Scand. J. Dent. Res. 1977: 85: 396-406 {Key words: immunization; .monkey; piilpUii)
Experimental pylpitis in immynized monkeys GUNNAR BERGENHOLTZ, STAFFAN AHLSTEDT AND JAN LINDHE Departments of Endodontics and Periodoniology and Institute of Medical Microbiology, University of Gothenburg, Gothenburg and Astra Liikemedel AB, Research and Development Laboratories, SedertSlje, Sweden
ABSTRACT - Bovine serum albumin (BSA) was topically applied to exposed dentin to assess whether inflammatory reactions can be induced in the pulp of monkeys immunized against BSA. Four cynomolgus monkeys received repeated injections of BSA emulsified with Freund's incomplete a.djuvant. Pulp challenge was performed by applying BSA in freshly cut dentin cavities prepared on the buccal surface in 34 teeth. In 29 control teeth ovalbumin (OVA) was applied. Control applications of BSA were also performed in 15 teeth prepared in three nonimmunized monkeys. Forty-eight hours after the initiation of the pulp challenge the monkeys were sacrificed and the ptilp tissue examined in the light microscope. Topical application of BSA to freshly exposed dentin in immunized monkeys resulted in severe inHamtnatory lesions in the pulp, characterized by bleeding and extravaScular infiltration of large numbers of leukocytes. Extensive tissue damage was an important feature in several pulps. Identical applications of OVA in control teeth and BSA applications in nonimmunized monkeys produced no such reactions. The results indicate that interactions between antigens and antibodies can occur within the dentin-pulp area and following the formation of immune-complexes, severe injury to the pulp can be induced. (Accepted for publicatim. 6 November 1976)
Previous investigations have revealed that cultivated from human dental plaque, a crude extract from human dental plaque similar phenomena developed in the pulp contains components with the capacity (BERGENHOLTZ 1977). The studies referred to induce or mediate acute inflamma- to show that freshly cut coronal dentin tory reactions in the dental pulp has a poor capacity to protect the pulp (BERGENHOLTZ 8c LiNDHE 1975). The from noxious agents such as microbial extract was applied in freshly cut dentin plaque. It is therefore not surprising to cavities in monkeys. Obvious signs of find that pathologic reactions can already vasculitis and leukocyte accumulation be detected in the pulp when a carious could be observed 10-32 h later in the lesion, which is infectious in nature, has pulp adjacent to the cavity preparation, reached the outer layer of dentin When the plaque extract was substituted (BRANNSTROM & LiND 1965, LANGELAND by material produced by microorganisms & LANGELAND 1968, BAUME 1970).
PULPITIS IN MONKEYS It has been suggested that immunologic mechanisms may contribute to the tissue breakdown in pulp inflammation (LANGELAND
1 9 7 2 , SELTZER & BENDER
1975). Considering the excessive amounts of antigens produced by the microorganisms involved in the caries process atid the presence of plasma cells and lymphocytes in the pulp tissue, SELTZER & BENDER (1975) assumed that synthesis of immunoglobulins may occur in the pulp. The formation of antibodies against the intruding antigens may protect the pulp, but the subsequently formed antigenantibody complexes may also, following complement activation, cause h^persensitivity reactions and destroy the pulp tissue.
397
immunization. Egg albumin (OVA; BDH Chetnicals Ltd., England) was used as a control substance. IMMUNIZATION PROCEDURE
Four monkeys (two males and t^vo females} received subcutaneous and intramuscular injections of BSA dissolved in saline and emulsified in equal volumes of Freund's incomplete adjuvant (FIA). At four different occasions 1-2 weeks apart a dose of 50 mg BSA in 2 ml saline was injected in multiple sites in the thighs and/or the back close tO' the lymph nodes. Six weeks after the last administration of BSA a booster injection of the same amount of BSA in FIA was given. Two of the monkeys (C 17 and CIS; Tables 1 and 2) had at this time poitit low antibody titers and were given one additional booster injection; C 17 with BSA in Freund's complete adjuvant (FCA). The aim of the present investigation was Six auiniais [three males and three females) to study whether bovine serum albumin were not treated and were used as controls. Scrum was sampled immediately before the (BSA) topically applied in freshly cut first injection, 1 week after the booster indentin cavities can induce inflammatory jections, and at sacrifice to assess the presence reactions in the ptilp of monkeys and quantity of antibodies to BSA and OVA immunized against BSA. BSA was utilized (see below). Cutaneous reactions were tested by inas test substance because it is a potent immunogen to induce antibody formation tradermal injections of 0.5, 0.25, and 0.12mg BSA dissolved in 0.1 ml saline in the shaved and because it has poor chemotactic abdominal skin. Adjacent sites were injected activity. with saline. As ati additional control the immunized animals were also skin tested to OVA (0.5, 0.25, and 0.12mg). The skin reactions, were examined at 1/2, 2, 4, 8, 24, and Material and methods 48 h after challenge for the presence of edema. To further study an immune-complexANIMALS mediated response, antisera obtained from the immunized monkeys were diluted in four twoThe experiments were carrieti out in 10 young fold step.s and incubated for 2 h at room adult niotikeys iMacaca cynomolgus) .of both sexestemperature with BSA (5 mg/ml). The mixtures (iive males and five Females) weighing (0.1 ml) were then injected intradermally in 2.5-3.5 kg. The monkeys were obtained from three untreated monkeys not otherwise the National Bacteriological Laboratory, participating in the experiments. Control inSweden, after they had been de^vormed and jections were done with BSA incubated with quarantined for 6 weeks following their arrival serum taken from the monkeys before imfrom Indonesia. They appeared in good munization. The injection sites were read as general health with no signs of infections. described above. ANTIGENS
INDUCTION OF PULPiTIS
Cr>'stallized bovine serum albumin (BSA; fraction V, Miles Labs, Inc., USA) was used for
During the experimental procedures the animals were anesthetized by intramuscular
398
BERGENHOLTZ, AHLSTEDT AND LINDHE
administration of Sernylan® (Bio-Ceutic Labs, Inc., USA) and Narkotal® (Astra, Sweden). Within 2 weeks after the booster injection, cavitj' preparations were made into the dentin of the permanent teeth essentially in the way described by BERGE.NHOLTZ & LINDHE (1975).
