characterizing the various cellsin inflammatory infiltrates have becomeavailable. Receptors have been used asmarkers to study the inflammatory infil-trates in various diseases of tbe skin andother tissues (Bjerke 1982. Nyland &Nilsen 1982) including periodontai dis-ease (Kristoffersen et al. 1985). Mono-clonal antibodies bave proved valuablein the study of gingival inflammatoryinfiltrates (Johannessen 1986, Sey-mour & Greenspan 1979, Seymour et

336 Aicoforado et al.

al, 1982, Okada et al. 1983, Walsh et al,1987), This method has the advantageof a high degree of specificity.It requires very gentle handling of thetissues in the laboratory, preferablyusing frozen sections, and only 1 or 2 celltypes can be marked with such specificityin the same section. Staining for a-na-phthyl acetate esterase (ANAE) (Mulleret al, 1975, Ranki & Hayry 1979) has alsoproved to be valuable in differentiatingbetween various cell types found in in-flammatory infiltrates, and this methodhas been used to study cell types involvedin periapical granulomas (Johannessen1986, Johannessen et al, 1984) and inaduit CIPD (Johaunessen et al, 1990),The method has the advantages that sev-eral cell types can be marked with a cer-tain specificity in the same section, fixedtissues can be used and tissue and cellmorphology are well preserved.

The aim of this study was to charac-terize the cell populations in gingivalinflammatory infiltrates in children,panicularly with respect to lymphoidcell types, by means of well-establishedenzyme histochemical methods whichallow for the identification of severalcell types in the same section.

Material and MethodsTissue

Biopsies were obtained from 23 patients,with a mean age of 8,6 years (SD = 1.8.range 5-11 years), undergoing extractionof primary or permanent teeth, andhaving a gingival mdex (GI). (Loe & Sil-ness 1963) of at least score 1 adjacent tothe tooth to be extracted. Cases of juven-iie periodontitis were not included in thisstudy. Informed consent from a guard-ian was obtained in each case.

Biopsies were taken under local anes-thesia and prior to the extraction, usinga disposable no, 15 scalpel, utilizing 2verticaJ incisions and 1 horizontal in-cision at the level of the bottom of theperiodontal pocket. For comparison,some biopsies were taken from patientsundergoing dental extraction andhaving a Gl score 0 at the site. In ad-dition, specimens of human spleen andlymph node obtained at autopsy wereused as controls.

Fixation, sectioning, staining

The biopsies were cut axially in severalpieces in such a way that the junctionalor pocket epithelium, the connective

tissue and the oral gingival epitheliumwould be present in the same section.

The specimens were fixed overnightin a solution of ) % glutaraldehyde di-luted in 0,1 M sodium cacodylate buffer.pH 6,3, with !% sucrose (Bozdech &Bainton 1981) at 4°C, The fixed tissueswere then kept in 0,1 M cacodylate buf-fer (pH 6,3) with 7% sucrose for 24h, dehydrated with acetone, cleared intoluene and embedded in paraffin. Sec-tions were cut at 3-4 ^m. collected ongelatin-coated glass slides and deparaf-finized in toluene.

Sections were stained for ANAE atpH 5,8 according to Miilier et al, (1975),The sections were incubated in a moistchamber for 3 h at room temperature.After incubation, the sections werewashed in phosphate-buffered saline pH7,2 (PBS) for 20 min, dipped in distilledwater, counterstained for 10-15 s withmethyl green, washed in PBS, dehy-drated in ethanol and cleared in toluene.The sections were then mounted in Dep-ex® mounting medium (Difco Labor-atories, England) and examined bytransmitted light.

Sections from all specimens taken atvarious levels during sectioning werestained with hematoxylin, eosin and saf-ranin (HES) for the purpose of orien-tation.

Differential cell counts

Differential cell counts were performedon 1 or more sections from each of 10biopsy specimens stained for ANAE,The sections were selected on the basisof the presence of a well-defined inflam-matory infiltrate, clear morphology andgood quality of the ANAE stainingthroughout. Sources of specimens se-lected for counting are given in Table 2,The mean age of these patients was 9,0years, range 5,0-11,0 years. Non-in-flammatory cells such as fibroblasts, fib-rocytes and endothelial cells were notincluded. Counting was done "blind"and was performed at a magnificationof 400 X , The microscope was connect-ed to a digitizer (Bit Pad One TM, Sum-magraphics, Connecticut, USA) whichwas in turn connected to a computer(Commodore CBM. Model 3032, SantaClara, CA, USA), The computer wasprogrammed (Medizinisch TechnischeApparate, Tubingen, FRG) to recordthe number of ANAE positive Tlymphocytes, plasma cells andmonocytes/macrophages as well asANAE negative cells. Results are re-

Fig. 1. The 3 areas in which differentialcounting of a-naphthyl acetate eslerase(ANAE) positive and negative cells was per-formed. ICT: the most densely infiltratedarea subjacent to the pocket epithelium,SOGE: Connective tissue immediately sub-jacent to the oral gingival epithelium ( x 100),(Drawing: courtesy of Dr, Ivar Holf.)

ported as mean %s of each cell type forthe respective areas.

