Effect of experimental neutropenia on initial gingivitis in dogs R. ATTSTROM AND H. E. SCHROEDFR Department of Periodontolo^, Royal Denial College, Aarhus, Denmark, and Department of Oral Structural Biology, Dental Institute, University of Zurich, Zurich, Switzerland-

Attstrom, R. & Schroeder, H. E.: Effect of experimental neutropenia on initial gingivitis in dogs. Sciind.J. Dent. Res. 1979: 87: 7-23 Abstract — The role of neutrophilic granulocytes in the loss of gingival collagen has been studied by inducing experimental neutropenia during initial gingivitis in beagle dogs. Neutropenia was induced for 4 d in three animals with normal gingiva by repeated injections oi rabbit anti-neutrophil serum. During neutropenia microbial plaque was allowed to form on the teeih. Samples of junctional [crevicular) leukocytes and gingival fluid were taken on days 0 and 4. Block hiopsies of buccal gingiva were obtained on day 4. Stained .semi- and ultrathin sections were used for histometric and stereologic tissue analysis. Gingival fluid flow increased from day 0 to day 4 in all dogs while junctional leukocytes increased in one dog only. Subgingival plaque had formed in most biopsies, and in the junctional epithelium very few neutrophilic granulocytes were present. In the coronal connective dssue subjacent to the junctional epithelium lymphoid cells, structurally abnormal neutrophiiic granulocytes and monocytes/macrophages were diffusely scattered. The gingival collagen appeared mainly displaced hy the inflammatorj' cells rather than dissoh'ed. The data suggest that neutrophilic granulocytes may contribute to the loss of gingival collagen during initial gingivitis in dogs. The neutrophils also seem to be of importance for the limitation of subgingival plaque growth along the tooth surface. Keywords: dogs; gingivitis; experimental; neutropenia. R. Attstrom, Department of Periodontolog)-, Royal Detital College, Aarhus, Vennelyst Boulevard, DK-8000 Aarhus, Denmark Accepted for pubhcation 9 October 1978

An optimally normal and healthy gingiva can be achieved in dogs by regular prophylaxis over a period of about 3 months. When plaque is allowed to accumulate

along the gingival margin under experimental conditions, significant histopathologic alterations occur during the first 4 days of the inflammatory

ATTSTROM AND SCHROEDER response (14, 25). The reaction arises in the coronal pan ot the marginal gingiva and is characterized by an acute exudation, a 60% loss of collageti fibers at the reaction site, and the development of a lyinphoid cell infiltrate (14, 19, 25). Furthermore, a gingival sulcus is formed, and leukocyte migration is enhanced. The pathogenic mechanisms generating" this collagen loss have not been elucidated although neutrophilic granulocytes and niorioc\'tes/macrophages have been stispected to play a role. These cells selectively release or secrete lysosomal enzymes when exposed to bacteria in dental plaque or their products in vitro (5, 15-17, 29). In addition, neutrophilic granulocytes are known to play a key role in peripheral defense against microbial infection {21, 22, 28). Severe inflammator)' periodontal disease may develop under neutropenic conditions or functional deficiency of neutrophilic granulocytes (7). Thus the aim of the present investigation has been to study the role of gingival neutrophilic granulocytes in initial gingivitis by experimental reduction of these cells utilizing anti-neutrophil serum.

Material and methods ANIMALS Three healthy male beagle dogs were used. The animals were from the same litter and 1 year old at the start of the experiment. Ninety days prior to experimentation the teeth of the dogs were thoroughly cleaned by scaling and polishing and thereafter subjected to systematic toothcleaning three times weekly (3). Gingival normaiit)' (i.e. less than 1096 of the gingival units showing some exudation of gingival fluid (3)) was established some 2 weeks before the start of experimentation. During these 2 weeks, the teeth were cleaned daily, and the diet consisted of raw bovine trachea (8).

