Humoral antibody response in rabbits after intragingival injections of bovine serum albumin and Escherichia coli endotoxin

Archs oral Bid. Vol. 16, pp. 247-257,1971.PergamonPress.Printedin Great Britain.

HUMORAL ANTIBODY RESPONSE IN RABBITS AFTER INTRAGINGIVAL INJECTIONS OF BOVINE SERUM ALBUMIN AND ESCHERICHIA COLI ENDOTOXIN A. T. BALL and J. R. Department

TROTT

of Oral Pathology, Faculty of Dentistry, Winnipeg 3, Canada

University

of Manitoba,

Summary-The humoral antibody response in rabbits to primary, secondary and multiple intragingival injections of bovine serum albumin (BSA) or somatic lipopolysaccharide endotoxin from Escherichia coli have been analysed by molecular sieve chromatography, immunoelectrophoresis and passive haemagglutination. No adjuvants were used in any of the experiments. BSA induced mainly IgG class of antibodies with the exception of the early part of the primary response. A pure IgM response was noted following a primary injection of endotoxin. Both IgM and IgG antibodies were found in the secondary response and after repeated multiple injections of endotoxin, but IgM dominated all responses to endotoxin. The results also indicate that an enhanced secondary antibody response exists following a secondary injection of endotoxin. The IgM and IgG profiles following intragingival injections of BSA or endotoxin have now been established following three different methods of administration. INTRODUCTION

IN RECENTyears a considerable amount of work has been undertaken to investigate the possible role of immune mechanisms in the pathogenesis of chronic destructive periodontal disease. To do this, a number of different antigens and methods of application of these antigens have been used to produce a model to study immune mechanisms at the local gingival site. Two broad groups of antigens have so far been used. First, protein antigens and secondly lipopoplysaccharide endotoxins. RIZZO and MITCHELL(1966) established that egg albumin placed repeatedly in the labial gingival pockets of rabbits produced a chronic inflammatory reaction in the gingiva and specific humoral antibody to egg albumin. RANNEY(1970) sensitized squirrel monkeys with ovalbumin by placing floss silk impregnated with the antigen, three times a week for three months in the gingival crevice of the animals. He found specific fluorescent staining, antibody-producing cells in the gingiva and the regional lymph nodes, as well as specific serum antibody to the antigen. BERGLUNDet al. (1969) injected 0.1 pg of lipopolysaccharide from Escherichia coli into the palatal mucosa of rabbits and found on the hfth day that only the ipsilateral lymph nodes showed a marked increase in plaque-forming cells, and that they produced a marked bactericidal antibody. The primary immune response of mice to BSA and horse ferritin antigens injected in the attached gingiva of the mandible has been reported by YOUNG et al. (1969). A systematic study has not been reported of the 247

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A. T. BALL and J. R. TROTT

humoral response following intragingival injection of either a protein or lipopolysaccharide, during a primary and secondary response. The aims of this study were to elucidate the humoral response under the following conditions :

(4 following a primary injection of either bovine serum albumin (BSA) or Escherichia coli endotoxin,

@I after a single challenge injection with either antigen, (c) following the secondary injection with an intermittent series of challenges every 3 or 4 days. The third series may be particularly important because it is conceivable that the periodontal tissues may be continuously exposed to potential antigens from the gingival crevice. MATERIALS