Dentin was removed to an extent that only a thin dentin wall separated the cavity from the
pulp. One drop of saline was added to crystallized BSA and mixed to paste-like consistency (ca. 2 g/ml). The pulp challenge was accomplished by sealing this mixture into freshly prepared deutin cavities in such a way that the cavity bottom was covered by a 0.5-mm-thick layer. Before the fitiai sealing of the cavities, surplus of BSA was carefully removed from the cavit)' margins. The BSA paste was then covered by a Teflon* disc (Habia, Sweden) and a zinc oxideeugenol dressing (ZOE; Fig. 1) according to
BERGENHOLTZ (1977). Gontrol cavities prepared in immunized animals received a similar treatment with OVA. Experimental and control teeth were selected at random. Among the immunized anjmals 34 teeth were treated with BSA and 29 teeth with OVA. In three nonimmunized animals (one male and two females) BSA was applied in 15 teeth. Forty-eight hours after the cavity sealing the animals were bled by heart puncttire and perfused with 10% neutral buffered formalin introduced through the carotic arteries. The teeth were separated from the jaws and processed for routine histology in a manner described previously (BEROENHOLTZ & LINDHE
1975). Longitudinal, 5-nm-thick serial sections were cut in a buccolingual direction through the cavity and the pulp. The sections were stained with hematoxylin-eosin and examined in the light microscope.
HISTOLOGIC EXAMINATION
BSA
The pulp tissue sections were coded and analyzed by one of the authors (G.B.). When assessing the inflammatory response of the pulps to treatment, the following parameters were considered r (1) the degree of inflammatory cell infiltration (polymorphonuclear and mononuclear leukocytes), (2) signs of hemorrhage (erythrocytes present in the connective tissue), and (3) the extent of tissue disorganization. Based on results from recent studies (BERGENHOLTZ fe LINDHE 1975, BERGENHOLTZ 1977) and the findings reported by HOLZ, FIORE-DONNO & BAUME (1968) the
1 Fig. 1. Outline of procedure used for application of antigen (BSA) and subsequent sealing of cavity with a Teflon disc and zinc oxideeugenol dressing (ZOE).
pulp tissue reactions were classified as slight, moderate, severe, and very severe. A slight reaction was characterized by the infiltration of a few leukocytes within the area of the pulp subjacent to the dentin rubules originating from the cavity prepared in the dentin (the "reaction area", BERGENHOLTZ & LINDHE 1975). In this area there was usually some reduction of the odontoblast layer (Fig. 2). A moderate reaction was characterized by a definite infiltration of leukocytes in the "reaction area". Occasionally, localized hyperemia with free erjthrocytes present in the surrounding connective tissue was .observed. The odontoblast layer was reduced.
PULPITIS IN MONKEYS
399
A severe reaction was characterized by an The shortest lengdi of the dentin tubules intense accumulation of neutrophils in the from the cavity bottom to the pulp was "reaction area" (Fig. 3 A, B). Abscesses were measured for each tooth (floor thickness). liequently seen (Fig. 3 C). Total destruction of the odontoblast layer was observed subjacent lo the cavity. In the peripher)' of the "reaction area" large numbers of mononuclear cells ANTIBODY DETERMINATION formed infiltrates of varying sizes and intensity. Congested vessels and hemorrhage could now The ammonium sulfate precipitating (ASP) and then also be noted. In apically located teclmique originally described by FARR (1958) regions the pulp had a normal appearance and modified according to AHLSTEDT, (Fig. 3 C). HOLMGREN & HANSON (1973) was used to antigen-binding A very severe reaction was characterized by an determine the primarj' extensive disorganization of the pulp tissue capacity of the sera. In these analyses 10 mg of the antigen (BSA or OVA) were'labeled with (Fig. 4). Numerous empty spaces were present 2 mCi '"I (AB Atomenergi, Sweden) using a and the entire odontoblast layer was destroyed modification of the chloramine T principle even in apically located portions. (HUNTER & GREENWOOD 1962)
described by
AHLSTEDT & RVLA.N'DER (1975). Five serial dilutions of each monkey serum in 1:3 steps were made and to each dilution step, labeled antigen (20ng/ml) was added. The titer was calculated as the reciprocal of the serum dilution which precipitated f,% of the antigen .added. .V A
.. •
Presence of IgG antibodies against BSA was tested by using an immunoradiometric assay according to the principle described by CATT & TREGEAR (1967) as modified by AHLSTEDT &
I ,
•
•
•
-
RYLANDER (1975). Anti-human IgG (Behriogewerke, West Germany), which showed cross reactions with monkey serum in immunoprecipitation-in-gel, was used in this test. The globulin fraction of 2 ml anti-IgG serum was precipitated with an equal volume of saturated ammonium sulfate and radioiodinated with '^^I (AHLSTEDT & RYLANDER
''g 2 Shght leaction in the area of pulp subjacent to dentin tubules originating from
keys
UI
E ,E '.5 c o Z 0
e
+1 c O u 0
bJD "^
2 c 0
s
PULPITIS IN MONKEYS
senced cutaneous reactions at the BSA injection sites. By 2 h cutaneous swellings were observed which by 4 h reached their maxima of intensity. Edema exceeding 20 mm in diameter characterized the sites injected with 0.5 mg BSA. By 24 and 48 h the tissue within the injection sites had retained its normal form and texture. Saline induced no overt reaction. Similar cutaneous reactions developed following injections of BSA incubated with serum taken from the immunized monkeys. The swellings, however, developed faster and [he maximum was reached within 2 h. Sites injected with BSA incubated with pre-immune serum showed no or only slight cutaneous reactions.