ANTl-NEtJTROPHIL SERUM lANS; ANS was raised in rabbits using neutrophilic granukxyteb collected from glycogen-induced peritoneal exudates in beagle dogs. The ANS was treated and adsorbed by dog er\'throcytes, livei-, kidney- and spleen cells, as described previously (4). The final preparation of ANS w-as adjusted to a leuko-agglutination titer of 1:500. This ANS' was. administered subcutaneously iti the back oi the dog's neck using dosages from 0.5 to 2.0 ml/kg body weight. The actual dosage selected for a particular dog at a given moment of time was dependent on tbe dog's response to the amount of ANS adinmistered previously; the first dosage given always being 1.5 tnl/kg body weight. The effect ol ANS was detertnined by total and diiierential coutiEs of blood leukoc^'tes, eiythrocytes and piatelets. The individual re.sponse to ANS-administration is shown in Fig. 1. EXPERIMENTAL PROCEDURES Approximately 2 weeks after gingival normality had been reached, ANS was injected for the hrst time in the morning of a given Monday (day 0). Injections and blood counts were repeated on days 1, 2, and 3 after day 0, as indicated in Fig. 1. During this latter period, the dogs were given a soft diet (8). Samples of junctional epithelial leukocytes (jEL; iormerly termed crevicular leukoc)'tes) and gingival fluid were collected in the mornings of days 0 and 4 Irom buccal gingival units of maxillary' right incisors (central region], canines, fourth premolars and first molars (mesial and distal regions), and of mandibular right canines and first and second molars (mesial and distal regions). These samples were analyzed with respect to the amount of leukocytes and fluid present (2). Around midday of day 4, block biopsies ol buccal gingival tissues were taken from the maxillary right first, second, and third premolars, and the mandibular right second, third, and fourth premolars (26).

BIOPSY PROCESSING Biopsies were processed under standardized conditions for light and electron microscopy (26). Stained semithin and uitrathin sections

EXPERIMENTAL NEUTROPENIA NUMBER OF N E U T R O P H I L S / m l BLOOD

f

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13000

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AM

PM DAY 1

DAY 0

PM

AM

DAY 2

PM

AM:

DAY 3

THE DAY DAY 4

Fig. I. Number ol neutrophilic granulocytes/ml blood prior to and during experimentation in ihree dogs. Table 1 Clinical score data (mean + s.e.) for gingival Jlutd (GF) and junctional epithelial leukocytes (JEL) al days 0 and 4 of experimenlai neutropenia in dogs GF Dog

DayO Mean±s.e.

Day 4 Mean + s.e.

DayO Mean± s.e.

Day 4 Mean + s.e.

3

4.3 2.5 3.4

2.2 1.4 1.6

15.1 3.2* 25.2 5.6" 11.1 1.7*

38.3 7.0 44.8 7.0 31.8 5.2

64.2 9.5* 46.3 6.8 S8.0 5.9

12 12 12

C

2.4

O.I

52.6 9.6

27.8 4.4

58.2 4.5

48

1

2

C, control data for normal dogs (see (25)). N, number of buccal gingival units included (see (3)). " Signiiicantly (P < 0.01) different from day 0 value. Table 2 Average proportions (percent) of neutrophilic granuhcytes (NG), lymphocytes (L) and momcytes/macrophagei (MM) among junctionat epithelial leukocytes on days 0 and 4 of experimental neutropenia in dogs

Day 4

DayO Dog 1

2 S

NG Mean+ s.e. 97 .4 0.5 97 .6 « . 4 97 .2 0.4

L Mean + s.e.

MM Mean + s.e.

NG Mean ± s.e.

L Mean + s.e.

MM Mean+s.e.

0

2. 6 0.5 2. 2 0.4 2. 5 0.4

14.6 1.2* 14.8 l.S* 3.8 0:9'

8 .0 12 .9 3 .8

1.4* 3.1* 0 .9*

7 7.4 1.7* 72.3 2.6* 92.4 1.2-

0.2 O.S

0. 1 0. i

' Significantly (P < 0.001) different from day 0 value.