The counting was performed separ-ately in 2 different areas of the section(Fig, 1):

ICT the inflammatory infiltrate sub-jacent to the junctional orpocket epithehum

SOGE connective tissue immediatelysubjacent to the oral gingivalepithelium.

The various cell types were identifiedaccording to the criteria given in Table1 and all cells within each specified areawere counted.

Statisticai methods

Statistical significance of differences be-tween the ICT and the SOGE in the %distribution of various cell types wasevaluated by the appropriate f-test orby analysis of variance, using the SPSSstatistical package (SPSS Inc, Chicago,III),

ANAE cells in childrens' gingivitis 337

Table 1. Characterization of mononuclear cells according to their ANAE staining pattern

Cell types ANAE staining pattertis Ref

T lymphocytes

monocytes/macrophages

plasma cells

large granular lymphocytes (NKcells)

one or a few discrete cytoplasmic spots

medium to strong difl-use cytoplasmic staining

multiple cytoplasmic granules or diffuse cyto-plasmic staining with spearing cf a perinuclearareamultiple cytopiasrtuc granules

Miiller et aJ. (1975). Raaki et al. (1976), Know-les & Hoick (1978)Ranki & Hayri (1979), Kulenkampff et al.(1977)Hermansky et al. (1980), Muiler el ai. (1981)

Timonen el al. (1979), Huhn et ai. (1982)

Results

The areas to be differentially counted(Fig. 1) and the various types of cells(Fig, 2) were readily distinguished. Tlymphocytes, containing 1 to 3 reddishdots, were readily recognized (Fig, 3).and so were monocytes/macrophages(Fig, 3). Tbe latter showed considerablevariability both with respect to size andmorphology and to staining intensity.Plasma cells showed variable intensityof staining. Scattered cells with the mor-phology and ANAE staining patterncharacteristic of NK cells were oc-casionally observed along the peripheryof the ICT (Fig. 5),

In some discrete regions immediatelysubjacent to the junctional or pocketepithelium, T lymphocytes were particu-larly numerous, and these areas alsocontained many cells identified asmacrophages or monocytes,

T lymphocytes were also frequentlyseen within the pocket epithelium (Fig.4). However, the infiammatory infiltratein most regions contained many cellswhich did not give a staining reactionwith the ANAE reagents. Such cells ap-peared to be somewhat more numerousalong the periphery of the ICT, In thisarea, plasma celts, although few in num-ber, were more frequently encountered.

In the connective tissue subjacent tothe oral gingivai epithelium (SOGE),the scattered inflammatory cell infiltrateexhibited a predominance of macro-phages or monocytes (Table 3), Thesecells were most often relatively smalland round, lacking dendritic projec-tions. Cells of similar appearance werealso seen within the oral gingival epithe-lium. Clusters of T lymphocytes alsooccurred in this area, but both Tlymphocytes and ANAE-negative cellswere proportionally fewer than in theICT

The density of inflammatory cells(number of cells per microscopic field)was approximately twice as bigh in the

Fig. 2. ANAE positive cells in the infiltrated connective tissue subjacent to the pocketepithehum. The infiltrate is predominated by T lymphocytes with 1 or a few dots (arrows)( X 800).

Fig. 3. T lymphocytes (open arrows) and monocytes/macrophages (arrow) in the infiltratedconnective tissue subjacent to the pocket epithelium (x 1120).

338 Alcoforado et a!.

Tr<j:i^

Fig. 4. T lymphocytes (arrows) within the pocket epithelium (PE). ICT, most densely infiltratedarea ( x 860).

central portion of the ICT compared tothe peripheral portion and the SOGE.Different microscopic fields in the vari-ous areas often exhibited remarkablysitnilar distribtition of the various typesof inflammatory cells, and repeatedblind countings of related areas showedho significant variability of results.

In Table 3, the mean %s of the 4types of cells in the different areas arepresented. Statistical significance of dif-ferences in proportions of the variouscell types between the 2 main areas areaiso noted.