ANTIBODY DETERMINATION
In sera sampled before the first BSA injection or in sera drawn from the control monkeys no antibody titer could be detected, regardless of assay used. No OVA antibodies were found in sera from the BSA-immunized monkeys. The immunized monkeys yielded elevated levels of serum antibodies to BSA as measured by the ASP technique. The antibody titer in comparison with monkeys however, between different animals (Table 2). Hence, in monkey C 18 there was only a moderate increase of the antibody titer in comparison to monkeys C U and C 12, although positive skin reactions as well as severe pulp inflammations were developed on challenge. The titers were 30, 675, and 810, respectively. Using the IRMA technique, all sera sampled at sacrifice from BSA-immunized monkeys showed elevated levels of antibodies of IgG class to BSA (Table 2). Furthermore, analyses of serum samples from monkeys C U , C 12, and C 17
403
Table 2 Antibody levels in sera sampled at sacrifice from the immunized monkeys. The ASP titer is given .as the reciprocal of serum dilution which causes 6% of labeied BSA added to precipitate. IRMA determinations af IgG antibodies to BSA are expressed as percentages of serum drawn before immunization in serum dilution 1:100. Precipitation titer ii given as the highest serum dilution reciprocal causing precipitation line
Monkey
ASP
trter C 11 C 12 C 17 G IS
675 810 225 30
IRMA Precipitation IgG titer tjter 810 920 570 310
8 8 8 !
showed positive immunoprecipitation results to BSA in 1:8 dilution of serum, but the serum from monkey C 18 gave only a faint line without dilution.
Discussion The present investigation demonstrated that topical application of bovine serum albumin (BSA) to freshly prepared dentin cavities in teeth of normal monkeys produced no obvious pathologic reaction yvithin the adjacent pulp tissue. In monkeys immunized to BSA, however, identical applications of BSA resulted in severe inflaintnatory lesions within the pulp. The lesions were identified in histologie sections and were characterized by the presence of large numbers of leukocytes within the extravascuiar tissue compartments. Bleeding was also a frequent finding. In several pulps extensive tissue disorganization was the most prominent feature. Inmiunoprecipitation-in.-gel and ammonium sulfate precipitation (ASP) analyses and immunoradiometric assays
404
BERGENHOLTZ, AHLSTEDT AND LINDHE
(IRMA) revealed that the sera of the immunized monkeys contained high levels of antibodies to BSA, predominatitly IgG antibodies. It seems thus reasonable to suggest that these antibodies played a decisive role in mediating the pulp lesions observed. This is further indicated by the fact that ovaibumin (OVA), when applied in control teeth of the BSA-immunized monkeys (which had no demonstrable amounts of antibodies to OVA), did not cause frank pulp tissue inflammation (Table 1). The experimental procedure and the histologie alterations obser\'ed indicate that the reaction was provoked by inimune.-complexes, possibly by means of a Type III reaction (CoOMBS & GELL 1968). This assumption is based not only on the presence of serum antibodies in the immunized monkeys but also upon the following observations: (1) the character of the pulp lesions: vasculitis, neutrophil migration, bleeding; (2) the time onset and die evolution rate of the skin reactions; and (3) the development of similar skin reactions in nonimmunized monkeys by passive transfer of immune-complexes (consisting of BSA and anti-BSA antibodies obtained from the immunized monkeys and combined in vitro prior to injection). No attempts were made, however, to further elucidate the immune-complexmediated pathogenesis, e.g. by determining the presence of BSA antibody complexes within the pulp or by reproducing the pulp reactions by sealing preformed immune-complexes into dentin cavities. Hence, it cannot be excluded that immune factors other than immunecomplexes composed by IgG antibodies and BSA also may be involved in the pathogenesis of the pulp inflammations. It has been proposed that reagins may participate in the development of Type III hypersensitivity in the lungs (PEPYS 1969)
and diat the joint infl.ammation in a disorder such as rheumatoid arthritis, described as principally mediated by immune-complexes, may be dependent on the participation of immune thymusderived lymphocytes (for review see MESSNERi974). In monkey C 18 it was observed that, although the animal had low levels of serum antibodies to BSA and only weak indications of precipitins compared with the other immunized monkeys, the pulp reaction to topical application of BSA was equally pronounced. This finding seems to reject the hypothesis that serum antibodies to BSA were of decisive importance for the development of the pulp lesions. On the other hand, it has been shown that diseases proposed to he mediated by immune-complexes do not always show these features. Hence, PEPYS (1969) found in studies on hypersensitivity pneumonitis in bird breeders that Type III reactions in the skin could be induced in some symptomatic patients even though precipitin reactions against the challenging antigens could not be demonstrated. The results from the present experiment revealed that reactions between antigens and circulating antibodies can indeed occur within the dentin-pulp area. Neutrophilic leukocytes are probably attracted to the reaction site in the pulp following the activation of complement (C3a and C5a) by BSA-anti-BSA complexes. The finding that neutrophils had accumulated in the pulp tissue adjacent to the exposed dentin does not necessarih' imply that BSA molecules had penetrated ihe dentin tubules and entered the pulp. It has been shown that dentin contains immunogiobulins (STUEBEN & SPRETER V. KREUDENSTEIN 1957, THOMAS & LEAVER
1975). Hence, it may be argued that the formation of BSA antibody complexes
PULPITIS IN MONKEYS
405
occurred within the dentin tubules and/or in the cavity prepared in the dentin. If complement is present in dentin, fluid factors chemotactic for neutrophils may thus be released and neutrophils could be attracted to the "reaction area". The reactions provoked by the application of BSA in dentin cavities of immunized animals are very similar in nature to those elicited in pulps subjected to irritation by substances produced by plaque microorganisms and described by BERGENHOLTZ & LiNDHE (1975) and BERGENHOLTZ (1977). Following the application on freshly cut dentin of an extract from dental plaque or substances obtained from cultivated dental plaque bacteria, there was in the adjacent pulp tissue vascular alterations and an accumulation of neutrophils. This does not mean, however, that the tissue reactions observed in the present experiment and those referred to above were initiated by the sanne factors. Serum albumin is a nonchemotactic protein (KELLER 1966) which has frequendy been used as a negative control when studying the capacity of neutrophils to respond to yarious chemotactic materials. While BSA through interaction with antibodies probably activates complement, products troni bacteria may exert chemotactic effects directly (KELLER & SORKIN 1967). This means that microbial products can directly stimulate the migration of leukocytes or can liberate chemotactic material from humoral enzyme systems, e.g. the complement system, whereas BSA in this model can induce inflammation only via the complement system.