10

ATTSTROM AND SCHROEDER

sensed ior histometric measurements and a stereologica! analysis oi the gingival tissues. [3). HISTOMETRiC AND STEREOLOGIC A.NALYSiS Four biopsies per dog, i.e. from the second and third and the mandibular third and fourth right premolars, served for quantitative assessments of tissue composition. A standardized sample of four blocks (tissue sJites) per biopsy was selected. From each block one stained semithin section served for histometric and stereologic analysis on magnihcation levels 1—4, and one uitrathin section (comprising a coronal portion of the connective tissue adjacent to the jutictional epithelium and the gingival sulcus) was used (or stereologic analysis at magnification levels 5 and 6. Thus, a total of 16 semithin and 16 ultrathio sections per dog were available for analysis. In all semithin sections (a total of 48)^ the distance between the gingival margin and the apicai termination of subgingival plaque, i.e. subgingival plaque extension (SPE), was measured on the screen of a Wild sampling microscope M 501, using a magnification of X 200. This distance was expressed in fim and averaged per tooth and dog unit. In order to achieve a hierarchical system of additive volumetric data^ and because constitueot identification is dependent on magnification, stereologic analysis was performed on six different levels of magnification, using point-counting procedures. Analysis on levels 1—3 followed the sampling design described previously (26, 27). Uvd 1 - The cross-sectional image of free and attached gingiva was artificially divided inio a marginal (MGT representing the site of inHammator)' reaction) and a cetA'ical (CGT) gingival tissue portion by placing an imaginary horizontal line across the middle between the gingival margin and the cemento-enamel junction (Fig. 2f. A magnification of x 400 and the multi-purpose test system M-42 (31) was used in order to estimate the volume densities of oral and junctional epithelia, the marginal (MCT) and cervical (CCT) connective tissue portions belonging to MGT and CGT respectively. Level 2 - A magnification of X 750 and the M-42 test system was used for estimating the

volume densities of collagen fibers, vessels including their lumina, and the residual tissue residing in MCT and CCT. Preliminary tests demonstrated that with the position of test helds as shown in Fig. 2a an optima! estimation ol collagen density would result for both MCT and CCT (27). Level S — A magnification of x 1000 and a coherent doubie-latdce test system B-25 (31) was used for assessing the volume densities of netitrophilic granulocytes and mononuclear cells residing within the jutictional epithelium, using the 100 light points of this lattice (26). Level 4 — A magnihcation of x 1000 and the B-25 test system was again used to estimate tl]e volume densities of collagen fibers and vessels (27). The sampling of test fields at this level is shown in Fig. 2b. This sample served for analyzing two different areas of the MCTconnective tissue, subjacent to the junctional (zone 1 representing the center of inflammatory reaction) and adjacent to the t>ra] epithelium (zone 2). Level •> - A series of nine electron microgiaphs per block was recorded at a primary magnification of x 890 (Fig. 2c, large open squares). At a secondary magnification (table projection) of X 10 240 the tnultipurpose test system M-42 was used for estimating the volume and numerical density of various cellular constituents and the volume density of collagen fibrils situated within the MCT-portion adjacent to the junctional epithelium (3). This sample site coincided with that of zone 1 in level 4. Level 6 — The sample consisted of five electroti micrographs per block, recorded at a pritnary magnification of X 3080 (Fig. 2c, black squares). A secondary tnagnification of X 35 260 and a coherent double-lattice test system C-64 (31) was used to determine the volume density of collagen fibrils in the ground substance of perivascular connective tissue in the MCT-portion adjacent to the junctional epithelium.,

DATA RECORDING AND COMPUTATION Primary counts were collected through respective keyboard buttons in separate counters of an electronic unit (30), Volume (Vy) and numerical (Ny) density parameters were calculated by applying basic stereologic formulas (30, 32). Calculations were

EXPERIMENTAL NEUTROPENIA

Fig. 3. Schematic illustradon indicating size and localization of sampling lields used at six levels of magnification. a = level 2, b = level 4, c=levels 5 (large squares) and 6 [small full squares)..

ATTSTROM AND SCHROEDER

12

performed on a Hewlett-Packard 9S30A calculator, using three different ofi-Iine computer programs, designed for automatic processing ol the data. Average parameters were calculated per jaw and dog, and expressed as densit)' fractions per cm^ unit volume of gingival tissue (GT) and gingival tissue portions (MGT, CGT), or per cm' of gingival connective tissue {GT) and gingival connective tissue portions (MGT, CCT).

CONTROL EXPERIMENTS Control data for comparison with those of the present study originate from an identically performed investigation of initial gingivitis in normal beagle dogs (25). In planning the ANS experiments,, the difficulties in keeping the neutropenic dogs alive were not realized. The control dogs died before the experiment had been carried through. The large oral wounds which result from the biopsy procedure obviouslv facilitate bacterial dissemination to an extent not compatible with survival of the animal. Therefore, no gingival biopsies were taken in the dogs of the present study until completion of the ANS-period (day 4).