AND

METHODS

Animals Two groups of three adult New Zealand white female rabbits were used and kept on a balanced pellet diet and water ad lib. Antigens Bovine serum albumin (BSA) was obtained from Nutritional Biochemical Corporation, Cleveland, Ohio. The primary injection consisted of 5 mg BSA/kg body weight, and all subsequent injections had 2.5 mg BSA/kg. E. coli lipopolysaccharide 055:B5, extracted by the Boivin method, was obtained from Difco Laboratories, Detroit, Michigan. The primary injection was 1.0 rg and challenge injections were 0.1 pg. Immunization Two groups of animals were used to study the humoral antibody response to the two antigens: (4 A primary intragingival injection of BSA was given and blood ias collected on days 3,5,7, 10, 15,21 and 28 following the injection. The other group of animals received endotoxin by the same route and blood was collected on days 3, 7, 14, 21 and 28. (b) A second intragingival @action was given on the 28th day after the primary injection. Blood was collected from the rabbits given BSA on days 3, 4, 5, 7, 10, 14, 21, 28, 36 and 42 and from rabbits given endotoxin on days 3, 7, 10, 14,21,28, 36 and 42. (4 Repeated injections of BSA and endotoxin were started 42 days following the first injection. These were repeated every 3 or 4 days for 28 days, stopped for 40 days and then begun again for 15 days. The animals were bled on days 3, 7, 10, 14, 17, 21, 24, 28, 68, 76, 82 and 85. A volume of 0.1 ml of antigen, prepared in sterile 0.85 % NaCl, was administered with a 30 gauge needle under a light general anaesthetic, into the right labial marginal gingiva of the mandibular incisors 1 mm from the gingival crest. Great care was taken to inject antigen into the gingiva only and not to allow any leakage into the gingival sulcus. Approximately 5 ml of blood was taken from the marginal ear vein, allowed to clot at room temperature for 1 hr and then kept at 4°C for 12 hr. The blood was then centrifuged and the separated serum was stored at -20°C. Haemagglutination Whole sera and the separated fractions were tested for specific antibody activity by passive haemagglutination. All sera were complement-inactivated by incubation for 30 min at 56°C. Heterophile agglutinins were absorbed with fresh normal sheep red blood cells (SRC) of the same batch that was used in the test (WEIR, 1967). Anti-BSA estimations were carried out with chromic chloride treated SRC sensitized with BSA (WEATHERALL, 1969). SRC were washed three times in normal saline and made up to a 5 % suspension

RABBITANTIBODYRBSPONSETOBSAANDENDOTOXIN

249

in saline .They were again gently washed, spun down and the supernatant discarded. One volume of a 0.1% BSA solution was the added to 20 vol. of the washed SRC followed immediately by 1 vol. of a 0.1% solution of freshly prepared chromic chloride in normal saline. The mixture was left at room temperature for 1 hr and shaken gently several times. The coated, sensitized cells were then washed three times in cold normal saline and made up to a 1 per cent suspension in normal saline. To test the antiendotoxin sera SRC were sensitized with E. coli lipopolysaccharide. The alkylation method of CRUMFKJN et al. (1958) was used; the endotoxin was alkylated with O-02 M NaOH (1 mg/ml), left at room temperature for 18 hrand brought to a pH of 7-O with O-2 N HCI. An aliquot of 50 pg of alkylated endotoxin was added to 1 ml of 2.5 ‘A SRC in normal saline and incubated for 1 hr at 37°C. The mixture was then washed three times in normal saline and a 1% SRC solution was made up in normal saline for titration. Samples of 0.1 ml of BSA and endotoxin anti-sera were serially diluted with normal saline containing 1% normal absorbed and inactivated rabbit sera or normal saline. All sera were titrated in V microwell trays (Microbiological Associates, Bethesda, Maryland). Reduction with 2-mercaptoethanol was performed with all sera. Equal volumes of serum and 0.2 M 2-mercaptoethanol were incubated for 1 br at 37”C, followed by alkylation with an equal volume of O-2 M iodoacetamide; this was then titrated according to the method of DRAPER and HIRATA (1968). Haemagglutination titres were recorded after standing overnight at room temperature in a humid chamber; they were expressed in terms of the log2 of the reciprocal dilution. The starting dilution was 1:2 for all sera assayed. The data presented are the arithmetic means of the haemagglutinating titres from three rabbits for each time period. Antibody separation Molecular sieve chromatography (KIM et al., 1968) for the separation of IgM and IgG was performed on Sephadex G200 (Pharmacia Uppsala, Sweden) in a 100 x 2-S cm column (Fig. 1). A 1 ml 2.0

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Fro. 1. Serum separation after passage through Sephadex G200, in a 100 x 2.5 cm column, with a flow rate of tris-HCl buffer at 5 ml/hr.