caused a significant increase in the number of leukocytes in the junctional epithelium and an altered permeability of the dentogingival vessels. The present data show diat freshly exposed coronal dentin constitutes poor protection for the ptilp against antigenic material. On the basis of these findings it seems reasonable to assume that some pulp reactions, e.g. during the caries process, are in fact the result of antigenic stimulation, possibly causing antibody formation leading to pathologic immune-complexes.
In recent experiments, KAHNBERG, LINDHE & ATTSTROM (1977) showed that
COOMBS,
iopical application of a dental plaque '.'xtract to the marginal gingiva in dogs immunized against plaque, in comparison v'ith the reactions of nonimmunized dogs.
References
AHLSTEDT, S., HOLMGREN, J. & HANSON, L. A.:
The validity of the atrimoiiiura sulphate precipitation technique of estimation of antibody amount and avidity. ImTnunology 1973:25:917-922. AHLSTEDT,
S. SC RYLANDER, H . :
Immuno-
radiometric assay for quantification of serum antibodies to dental plaque antigen in immunized dogs. / . Periodontal Res. 1975: 10: 224-229. BAUME, L. J.: Dental pulp conditions in relation to carious lesions. Int. Dent. J. 1970: 20; 309-337. BERCENHOLTZ, G. : Etfect of bacterial products on inflammatory reactions in the dental pulp. Scand. f. Dent. Res. 197 7: 85: 122-129. BERGENHOLTZ, G. & LiNDHE, J.: Effect of soluble plaque factors on inOammatory reactions in the dentai pulp. Scand. J. Dent. Res. 1975: 83: 153-158. BRANNSTROM,
M . & LIND,
P.
O.:
Pulpal
response to early dental caries. / . Deni. Res. 1965: 44: 1045-1050. GATT, K. J . SC TREGEAR, G . W. : Solid-phase
radioimmunoassay in andbody-coated tubes. Science 1967: 158: 1570-1572. R.
R. A.
& GELL,
P.
G.
H.:
Glassification of allergic reactions responsible for clinical hypersensitivity and disease, ln: GELL, P. G. H. & GOOMBS, R. R. A. (ed.): Clinical .aspects of immunology. Blackwell
Scientific Publications, Oxford, Edinburgh 1968, p. 575.
406
BERGENHOLTZ, AHLSTEDT AND LINDHE
FARR, R. S.: A quantitative immunochemical LANGELAND, K. : Prevention of pulpal damage. measure of the primary interaction between DetU. Clin. North Am. 1972: 16: 709-732. I BSA and antibody.'/. Infect. Dis. 1958: LANGELAND, K. SC LANGELAND, L. K. : Indirect 103: 2.39-262 capping and the treatment of deep carious lesions. Int. Dent.f. 1968: 18: 326-380. HOLZ, J., FIORE-DONNO, G . & BAUME, L. J.: Gontroles biologiques des materiaux MESSNER, R. P. : Clinical aspects of T- and Blymphocytes in rheumatic diseases. Arthritis d'obturation: normalisation des methodes Rheum. 1974:17:339-346. experimentales et des criteres d'evaluadon. Schweiz. Monatsschr. Zahnheiikd. 1968: 78:PEPYS, J.: Hypersensicivity diseases of the lungs due to fungi and organic dusts. Mmiogr. 307-351. Allergy 1969:4. Hu.NTER, W. M. Sc GREENWOOD, F. G . : Preparation of iodine-131 labelled human SELTZER, S. & BENDER, I. B.: The dental pulp. • Biologic considerations in dentai procedures. J. B. growth hormone with specific activity. Lippincott, Philadelphia, Toronto 1975. Nature' 1962: 194: 495-496. KAHNEERG, K.-E., LINDHE, J . & ATTSTROM, R. :
The effect of decomplementation by carragheenan on experimental initial gingivitis in hyperimmune dogs. / . Periodontal Res. 197 7: in press. KELLER, H . U . : Studies on chemotaxis, IIL Modilication of Boyden's technique for the evaluation of chemotactic agetits. Immunotogy 1966: 10: 225-230. KELLER,
H . U . & SORKIN, V..: Studies on
chemotaxis, V. On the chemotactic effect of bacteria. Int. Arch. Allerg. Appl. Immunol. 1967:31:505-517. Address: Gunnar Bergenholtz Department of Endodontics Faculty of Odontology University of Gothenburg S'4OO 33 Gothenburg 33 Sweden
STEUBEN, J . & SPRETER VON KREUDENSTEIN, T. :
Dentinstoflw'echselstudien. VI. Papierelektrophoretische Untersuchungen ueber die Zusaramensetzting der Dentioliquorproteine. Dtsch. Zahnaerztl. Z. 1957: 12:' 500-507. THOMAS, M . & LEAVER, A. G.: Identification
and estimation of plasma proteins in human dentine. Arch. Oral Biol. 1975: 20: 217-218. WADSWORTH, G.: A slide microtechnique for the analysis of immune precipitates in gel. Int. Arch. Allerg. Appl. Immunol. 1957: 10: 355-360.