Results INDUCTION OE NEUTROPENIA

The injection of ANS resulted in a rapid decrease in the number of blood neutrophils/ml of blood, eliminating approximately 99% of the neutrophilic granulocytes normally present (Fig. 1), Some variations in the reduced number of blood neutrophils could, however, be noted in each dog, although the state of relative neutropenia was maintained throughout the experimental period in all dogs. The neutropenic state was most pronounced in dog 3. The numbers of erythrocytes and platelets never deviated from normal values.

CLINICAL OBSERVATIONS

On day 0 the buccal gingival tissues were normal. Gingival fluid flow was minute and JEL were Few (Table 1). At day 4 the gingiva and part of the alveolar mucosa were deep red. These changes were most pronounced in dog 3, which had also developed isolated areas of tnucosal ulcerations. Spontaneous gingival bleeding occttrred in all dogs but most frequently in dog 3. Gingival fluid flow increased signihcantly from day 0 to day 4 in all dogs while JEL increased significantly in dog 1 only. On day 0, the JEL were composed of around 97% neutrophils, 0.5% lymphocytes and 2-3% monocytes/macrophages. These proportions had changed to 5—15% neutrophils, 4—13% !ymphoc^'tes and 7 7—92% monocytes/macrophages on day 4 (Table 2). GE.NERAL MORPHOLOGY

The appearance of the gingival tissues of dogs 1 and 2 was different from that of dog 3 at day 4. In dogs 1 and 2 a gingival sulcus of varying depth was found. The connective tissue adjacent to the coronal part of the junctional epithelium comprised numerous vessels, some inflammatory cells and moderately dense collagen fibers (Fig. 3a and b). The inflammatory cells were scattered and did not form a definable infiltrate. Dental plaque extended on the average 290+ 150fim subgingivally. Neutrophilic granulocytes and monocytes/macrophages were seldom observed between the plaque and the junctional epithelium. The junctional epithelium exhibited some rete peg formation in the coronal part (Fig. 3b). Ultrastructurally, the marginal con-

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ATTSTROM AND SCHROEDER

14

nective tissue adjacent to the junctional epithelium comprised dense collagen fiber bundles and various mononuclear cells. The coUagenous fiber sheath around the vessels was tnostly still intact. Only minute signs of an acute inflammatory exacerbation were present. Thus, fibrin deposition or collagen fibril dissolution were rarely observed, Intraand extravascular neutrophils were abnormally structured. These ceils, often seen between densely arranged collagen fiber bundles, contained few atypical lysosomes, some endoplasmic reticulum and large, multiple glycogen granules (Fig. 4a), In addition, signs of autolysis were observed in these cells and neutrophil fragments were also observed inside macrophages.

Tlie gingival tissues of dog 3 showed a shallow gingival sulcus. No accumulations of round cells were found in the connective tissue. Numerous dilated blood vessels occurred between the densely organized collagen fiber bundles (Figs. 3c, d; 4). Subgingival plaque was less frequent than in dogs 1 antl 2 and extended on the average 50 + 80 fim subgingivally. No signs of an inflammatory exudation or infiltration were found ultrastructurally in the tissues of dog 3. Dense bundles of collagen fibrils filled the extravascular spaces.

Free lysosomes of neutrophil origin did not occur in the tissues. The very few neutrophilic granulocytes and motiocytes/macrophages encountered close to the subgingival plaque surface rarely demonstrated signs of phagocytosis (Fig. 4c).

The tissue of the maxilla and mandible of dogs 1 atid 2 did not differ in composition. Therefore, the data for dogs 1 and 2 have been pooled while the data of dog 3, being different,, are presented separately.