Discussion

The ANAE staining reaction togetherwith cotinterstaining and good cellularmorphology permitted evaluation of thelocalization and distribution of the vari-ous cell types. Some cells which wereslightly larger than lymphocytes butwhich exhibited a granular ANAEstaitiing pattern of their cytoplasm orhad more than 3 ANAE reactive dots,could possibly represent NK cells. Suchcells are difficult to identify with cer-tainty on the basis of morphology and

staining reaction and were not includedduring counting. Thus, some cells classi-fied as plasma cells or T lymphocytesmay have been NK cells. Preliminarystudies in our laboratory using mono-clonal antibody to NK cells indicatethat they are present as scattered cellsin gingival inflammatory infiltrates.

Only cells with the characteristic mor-phology and a positive ANAE stainingreaction were counted as plasma cells.The number of plasma cells recordedmay therefore be somewhat low. sinceit is known that some plasma cells donot give positive ANAE staining(Johannessen, Nilsen & Skaug 1984).

The majority of inflammatory cellscounted as "ANAE negative cells" ex-hibited lymphocyte morphology andprobably represent B lymphocytes.However, some of these ANAE negativecells may represent plasma cells. A pro-portion of the ANAE negative cells mayactually have been polymorphonuclearleukocytes. In their study of lymphoidcell subpopulations in gingivitis inchildren, Seymour et al. (1981) reportedthat less than 1% of the infiltrating cellswere monocytes, polymorphonuclearleukocytes or plasma celis. Their esti-mate appears to be in reasonably goodagreement with the findings in the pres-ent study.

Our findings using enzyme histo-chemical markers have corroboratedand extended the observations ofLonghurst et al. (1977). Using methylgreen-pyronin stain on sections of in-flamed gingiva from adults andchildren, they found that small and me-dium-sized lymphocytes predominatedin the latter group. Our findings are,however, at some variance with the ob-servations of Longhurst et al. (1980).On the basis of electron microscopic ob-servations which demonstrated a rela-tive lack of transforming T lymphocytes

Table 2. Age, sex, area of biopsy with GIscore for that area for 10 patients for whomdifferential countings were performed

Fig. 5. ANAE positive cell (arrow) with a staining pattern characteristic of natural killer (NK)ceils (X 1200).

Patientno.

I23456789

10

Age(years)

101179

118

105

118

Sex

MMFFFFMFFM

Area of biopsy

74 - buccal53 - buccal

51, 52-buccal24 ~ buccal55 - buccal85 - buccal46 - buccal75 - buccal54 - lingua!

74, 75 - buccal

GI

2113212222

ANAE ceiis in chiidrens' gingivitis 339

and a rather high proportion of plasmacells, they cotnpared the inflatnmatorylesion of chiidrens' gingivitis with astage between the early and estabhshedlesion as seen in adults (Page & Schroe-der 1976). Our findings are also appar-ently in conflict with the recent report byGillett et al, (1986), which indicated thatthe lymphocytes associated with gingi-vitis in children are primarily B lytnpho-cytes. They based their findings on theuse of a monoclonal atiti-HLADr anti-body. This antibody would mark both(certain) T and (certain) B lymphocytesand their results would consequently notbe directly comparable with ours.

To a large extent, our observationscorroborate the fmdings of Seymour etal. (1981) and Walsh et al. (1987) withrespect to the composition of the lymph-oid cell population in gingivitis inchildren. Seymour et al. (1981) alsofound that lymphocytes predominatedin these lesions and estimated that ap-proximately 70% of these lymphocyteswere T celis as evidenced by their non-specific esterase activity. Using a mono-clonal antibody reacting with mature Tcells and some thymocytes on sectionsfrom 2 individuals. Seymour et al,(1982) found that 75-80% of thelymphocyte population were T cells,Walsh et al, (1987) using a panel of anti-T-cell antibodies on gmgival sectionsfrom 10 children found that the gingivalinflammatory lesions consisted largely(80-88%) of T lymphocytes. In ourstudy, the proportion of lymphocytesrecorded as T cells was somewhat lower,and ANAE negative cells were actually

equally numerous in the most denselyinfiltrated area. However, these ANAEnegative cells also include a few poly-morphonuclear cells, plasma cells andpossibly mast cells. Our observationsthat plasma ceils constituted a verysmall proportion of the inflammatoryinfiltrate in the inflamed gingiva of thechildren studied, are also in accordancewith the observations of Seymour et al,(1981, 1982) and Walsh et al, (1987),

Although direct comparisons can notbe made, we got the impression that thevariability from section to section andfrom biopsy to biopsy was less pro-nounced in children than in adult CIPD(Johannessen et al, 1986, 1990), Furtherstudies of gingival inflammatory infil-trates in children are called for. Suchstudies should aim to elucidate boththeir cellular composition with morespecific markers and their variabilityfrom individual to individual and fromsection to section in the same specimen.