250

A. T. BALL and J. R. TR~XT

sample of rabbit serum was applied to the column through a three-way valve without stopping the flow through the gel. All rabbit antisera were fractionated using 0.1 M tris-HCI buffer at pH 8-2 containing 0.2 M NaCI. Column eluents were continuously monitored and automatically recorded at 280 ma on a Uvicord II L.K.B. (Canlab, Toronto). A flow rate of 5 ml/hr of buffer was used and 5 ml samples were collected. A void volume of 110 ml was determined by passing Blue dextran 2000 (Pharmacia, Uppsala, Sweden) through the column. Then 10 ml of eluent, corresponding to the ascending curves of the first two peaks, were collected separately, concentrated twenty times by pervaporation in a dialysis bag and kept at -20°C. Immunoelectrophoresis The IgM and IgG antibody containing fractions were analysed for homogeneity by immunoelectrophoresis, with Gelman L.K.B. apparatus (Gelman Inst. Co., U.S.A.). After electrophoxesis, the IgM and IgG fractions were reacted with goat anti-rabbit globulins, (Pentex, Illinois), and were read 24-72 hr later to ensure complete development of the immunoprecipitates (Fig. 2). RESULTS

Bovine serum albumin

A typical primary response to a protein antigen was seen from the haemagglutination estimations of whole sera. All rabbits responded for each time period. The first indication of an antibody response occurred after a lag period of 5 days, with a peak at 14 days and then a slow decline to 28 days (Fig. 3). The separated fractions showed that the response at 5 days was purely IgM in nature and this was followed rapidly

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ANTIBODY

RESPONSE

TO BSA AND

251

ENDOTOXIN

at 7 days by an IgG response. The two immunoglobulins rose together until day 14. After this time, IgG class of antibodies rose to a peak at day 21 and then remained the predominant immunoglobulin. The response to a secondary injection of antigen at 28 days showed a short lag period before an exponential rise of antibodies, reaching a peak on the fifth day (Fig. 4). This was followed by a gradual decline during the next 40 days; even at the end of this time there was a high antibody titre. The antibodies belonged to the IgG class and, although IgM was present throughout, it-was only found in small amounts after the fifth day. The immediate and rapid rise of the IgM fraction paralleled that of the IgG response until they peaked at day 5. After this time, there was a more marked decline in the IgM response during the next 40 days. --20 1.9 16 14

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Forty-two days after the first challenge injection there were still significant amounts of haemagglutinating antibodies to BSA which belonged mainly to the IgG class. With repeated injections, both whole sera and the chromatographic fractions showed, after an initial rise in the first 7 days, a plateau that was reached with both IgM and IgG (Fig. 5). After a further injection on day 68, the whole serum antibody response was similar in nature to that found at the beginning of the schedule of repeated injections. Analysis of the separated sera showed that IgG and IgM response was similar to that found at the beginning of the regime.

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FIO. 5. IgG and IgM class of antibodies remained elevated during 28 days of repeated intragingival injections of 2.5 mg/kg of BSA every 3 or 4 days.

Endotoxin Antibody was detected 3 days after a primary injection of 1-O pg of E. coli endotoxin. A peak antibody titre was reached at 7 days and this was maintained until day 21 when there was a slight fall in titre (Fig. 6). Specific antibody activity was found entirely in the IgM fraction. 2-Mercaptoethanol reduction of whole sera confirmed that the entire antibody activity was of the IgM class. Analysis of whole sera following the challenge injection of O-1 pg of endotoxin at 28 days showed a slow but noticeable rise over the first 14 days of the secondary response. This was followed by a slow decline over the next 28 days. Throughout this response, the antibody titres were greater than those found in the primary endotoxin response and belonged mostly to the IgM class throughout the 40 day period (Fig. 7). All rabbits responded during the 42 day period. The titres of IgG class of antibodies at days 7, 10, 14 and 21 were very low and it was not until day 28 that all 3 rabbits showed a more appreciable IgG response that was maintained until the end of the 40 day period. Forty-two days after the first challenge injection a well-marked IgM response remained as well as a small but detectable level of IgG class of antibody. After a series of repeated injections of O- 1 ,ug of endotoxin, there was a progressive rise of