STEREOLOGIC DATA

Dogi

1

microscopic

and

2:

Levels

analysis) -

1-4

The

(light

gingivai

Table 3 Volume density (mm'lcm^ gingival tissue; GT) of various gingival constituents of neutropenic dogs at end (day 4) of period of initial gingivitis

Dogs 1 and 2 Parameter

Symbol

Gingival tissue Epithelium Oral Junctional Junctional leukocytes Connective tissue (CT) Collagen fibers Vessels Residual Marginal CT Collagen fibers in MCT Vessels in MCT Residual in MCT Cervical CT

GT Vve

. Vvoe Vvje Vv]

Vvct Vvco Vvw

Vvre Vvmct Vcomct ^ Vvemct ^ Vremcc

Vvcc.

x±s.e. 1 488 389 99 2.4 512 342 68 102 140 64 18 58 S72

40 28 14 0.6 40 25 17 42 57 32 6 22 25

Dog 3 x± s.e.

Dimension

1 438 16 346 19 3 92 1.3 0.2 562 16 5 437 84 4 41 56 110 52 79 42 11 8 1 20 452 36

an^ tnm'/cm^ rom'/cm? mm'/cm' tnm'/ctn' mmVcm* mmVcm^ mm'/cm^ mm Van' mmVcm' mm'/cm' mm'/cm" mm^/cm^ mm^/cm"

EXPERIMENTAL NEUTROPENIA

tissues consisted of 49% epithelium and 51 % connective tissue, 67 % of which was occupied by collagen hbers (Table 3). The marginal connective tissue (MCT) made up 27% and the cervical connective tissue (CCT) 73 96 of the total gingival connective tissue. The MCT comprised 46 96 collagen fibers and 13% vessels, the residual occupied by cells and interstitium (41%) (Table 3). In MCT-zone 1 adjacent to the

15

juiictional epithelium, collagen fiber density was 25% and in MCT-zone 2 adjacent to the oral epithelium, 64 S6. Blood vessel density were similar in these two zones (Table 5). In the CCT region the collagen fibers occupied 73 %. In the junctional epithelium there were 2.4% leukocytes, &% of which were neutrophils, and 92% mononudear cells (Table 4). Dog J: Levels 1-4 (light microscopic

Table 4 Volume density (mm'/cm' gingival tissue) ofjunctional epithelium (VijJ and neutrophiiic granulocytes fyvngj ""'^ mononudear cells (Vy^^J residing within JE of nattropenic dogs at end (day 4) of period oj initial gingivitis Dog

N

Vvje

* Vngc

x±s.d.

S + s.d.

Ratio

V vi

Vmoe

9ty

x±s.d.

%

9fD

1/2 3

8 4

99± 14 92 ± 3

0.2±0.1 0.1 ± 0.1

7.5 8.0

2.2 + 0.6 1.2±0.1

92.5 92.0

2. 4 1.4

1:11.0 1:12.0

C

6

94+ 17

1.2±0.3

40 .0

1.7 ±0.5

60.0

3. 0

1:1.50

C, control data lor normal dogs (see (25)). N, number ol biopsies; data expressed as means + standard deviations.

Table 5 Volume density of collagen (co) and vessels (ve) in mm?lcm^ af marginal connective tissue (met) and cervical connective tissue (ccl) respectively in gingiva of netUropenic dogs at end (day 4) of period of initial gingivitis. Data are expressed as means ± standard deviations. Number of biopsies were four per dog

Dogs 1 and 2

Dog 3 i.d.

X+i

Parameter

Symbol

x±s.d.

Collagen fibers in MCT Vessels in MCT Collagen fibers in zone 1 of MCT Collagen fibers in zone 2 of MCT Vessels in zone I of MCT

VVcoinct VVvemct VVco

495 34 196 64

zone 1 VVco zone 2

252 76

676 25 15 208 614 101

639 73

824

8

101 21

102

18

Vessels in zone 2 of M CT

VVve

25

VVcocct VVvccct

148 43 728 43 116 47

70

Collagen fibers in CCT Vessels in CCT

803 130

2 29

VVvc

zone 1 zone 2

'is'' -I .1 K-- - ^'^£f

Fig 4 Typical election micrographs illustrating collagen fibril densit)' m perivascuiar tegions of iubepithelial (junctional eptthelium) connective tissue in buccal giogiva of dog 2 (a) and dog 3 (b), and marginal subgingival plaque in dog 2 covered by mononuclear leukocytes (c). Neutrophils show an abnormal cytoplasmic structure (a). BV, blood vessel, M, monocyte. NG,, neutrophilic granuloc)'te. L, lymphocyte, a, b, x 6000; c, x 5850.