Zusammenfassung

Die Zusammensetzung des gingivaien entzund-licken Zeilinfiitrates bei Kindern, studiert mit-teis EnzymhistochemieBei 23 Kinderu im Alter voti 5-i i Jahren(8.6+ 1.8 ,Tahre) wurden Gingivabiopsien ge-nommen. Die Proben stammten von amZahn befindlicher Gingiva. Die Zahne wur-den wegen Karies oder ihrer Folgen extra-hiert und ihr kiinischer Gingiva-lndexwertbetrug wenigstens 1. Die Farbung der a-Naphtylazetatesterase mit unspezifischerEsterase bei pH 5.8 (ANAE) erlaubte dieIdentifizierung von T-Lymphoryten, Mono-zyten; Makrophagen. Plasmazellen und nicht-

reaktiven Zeiien (ANAE-negativen) ZeUen.Zellen die vorlaufig als "natlirliehe iCillerzel-len" (NK) identifiziert wurden, waren auchzu beobachten. Bei zehn Proben wurde aufder Basis eines gut deftnierteti entztindhchenInfiltrates, einer uberall klaren Morphologieund guter ANAB-Farbung. eine differenzier-te Zellauszahlung durehgefuhrt. Die Zellaus-zahlung bestatigte frlihere Studien, die zeig-ten, daB Lymphozyten im entzundlichen In-filtrat der Gingivitis bei Kindern dominieren,T-Lymphozyten dominieren leilweise in derPeripherie der am dichtesten infiltrierten Re-gionen. Relativ wenig Plasmazellen wurdenbeobachtet, Es wurde gefolgert, dafi T-Lym-phozyten im entziiEdliclien Infiltrat der Gin-givitis bei Kindern dominieren.

Resume

Composition des infiltrats de cellules inflam-matoires dans ia gencive ehez les enfants -etude histoehimique enzymatiqueDes biopsies gingivaies ont ete prelevees chez23 enfants ages de 5-11 ans (moyenne 8,6,ecart-type 1,8 ans), Les specimens ont etepreleves dans des zones oii la gencive attenaita des dents devant etre extraites pour catisede carie ou de complications de la carie, etoil les scores de l'indice gingival etaient> I,Une coloration pour a-naphtyl acetate estera-se avec esterase nor specifique a pH 5,8(ANAE)apermisd"identirierdes)}Tnphoc>lesT, des monocytes.'macrophages, des piasmo-cytes et des cellules sans reaction (ANAE-ne-gatif). On observait aussi des cellules qu'onclassait comme cellules NK dans une tentatived'identification, Des numerations differentiei-les ont ete pratiquees pour 10 specimens, choi-sis parce qu'ils presentaient un infiltrat inflam-matoire bien defini, une morphologie nettedans tout le specimen, et qu'ils prenaiem bienla coloration ANAE, Les resultats des nume-

Tabie 3. Mean %s of various cell types in the gingivai (ICT) and in the area underlying the orai gingivai epithelium (SOGE) for the 10 patientsfor whom differential countings were performed; total mean (± standard deviation = SD) and ranges

Patientno.

ICT SOGE

Cell types Cell t\pes

M/M T cells PI. cells neg. cells M.M T cells PI. cells neg. cells

123456789

10

9,726,412,610.727.017.312.837.619,921,2

40,638,046,764,845,053,554,538,943,446.1

2,02,71.25.44.02,74,54,82,10.4

47.832,939,419,124,026,528,118,834,532,4

27.645,857,966,542.540.549.135,245,050,5

39.133,627,721,933,134,530,439,136,426.4

5.72.30,21,11,62,43,61,91,4

27.618,314,210,422,822,617,023,817,220.9

mean

SD

range

19,5*

8,9

9,7-37,6

47.2*

S.3

38,0-64,8

3.0

1.6

0.4-5,4

30,4*

9.0

lS,8-t7,8

46.1

11.0

27,6-66.5

32.2

5.6

21.9-,39.!

1.5

0.2-5,7

19.5

5.1

10.4-27.6

M/M=monocytes. macrophages; T cells=T lymphocytes; PI. cells= plasma cells; neg. cells=ANAE negative cells, mainly B lymphocytes,• Significant difference (i '<0,05) between ICT and SOGE,

340 Alcoforado et al.

rations montraient que, dans la gingivite del'enfant, les lymphocytes predominaient dansles infiltrats inflammatoires. Les lymphocytesT dominaient particulierement a la peripheriedes zones presentant les infihrats les plus den-ses. On observait relativemem peu de plasmo-cytes. En conciusion, Tinfiltrat inflammatoiredans la gingivite dc I'enfant est domine par leslymphocytes T.

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Address:

Tore KristoffersenDepartment of PeriodonlologySehool of DentistryUniversity cf BergenArstadveien 17N-5009 BergenNorway