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IgM class of antibodies up to 14 days followed by a plateau (Fig. 8). The antibody level was slightly higher than that found after a single secondary injection. After the injections ceased at day 28, a rest period followed for 40 days and by that time the whole serum and the IgM response had returned to the level found at the end of the single secondary response. The IgG class of antibody response did not change but after three injections it rose at day 10 and reached a plateau at day 14. This was

254

A. T. BALL and J. R. TROTT

maintained until the end of the injection schedule; after a rest of 40 days, the IgG titre also returned to the same level as that found at the end of the initial secondary response. When injections were repeated after the 40 day rest period, both IgM and IgG class of antibodies rose gradually to levels similar to those observed before the rest period. ---

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DISCUSSION

The present study showed that after intragingival administration of a protein antigen (BSA), IgM and IgG class of antibodies were present simultaneously from day 7 and then throughout the primary response. The inability to detect IgG class of antibodies on day 5 when IgM antibodies were first found was probably due to the haemagglutination technique favouring the IgM type of response. Indeed, WEI and STAVITSKY(1967), using antigen binding and radio-immunoelectrophoresis techniques, showed that IgG was present simultaneously with IgM in the earliest antibody response of rabbits to human serum albumin. The exponential rise of IgM and IgG class of antibodies paralleled each other and both reached maximum titres simultaneously. The titres of IgM class of antibody appeared to decline more rapidly in the primary response than did IgG antibody titres. This may have been due to their half lives of 0.5 and 5 *4 days (Brozzr et al., 1968), or it could be due to negative feedback from the increasing amount of IgG (NOSSALand ABBOTT, 1968).

RABBIT

ANTIBODY

RESPONSE

TO BSA AND ENDOTOXIN

255

The accelerated and elevated response of IgG to a secondary protein injection is widely accepted but the contribution made by IgM is not. IgM in the secondary response in rabbits is found in very small amounts (DRAPERand HIRATA, 1968), or in amounts equal to the initial IgM primary peak (BAUERet al., 1963) or IgM can be totally absent (SCHELLINGand SILVERMAN, 1968). Much of this inconsistency may be due to the reliance on 2-mercaptoethanol reduction of whole sera as a test for IgM activity. Robbins et al. (1965) stressed the necessity for studying isolated antibodies and urged re-examination of the use of 2-mercaptoethanol as the sole criterion in distinguishing IgM and IgG antibodies. In the present study, secondary injections of BSA produced not only an expected IgG response but also a substantial IgM response that was similar to the peak found in the primary response. Significant IgM titres were found throughout the secondary response. Multiple BSA injections may prolong the IgM response in rabbits (FREEMAN, 1968). This was confirmed in this study and so was the observation that the antibody titres are only slightly higher than those found after a single secondary injection. Both IgG and IgM antibodies soon reached a plateau, above which a further response could not be elicited, despite continual BSA injections. Endotoxins are one of the most potent and controversial antigens (BRITTON,1969). Doses of I.0 pg and 0.1 pg of E. coli somatic antigen were sufficient to provoke significant antibody responses in all rabbits studied. Intragingival endotoxin administration elicited only IgM antibodies in the primary response. The inability to detect IgG antibodies by means of serum fractionation and haemagglutination, immunoelectrophoresis or 2-mercaptoethanol reduction, does not, however, preclude the possibility that these antibodies may be present (ALTEMIERet al., 1966). When a second dose of 0 - 1 pg of endotoxin was administered, an increase in antibody activity occurred which appeared sooner, reached higher levels and was maintained longer than the primary response. Doubts have been expressed as to whether endotoxins are capable of inducing a true secondary response (BAUERet al., 1963 ; LANDYet al., 1965). The two-fold rise of IgM titres over that of the primary response found in the present investigation suggests that a secondary response occurred in spite of an absence of an exponential rise. The response was mainly of the IgM class although a small but significant amount of IgG was seen later in the secondary response. LANDYet al. (1965) and ROBBINSet al. (1965) also demonstrated IgG class of antibodies in the secondary response. Repeated injections of endotoxin resulted in a plateau being reached with both IgM and IgG antibodies; the titres were only slightly higher than those after a single secondary injection. Similar results were reported by MULHOLLAND et al. (1965). The antibody levels to BSA were consistently higher than those to endotoxin and whereas BSA induced mostly IgG class of antibodies, the response to endotoxin was predominantly of the IgM class. Acknowledgements-We wish to acknowledge the help of Mr. A. DOMOKOSfor preparation of the graphs and Mrs. B. TILLERfor typing the manuscript. This work was supported by the M.R.C. of Canada Grant MA 3707.