EXPERIMENTAL NEUTROPENIA analysis) - The gingival tissues were composed of 44% epithelium and 56% connective tissue, 78% of which was occupied by collagen fibers and 15 96 by vessels. The MCT was 20% and CCT 80% of the total gingival connective tissue. Collagen fibers constituted 72%, blood vessels. 10% and the residual 18% of the MCT (Table 3). The difference in collagen density between MCT zones 1 and 2 was smail, that of the latter being similar to die collagen density in the cervical connective tissue (Table 5). The junctiona! epithelium contained 1.4% leukocytes, of which 8% were neutrophils and 92% mononuclear cells (Table 4-). Dogs 1 and 2: Levels i and 6 (electron

microscopic analysis) — In the connective tissue adjacent to the coronal part of the junctional epithelium, collagen fibrils made up 1 7 % of the volume, cellular constituents 43% and residual tissue 40% (Table 6). The density of the perivascular collagen fibrils per connective tissue ground substance (Level 6) amounted to 20 + 4%. Six percent of the volume was neutrophils, and 11 % monocytes/macrophages. The medium-sized lymphocyte was the most numerous lymphoid cell, the remaining immunocompetent cells being 3% or less each. In total around 830 x 10* cells were present per cm' of this connective tissue portion. Dog J:

Levels

5

and

6

(electron

microscopic analysis) - The connective tissue adjacent to the jtuictional epithelium comprised 52% collagen fibrils, 20% cells and 28% residual dssue. The density of perivascular collagen fibrils per connective dssue ground substance was 34 + 8% (Table 6). The volume density of neutrophils was 0.4%, of monocytes/macrophages 596, and of medium-sized lymphocytes 4%. The 2

17

remaining lymphoid cells contributed around 1 % each to the volume. In total, 410x 10^ cells were present per cm^ of this connective tissue area.

Discussion The repeated injections of ANS resulted in a relative and profound neutropenia in 3:11 animals. However, some variations in the number of blood neutrophils were observed both within the same dog and between the dogs. This reaction pattern towards the ANS was in accord with observations from previous studies utilizing ANS as an experimental tool (1, 4, 24). The effects of the ANS on the morphology and number of intra- and extravascular neutrophils together with the decrease of neutrophils among JEL indicate that the antiserum severely interfered with neutrophil function in the experimental animals. The response of normal gingiva towards plaque formation in a state of relative neutropenia was clinically characterized by an intense vascular reaction and by a low exudation of gingival fluid. Microbial pfaque formed and rapidly extended subgingivally while neutrophils were scarce in the junctional epithelium. In dogs 1 and 2 only minute signs of both an acute exudative process and collagen dissolution were noted in the connective tissue adjacent to the coronal part of the junctional epithelium. Most of the coliagenous framework was retained, and infiammatory cells had accumulated in between the collagen fiber bundles, although without forming a dense connective tissue infiltrate. Numerous abnormally structured neutrophils were present in the connective tissues of dogs 1 and 2 and macrophage phagocytosis of neutrophil remnants was li-equently seen.

18

ATTSTROM AND SCHROEDER

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EXPERIMENTAL NEUTROPENIA

Jn dog 3, no signs of exudative phenomena were found in the connective tissue. The rapidity of subgingival plaque proliferation was remarkable. The maxima of subgingival plaque extension were between 0.4 and 0.8 mm, implying a daily advancement of 0.1—0.2 mm. The mode of microbial advancement, i.e. proliferation, locomotion, transport, is unknown at present. However, it seems obvious that nonnally functioning neutrophils and other exudative phenomena operate to prevent subgingival advancement of microorganisms. Long-term experimental neutropenia in rats has been shown to result in necrosis of the periodondum ultimately leading to complete tissue destruction (20). Furthermore, patients with juvenile periodontitis seem to suffer from neutrophils with a reduced phagocytic capacity in comparison with neutrophils ol normal individuals (7). Neutrophil phagocytosis does occur in the dentogingival region (9, 13). Nevertheless, other mechanisms, e.g. release of lysosoma! enzymes from neutrophils accumulating at the tooth surface, exudation of immunoglobulios, complement, may aid in controlling bacterial advancement. Despite deep plaque extension along the tooth surface, and despite the paucity of neutrophils in the junctional epithelium of the neutropenic dogs, no bacterial penetration into the tissues was seen. This lack of bacterial tissue penetration was also observed in a previous study on the effect of ANS on established chronic gingivitis in dogs (24). It is conceivable that mononuclear cells which, in contrast to cells in normal dogs during initial' gingivitis, were numerous in the junctional epithelium of the neutropenic dogs and still capable of phagocytosis and lysosomal enzyme