Experimental pylpitis in immynized monkeys GUNNAR BERGENHOLTZ, STAFFAN AHLSTEDT AND JAN LINDHE Departments of Endodontics and Periodoniology and Institute of Medical Microbiology, University of Gothenburg, Gothenburg and Astra Liikemedel AB, Research and Development Laboratories, SedertSlje, Sweden
ABSTRACT - Bovine serum albumin (BSA) was topically applied to exposed dentin to assess whether inflammatory reactions can be induced in the pulp of monkeys immunized against BSA. Four cynomolgus monkeys received repeated injections of BSA emulsified with Freund's incomplete a.djuvant. Pulp challenge was performed by applying BSA in freshly cut dentin cavities prepared on the buccal surface in 34 teeth. In 29 control teeth ovalbumin (OVA) was applied. Control applications of BSA were also performed in 15 teeth prepared in three nonimmunized monkeys. Forty-eight hours after the initiation of the pulp challenge the monkeys were sacrificed and the ptilp tissue examined in the light microscope. Topical application of BSA to freshly exposed dentin in immunized monkeys resulted in severe inHamtnatory lesions in the pulp, characterized by bleeding and extravaScular infiltration of large numbers of leukocytes. Extensive tissue damage was an important feature in several pulps. Identical applications of OVA in control teeth and BSA applications in nonimmunized monkeys produced no such reactions. The results indicate that interactions between antigens and antibodies can occur within the dentin-pulp area and following the formation of immune-complexes, severe injury to the pulp can be induced. (Accepted for publicatim. 6 November 1976)
Previous investigations have revealed that cultivated from human dental plaque, a crude extract from human dental plaque similar phenomena developed in the pulp contains components with the capacity (BERGENHOLTZ 1977). The studies referred to induce or mediate acute inflamma- to show that freshly cut coronal dentin tory reactions in the dental pulp has a poor capacity to protect the pulp (BERGENHOLTZ 8c LiNDHE 1975). The from noxious agents such as microbial extract was applied in freshly cut dentin plaque. It is therefore not surprising to cavities in monkeys. Obvious signs of find that pathologic reactions can already vasculitis and leukocyte accumulation be detected in the pulp when a carious could be observed 10-32 h later in the lesion, which is infectious in nature, has pulp adjacent to the cavity preparation, reached the outer layer of dentin When the plaque extract was substituted (BRANNSTROM & LiND 1965, LANGELAND by material produced by microorganisms & LANGELAND 1968, BAUME 1970).
PULPITIS IN MONKEYS It has been suggested that immunologic mechanisms may contribute to the tissue breakdown in pulp inflammation (LANGELAND
1 9 7 2 , SELTZER & BENDER
1975). Considering the excessive amounts of antigens produced by the microorganisms involved in the caries process atid the presence of plasma cells and lymphocytes in the pulp tissue, SELTZER & BENDER (1975) assumed that synthesis of immunoglobulins may occur in the pulp. The formation of antibodies against the intruding antigens may protect the pulp, but the subsequently formed antigenantibody complexes may also, following complement activation, cause h^persensitivity reactions and destroy the pulp tissue.
397
immunization. Egg albumin (OVA; BDH Chetnicals Ltd., England) was used as a control substance. IMMUNIZATION PROCEDURE
Four monkeys (two males and t^vo females} received subcutaneous and intramuscular injections of BSA dissolved in saline and emulsified in equal volumes of Freund's incomplete adjuvant (FIA). At four different occasions 1-2 weeks apart a dose of 50 mg BSA in 2 ml saline was injected in multiple sites in the thighs and/or the back close tO' the lymph nodes. Six weeks after the last administration of BSA a booster injection of the same amount of BSA in FIA was given. Two of the monkeys (C 17 and CIS; Tables 1 and 2) had at this time poitit low antibody titers and were given one additional booster injection; C 17 with BSA in Freund's complete adjuvant (FCA). The aim of the present investigation was Six auiniais [three males and three females) to study whether bovine serum albumin were not treated and were used as controls. Scrum was sampled immediately before the (BSA) topically applied in freshly cut first injection, 1 week after the booster indentin cavities can induce inflammatory jections, and at sacrifice to assess the presence reactions in the ptilp of monkeys and quantity of antibodies to BSA and OVA immunized against BSA. BSA was utilized (see below). Cutaneous reactions were tested by inas test substance because it is a potent immunogen to induce antibody formation tradermal injections of 0.5, 0.25, and 0.12mg BSA dissolved in 0.1 ml saline in the shaved and because it has poor chemotactic abdominal skin. Adjacent sites were injected activity. with saline. As ati additional control the immunized animals were also skin tested to OVA (0.5, 0.25, and 0.12mg). The skin reactions, were examined at 1/2, 2, 4, 8, 24, and Material and methods 48 h after challenge for the presence of edema. To further study an immune-complexANIMALS mediated response, antisera obtained from the immunized monkeys were diluted in four twoThe experiments were carrieti out in 10 young fold step.s and incubated for 2 h at room adult niotikeys iMacaca cynomolgus) .of both sexestemperature with BSA (5 mg/ml). The mixtures (iive males and five Females) weighing (0.1 ml) were then injected intradermally in 2.5-3.5 kg. The monkeys were obtained from three untreated monkeys not otherwise the National Bacteriological Laboratory, participating in the experiments. Control inSweden, after they had been de^vormed and jections were done with BSA incubated with quarantined for 6 weeks following their arrival serum taken from the monkeys before imfrom Indonesia. They appeared in good munization. The injection sites were read as general health with no signs of infections. described above. ANTIGENS
INDUCTION OF PULPiTIS
Cr>'stallized bovine serum albumin (BSA; fraction V, Miles Labs, Inc., USA) was used for
During the experimental procedures the animals were anesthetized by intramuscular
398
BERGENHOLTZ, AHLSTEDT AND LINDHE
administration of Sernylan® (Bio-Ceutic Labs, Inc., USA) and Narkotal® (Astra, Sweden). Within 2 weeks after the booster injection, cavitj' preparations were made into the dentin of the permanent teeth essentially in the way described by BERGE.NHOLTZ & LINDHE (1975).