19

release, may have subserved the function of neutrophils in the prevention of bacterial penetration into the gingival tissue (6, 11). Although an intense vascular reaction was noted in all neutropenic dogs of the present study, edema formation and gingival fluid Row were less pronounced than in normal dogs during initial gingivitis. Experimental reduction of gingival neutrophils using ANS in dogs with chronic gingivitis lowers the concentration of lysosomal enzymes of neutrophil origin in the dentogingival region. Also gingival fluid flow decreases during the neutropenic state (4, 24). The present findings confirmed these statements and indicated that they are valid for initial gingivitis as well. The exaggerated vascular response resulting in pronounced gingival redness of the neutropenic dogs is corroborated by findings in humans suffering from neurropenia (12, 23). Possibly, the vascular reaction during neutropenia is an attempt to compensate for the lack of neutrophils by increasing the blood supply to the gingival margin. On the other hand, no vascular reaction of this kind was found during neutropenia in dogs with chronic gingivitis (24). In normal dogs undergoing initial gingivitis, the reaction site iTi the connective tissue adjacent to the junctional epithelium is characterized by an acute inflammatory process breaking up the dense network of collagen 6ber bundles in this area. The inflamed tissue compiises exudative material including normal and disintegrating inflammatory cells, isolated free lysosomal bodies and fibrin deposition. Dissolution of collagen fibrils is a prominent feature of this lesion (14, 18, 25). In the neutropenic dogs the gingival connective tissue revealed only small signs of acute inflammation, and collagen fibril

ATTSTROM AND SCHROEDER

20

dissolution was minute. These findings suggest that neutrophilic granulocytes may, through their lysosomal material, contribute to the generation of the intense inflammatory reaction of initial gingivitis. The volume density of inflammatory cells other than neutrophils was as large, equal or even higher in the neutropenic dogs than in normal dogs, indicating that the gingival immune reaction in neutropenic dogs was similar to that obseri'ed in normal dogs during initial gingivitis (Table 7). A high density of extravascular neutrophilic granulocytes was observed in the connective tissues of dogs 1 and 2 while these cells were virtually absent in dog 3. In dogs 1 and 2 these cells were abnormally structured and possibly had a severely reduced functional capacity'. In vitro studies have shown that antineutrophil antibodies can damage the cells and cause release of lysosomal enzymes (10). If this had occurred to neutrophils accumulated in the gingiva, a high degree of injurious effects would have been expected. Therefore, the

limited tissue damage observed in dogs 1 and 2 may imply that the neutrophils had been functionally defective already prior to entering the gingival connective tissue. Furthermore, these cells also appeared to have a reduced capacity to respond chemotactically to the subgingival bacteria as most of the neutrophils accumulated in the connective tissue rather than emigrating via the junctional epithelium. The reaction sites in dogs I and 2 at day 4 consisted of approximately 17 % collagen fibrils, 43% cellular constituents and 40% residual tissues. The corresponding figures lor normal dogs were 24%, 24%, and 52% (Table 6). Thus, in normal dogs the volume densities of cells and collagen librils are equal while the residual occupies twice the size of the collagen volume. In the neutropenic dogs 1 and 2, however, the cells occupied a 2.5 times larger volume than the collagen fibrils while the residual volume, mainly of blood vessels, was equal to the volume of collagen fibrils (Table 8). These data suggest that in normal dogs the collagen fibril reduction during initial gingivitis is

Table 7 Percent contribution of various celts in lymphoid cell population in connective tissue adjacent to junctional epithelium at day 4 of initial gingivitis in neutropenic dogs

Neutropenic dogs Day 4 Cell t\'pe

Day 4

Dogs 1 and 2

Dog 3

3 62 7 7 21

8 58 10 3 21

S 62 1 8 21

21 51 10 6 12

152

66

37

77

Small lymphocytes Medium lymphocytes Immunoblasts X-cells Plasma cells Lymphoid cell Volume mmVcm^

Normal dogs' DayO

Data from SCHROEDER et al. (25).

21

EXPERIMENTAL NEUTROPENIA Table 8 Ratios between total cell volume and collagen fibril volume and between residual volume and collagen fibril volume in connective tissue adjacent to junctional epithelium in neutropenic dogs at day 4 and in nwmal dogs al days 0 and 4 during initial gingivitis

Neutropenic dogs Day 4 Dogs 1 and 2 Cells/ collagen fibrils Residual/ collagen fibrils

Normal dogs' Day 0

Day 4

Dog 3

2.6

0.4

0.4

1

2..4

0.5

0.4

2

* Dala from SCHROEDER et al. (25).

almost entirely compensated for by an increase in the residual tissue, while in the neutropenic dogs 1 and 2 the reduction in collagen density was compensated for primarily by aa increase in the cellular constituents. Similar calculations for dog 3' showed no deviation from the tissue composition in normal healthy dog gingiva (Table S). These data in combination with tissue morpholog)' at the reaction site imply that in the neutropenic dogs collagen was mainly displaced rather than dissolved. Taken al! together the results seem to indicate that neutrophiiic granulocytes moderate the acute inflammatory process and may contribute to the collagen dissolution normally observed during initial gingivitis. Neutrophils also seem to counteract microbial advancement along the tooth surface subgingivally. Further studies into this latter phenomenon might elucidate the importance of neutrophiiic granulocytes in the prevention of subgingival plaque formation.

Acknowledgemerlts — We wish to thank Mrs. M. GRAF-DE-BEER for designing additional coniputer programs for automatic data processing and for her invaluable help in perform-

ing part oi the morphometrie analysis. Furthermore, we are indebted to Mrs. S. MONZEL-PEDRAZZOLLI and Miss K. ROSSINSKY

for skillful technical assistance.

References 1. ANDERSEN, L., ATTSTROM, R. & FEJERSKOV,

O.: The effect of experimental neutropenia on oral wound healing in guinea pigs. Scand. J. Dent. Res. 1978; 86: 237-247 2. ATTSTROM, R . & EGELBERG, J.: Presence of

leutocjtes within the gingival crevices during developing gingivitis in dogs. / . Periodontal Res. 1 9 7 1 : 6 : 110-114 S. ATTSTROM, R., GRAF-DE-BEER,

M.

fc

SCHROEDER, H. E.: Clinical and histologie characteristics of normal gingiva in dogs. J. Periodontal Res. 1975: 10: 115-127 4, ATTSTROM, R., TYNELIUS-BRATTHALL, G , &

EGELBERG, J.: Effect of experimental leukopenia on chronic gingival inflammation in dogs. n . Induction of leukopenia by heteroiogous anti-neutrophil serum, J. Periodontal Res. 1971: 6: 200-210 5, BAEHNI, P., TSAI, C , TAICHMAN, N . 8C

MCARTHUR, N . P . : Interacdon of inflammatory cells and oral microorganisms, V, Electron microscopic and biochemical study on the mechanisms of release of lysosomai constituents from human poljinorphonuclear leukocytes exposed to denul plaque,/. Periodontal Res. 1978: 13: 333-348

ATTSTROM AND SCHROEDER

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EXPERIMENTAL NEUTROPENIA 28. StNN, H. J. & JUNGI, W. F.: Neutrophil migration in health and disease. In: HtlMBERT, J. R., MlESCHER, P. A. & JAFFE. E. R. (eds.): .Neutrophil phymlogj and pathology. Grune & Stratton, New York 1975, pp. 25-43 29. T.AiCHMAN, N. S. & MCARTHUR, W . P.: Interaction of inflammatory ceiU and oral bacteria: Release of lysosomal hydrolases From rabbit poljinorphonuclear leukocytes exposed to gram-posidve plaque bacteria. Arch. OralBwl. 1976: 21: 257-263 30. WEIBEL, E. R. : Stereologk principles for

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Effect of experimental neutropenia on initial gingivitis in dogs.

Effect of experimental neutropenia on initial gingivitis in dogs R. ATTSTROM AND H. E. SCHROEDFR Department of Periodontolo^, Royal Denial College, Aa...
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