Dentin was removed to an extent that only a thin dentin wall separated the cavity from the
pulp. One drop of saline was added to crystallized BSA and mixed to paste-like consistency (ca. 2 g/ml). The pulp challenge was accomplished by sealing this mixture into freshly prepared deutin cavities in such a way that the cavity bottom was covered by a 0.5-mm-thick layer. Before the fitiai sealing of the cavities, surplus of BSA was carefully removed from the cavit)' margins. The BSA paste was then covered by a Teflon* disc (Habia, Sweden) and a zinc oxideeugenol dressing (ZOE; Fig. 1) according to
BERGENHOLTZ (1977). Gontrol cavities prepared in immunized animals received a similar treatment with OVA. Experimental and control teeth were selected at random. Among the immunized anjmals 34 teeth were treated with BSA and 29 teeth with OVA. In three nonimmunized animals (one male and two females) BSA was applied in 15 teeth. Forty-eight hours after the cavity sealing the animals were bled by heart puncttire and perfused with 10% neutral buffered formalin introduced through the carotic arteries. The teeth were separated from the jaws and processed for routine histology in a manner described previously (BEROENHOLTZ & LINDHE
1975). Longitudinal, 5-nm-thick serial sections were cut in a buccolingual direction through the cavity and the pulp. The sections were stained with hematoxylin-eosin and examined in the light microscope.
HISTOLOGIC EXAMINATION
BSA
The pulp tissue sections were coded and analyzed by one of the authors (G.B.). When assessing the inflammatory response of the pulps to treatment, the following parameters were considered r (1) the degree of inflammatory cell infiltration (polymorphonuclear and mononuclear leukocytes), (2) signs of hemorrhage (erythrocytes present in the connective tissue), and (3) the extent of tissue disorganization. Based on results from recent studies (BERGENHOLTZ fe LINDHE 1975, BERGENHOLTZ 1977) and the findings reported by HOLZ, FIORE-DONNO & BAUME (1968) the
1 Fig. 1. Outline of procedure used for application of antigen (BSA) and subsequent sealing of cavity with a Teflon disc and zinc oxideeugenol dressing (ZOE).
pulp tissue reactions were classified as slight, moderate, severe, and very severe. A slight reaction was characterized by the infiltration of a few leukocytes within the area of the pulp subjacent to the dentin rubules originating from the cavity prepared in the dentin (the "reaction area", BERGENHOLTZ & LINDHE 1975). In this area there was usually some reduction of the odontoblast layer (Fig. 2). A moderate reaction was characterized by a definite infiltration of leukocytes in the "reaction area". Occasionally, localized hyperemia with free erjthrocytes present in the surrounding connective tissue was .observed. The odontoblast layer was reduced.
PULPITIS IN MONKEYS
399
A severe reaction was characterized by an The shortest lengdi of the dentin tubules intense accumulation of neutrophils in the from the cavity bottom to the pulp was "reaction area" (Fig. 3 A, B). Abscesses were measured for each tooth (floor thickness). liequently seen (Fig. 3 C). Total destruction of the odontoblast layer was observed subjacent lo the cavity. In the peripher)' of the "reaction area" large numbers of mononuclear cells ANTIBODY DETERMINATION formed infiltrates of varying sizes and intensity. Congested vessels and hemorrhage could now The ammonium sulfate precipitating (ASP) and then also be noted. In apically located teclmique originally described by FARR (1958) regions the pulp had a normal appearance and modified according to AHLSTEDT, (Fig. 3 C). HOLMGREN & HANSON (1973) was used to antigen-binding A very severe reaction was characterized by an determine the primarj' extensive disorganization of the pulp tissue capacity of the sera. In these analyses 10 mg of the antigen (BSA or OVA) were'labeled with (Fig. 4). Numerous empty spaces were present 2 mCi '"I (AB Atomenergi, Sweden) using a and the entire odontoblast layer was destroyed modification of the chloramine T principle even in apically located portions. (HUNTER & GREENWOOD 1962)
described by
AHLSTEDT & RVLA.N'DER (1975). Five serial dilutions of each monkey serum in 1:3 steps were made and to each dilution step, labeled antigen (20ng/ml) was added. The titer was calculated as the reciprocal of the serum dilution which precipitated f,% of the antigen .added. .V A
.. •
Presence of IgG antibodies against BSA was tested by using an immunoradiometric assay according to the principle described by CATT & TREGEAR (1967) as modified by AHLSTEDT &
I ,
•
•
•
-
RYLANDER (1975). Anti-human IgG (Behriogewerke, West Germany), which showed cross reactions with monkey serum in immunoprecipitation-in-gel, was used in this test. The globulin fraction of 2 ml anti-IgG serum was precipitated with an equal volume of saturated ammonium sulfate and radioiodinated with '^^I (AHLSTEDT & RYLANDER
''g 2 Shght leaction in the area of pulp subjacent to dentin tubules originating from
keys
UI
E ,E '.5 c o Z 0
e
+1 c O u 0
bJD "^
2 c 0
s
PULPITIS IN MONKEYS
senced cutaneous reactions at the BSA injection sites. By 2 h cutaneous swellings were observed which by 4 h reached their maxima of intensity. Edema exceeding 20 mm in diameter characterized the sites injected with 0.5 mg BSA. By 24 and 48 h the tissue within the injection sites had retained its normal form and texture. Saline induced no overt reaction. Similar cutaneous reactions developed following injections of BSA incubated with serum taken from the immunized monkeys. The swellings, however, developed faster and [he maximum was reached within 2 h. Sites injected with BSA incubated with pre-immune serum showed no or only slight cutaneous reactions.
ANTIBODY DETERMINATION
In sera sampled before the first BSA injection or in sera drawn from the control monkeys no antibody titer could be detected, regardless of assay used. No OVA antibodies were found in sera from the BSA-immunized monkeys. The immunized monkeys yielded elevated levels of serum antibodies to BSA as measured by the ASP technique. The antibody titer in comparison with monkeys however, between different animals (Table 2). Hence, in monkey C 18 there was only a moderate increase of the antibody titer in comparison to monkeys C U and C 12, although positive skin reactions as well as severe pulp inflammations were developed on challenge. The titers were 30, 675, and 810, respectively. Using the IRMA technique, all sera sampled at sacrifice from BSA-immunized monkeys showed elevated levels of antibodies of IgG class to BSA (Table 2). Furthermore, analyses of serum samples from monkeys C U , C 12, and C 17
403
Table 2 Antibody levels in sera sampled at sacrifice from the immunized monkeys. The ASP titer is given .as the reciprocal of serum dilution which causes 6% of labeied BSA added to precipitate. IRMA determinations af IgG antibodies to BSA are expressed as percentages of serum drawn before immunization in serum dilution 1:100. Precipitation titer ii given as the highest serum dilution reciprocal causing precipitation line
Monkey
ASP
trter C 11 C 12 C 17 G IS
675 810 225 30
IRMA Precipitation IgG titer tjter 810 920 570 310
8 8 8 !
showed positive immunoprecipitation results to BSA in 1:8 dilution of serum, but the serum from monkey C 18 gave only a faint line without dilution.
Discussion The present investigation demonstrated that topical application of bovine serum albumin (BSA) to freshly prepared dentin cavities in teeth of normal monkeys produced no obvious pathologic reaction yvithin the adjacent pulp tissue. In monkeys immunized to BSA, however, identical applications of BSA resulted in severe inflaintnatory lesions within the pulp. The lesions were identified in histologie sections and were characterized by the presence of large numbers of leukocytes within the extravascuiar tissue compartments. Bleeding was also a frequent finding. In several pulps extensive tissue disorganization was the most prominent feature. Inmiunoprecipitation-in.-gel and ammonium sulfate precipitation (ASP) analyses and immunoradiometric assays
404
BERGENHOLTZ, AHLSTEDT AND LINDHE
(IRMA) revealed that the sera of the immunized monkeys contained high levels of antibodies to BSA, predominatitly IgG antibodies. It seems thus reasonable to suggest that these antibodies played a decisive role in mediating the pulp lesions observed. This is further indicated by the fact that ovaibumin (OVA), when applied in control teeth of the BSA-immunized monkeys (which had no demonstrable amounts of antibodies to OVA), did not cause frank pulp tissue inflammation (Table 1). The experimental procedure and the histologie alterations obser\'ed indicate that the reaction was provoked by inimune.-complexes, possibly by means of a Type III reaction (CoOMBS & GELL 1968). This assumption is based not only on the presence of serum antibodies in the immunized monkeys but also upon the following observations: (1) the character of the pulp lesions: vasculitis, neutrophil migration, bleeding; (2) the time onset and die evolution rate of the skin reactions; and (3) the development of similar skin reactions in nonimmunized monkeys by passive transfer of immune-complexes (consisting of BSA and anti-BSA antibodies obtained from the immunized monkeys and combined in vitro prior to injection). No attempts were made, however, to further elucidate the immune-complexmediated pathogenesis, e.g. by determining the presence of BSA antibody complexes within the pulp or by reproducing the pulp reactions by sealing preformed immune-complexes into dentin cavities. Hence, it cannot be excluded that immune factors other than immunecomplexes composed by IgG antibodies and BSA also may be involved in the pathogenesis of the pulp inflammations. It has been proposed that reagins may participate in the development of Type III hypersensitivity in the lungs (PEPYS 1969)
and diat the joint infl.ammation in a disorder such as rheumatoid arthritis, described as principally mediated by immune-complexes, may be dependent on the participation of immune thymusderived lymphocytes (for review see MESSNERi974). In monkey C 18 it was observed that, although the animal had low levels of serum antibodies to BSA and only weak indications of precipitins compared with the other immunized monkeys, the pulp reaction to topical application of BSA was equally pronounced. This finding seems to reject the hypothesis that serum antibodies to BSA were of decisive importance for the development of the pulp lesions. On the other hand, it has been shown that diseases proposed to he mediated by immune-complexes do not always show these features. Hence, PEPYS (1969) found in studies on hypersensitivity pneumonitis in bird breeders that Type III reactions in the skin could be induced in some symptomatic patients even though precipitin reactions against the challenging antigens could not be demonstrated. The results from the present experiment revealed that reactions between antigens and circulating antibodies can indeed occur within the dentin-pulp area. Neutrophilic leukocytes are probably attracted to the reaction site in the pulp following the activation of complement (C3a and C5a) by BSA-anti-BSA complexes. The finding that neutrophils had accumulated in the pulp tissue adjacent to the exposed dentin does not necessarih' imply that BSA molecules had penetrated ihe dentin tubules and entered the pulp. It has been shown that dentin contains immunogiobulins (STUEBEN & SPRETER V. KREUDENSTEIN 1957, THOMAS & LEAVER
1975). Hence, it may be argued that the formation of BSA antibody complexes
PULPITIS IN MONKEYS
405
occurred within the dentin tubules and/or in the cavity prepared in the dentin. If complement is present in dentin, fluid factors chemotactic for neutrophils may thus be released and neutrophils could be attracted to the "reaction area". The reactions provoked by the application of BSA in dentin cavities of immunized animals are very similar in nature to those elicited in pulps subjected to irritation by substances produced by plaque microorganisms and described by BERGENHOLTZ & LiNDHE (1975) and BERGENHOLTZ (1977). Following the application on freshly cut dentin of an extract from dental plaque or substances obtained from cultivated dental plaque bacteria, there was in the adjacent pulp tissue vascular alterations and an accumulation of neutrophils. This does not mean, however, that the tissue reactions observed in the present experiment and those referred to above were initiated by the sanne factors. Serum albumin is a nonchemotactic protein (KELLER 1966) which has frequendy been used as a negative control when studying the capacity of neutrophils to respond to yarious chemotactic materials. While BSA through interaction with antibodies probably activates complement, products troni bacteria may exert chemotactic effects directly (KELLER & SORKIN 1967). This means that microbial products can directly stimulate the migration of leukocytes or can liberate chemotactic material from humoral enzyme systems, e.g. the complement system, whereas BSA in this model can induce inflammation only via the complement system.
caused a significant increase in the number of leukocytes in the junctional epithelium and an altered permeability of the dentogingival vessels. The present data show diat freshly exposed coronal dentin constitutes poor protection for the ptilp against antigenic material. On the basis of these findings it seems reasonable to assume that some pulp reactions, e.g. during the caries process, are in fact the result of antigenic stimulation, possibly causing antibody formation leading to pathologic immune-complexes.
In recent experiments, KAHNBERG, LINDHE & ATTSTROM (1977) showed that
COOMBS,
iopical application of a dental plaque '.'xtract to the marginal gingiva in dogs immunized against plaque, in comparison v'ith the reactions of nonimmunized dogs.
References
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The validity of the atrimoiiiura sulphate precipitation technique of estimation of antibody amount and avidity. ImTnunology 1973:25:917-922. AHLSTEDT,
S. SC RYLANDER, H . :
Immuno-
radiometric assay for quantification of serum antibodies to dental plaque antigen in immunized dogs. / . Periodontal Res. 1975: 10: 224-229. BAUME, L. J.: Dental pulp conditions in relation to carious lesions. Int. Dent. J. 1970: 20; 309-337. BERCENHOLTZ, G. : Etfect of bacterial products on inflammatory reactions in the dental pulp. Scand. f. Dent. Res. 197 7: 85: 122-129. BERGENHOLTZ, G. & LiNDHE, J.: Effect of soluble plaque factors on inOammatory reactions in the dentai pulp. Scand. J. Dent. Res. 1975: 83: 153-158. BRANNSTROM,
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O.:
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R. A.
& GELL,
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G.
H.:
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FARR, R. S.: A quantitative immunochemical LANGELAND, K. : Prevention of pulpal damage. measure of the primary interaction between DetU. Clin. North Am. 1972: 16: 709-732. I BSA and antibody.'/. Infect. Dis. 1958: LANGELAND, K. SC LANGELAND, L. K. : Indirect 103: 2.39-262 capping and the treatment of deep carious lesions. Int. Dent.f. 1968: 18: 326-380. HOLZ, J., FIORE-DONNO, G . & BAUME, L. J.: Gontroles biologiques des materiaux MESSNER, R. P. : Clinical aspects of T- and Blymphocytes in rheumatic diseases. Arthritis d'obturation: normalisation des methodes Rheum. 1974:17:339-346. experimentales et des criteres d'evaluadon. Schweiz. Monatsschr. Zahnheiikd. 1968: 78:PEPYS, J.: Hypersensicivity diseases of the lungs due to fungi and organic dusts. Mmiogr. 307-351. Allergy 1969:4. Hu.NTER, W. M. Sc GREENWOOD, F. G . : Preparation of iodine-131 labelled human SELTZER, S. & BENDER, I. B.: The dental pulp. • Biologic considerations in dentai procedures. J. B. growth hormone with specific activity. Lippincott, Philadelphia, Toronto 1975. Nature' 1962: 194: 495-496. KAHNEERG, K.-E., LINDHE, J . & ATTSTROM, R. :
The effect of decomplementation by carragheenan on experimental initial gingivitis in hyperimmune dogs. / . Periodontal Res. 197 7: in press. KELLER, H . U . : Studies on chemotaxis, IIL Modilication of Boyden's technique for the evaluation of chemotactic agetits. Immunotogy 1966: 10: 225-230. KELLER,
H . U . & SORKIN, V..: Studies on
chemotaxis, V. On the chemotactic effect of bacteria. Int. Arch. Allerg. Appl. Immunol. 1967:31:505-517. Address: Gunnar Bergenholtz Department of Endodontics Faculty of Odontology University of Gothenburg S'4OO 33 Gothenburg 33 Sweden
STEUBEN, J . & SPRETER VON KREUDENSTEIN, T. :
Dentinstoflw'echselstudien. VI. Papierelektrophoretische Untersuchungen ueber die Zusaramensetzting der Dentioliquorproteine. Dtsch. Zahnaerztl. Z. 1957: 12:' 500-507. THOMAS, M . & LEAVER, A. G.: Identification
and estimation of plasma proteins in human dentine. Arch. Oral Biol. 1975: 20: 217-218. WADSWORTH, G.: A slide microtechnique for the analysis of immune precipitates in gel. Int. Arch. Allerg. Appl. Immunol. 1957: 10: 355-360.