256

A. T. BALL and J. R. TROTS R&sun&--La reaction d’anticorps humorale chez les lapins aux injections intragingivales primaires, secondaires et multiples de serum albumines bovin, (BSA) ou aux endotoxines lipopolysaccharides somatiques du colibacille a et6 analys& par chromatographie mol&culaire en papier, immuno&ctropho&se et hemoagglutination passive. Aucun adjuvant n’a CtCutilise dans ces experiences. Le BSAproduisit surtoutd’anticorps de la classe JgG a l’exception de la part initiale de la reaction primaire. Une reaction JgM pure a Btt notee suivant une premiere injection d’endotoxine. J_es deux anticorps JgM et JgG ont ete trouves dans la reaction secondaire et apt&s des injections multiples rep&es, mais le JgM dominait toutes les reactions a l’endotoxine. J_es r&sultats indiquent aussi qu’une reaction secondaire augment&e d’anticorps existe apres une injection secondaire d’endotoxine. Les protils JgM et JgG suivant les injections intragingivales deBSAontCttmaintenant&ablis, en suivant trois methodes differentes d’administration. zusammenfasstntg-Es wurde die humorale Abwehrreaktion bei Kaninchen gegen primlre, sekundare und mehrfache intragingivale Jnjektionen mit RinderserumAlbumin (RSA) oder somatischem Lipopolysaccharid-Endotoxin von Escherichiu coli mittels molekularer Siebchromatographie, immunoelektrophoresischer und passi ver Zusammenballung der Blutkorperchen analysiert. Bei keinem der Experimente wurde ein Adjuvans verwendet. Mit Ausnahme des friihen Stadiums der Primarreaktion liiste RSA haupttichlich Abwehrstoffe der JgG Gruppe aus. Nach einer prim&en Endotoxininjektion wurde keine JgM Reaktion beobachtet. Sowohl JgM als such IgG Abwehrstoffe wurden bei der Sedundlrreaktion und nach wiederholten, mehrfachen Endotoxininjektionen festgestellt, doch iiberschattete IgM alle Endotoxinmaktionen. Die Befunde weisen such darauf hin, dass nach einer sekund&ren Endotoxininjektion eine gesteigerte, sekundare Abwehrreaktion auftritt. Die Umrisse von JgM und JgG nach intragingivalen RSA Injektionen oder nach Endotoxin wurden nun nach drei verschiedenen Verabreichungsmethoden festgelegt. REFERENCES

AL~MEIER, W. A., ROBBINS,J. B. and SW, R. T. 1966. Quantitative studies of the immunoglobulin sequence in the response of the rabbit to a somatic antigen. J. exp. Med. 124,443-W. BAUER,D. C., MATHIES,M. J. and STA~~~KY, A. B. 1963. Sequences of synthesis of rl-macroglobulm and y2-globulin antibodies during primary and secondary responses to proteins, salmonella antigens and phage. J. exp. Med. 117, 889407. BBRGLUND,S. E., RIZZO, A. A. and MERGENHAOEN, S. E. 1969. The immune response in rabbits to bacterial somatic antigen administered via the oral mucosa. Archs oral Biol. 14, 7-17. Brozzr, G.. STIFFEL,C., MOIJT~N, D., BOUTHILLIER,Y. and DECREUSEFOND. C. 1968. A kinetic study of antibody producing cells in the spleen of mice immunixed intravenously with sheep erythrocytes. ImmuIwrogy 14,7-19. B~UTION,S. 1969. Regulation of antibody synthesis against E. coli endotoxin-IV. J. Immwwl. 129, 469482. CRUMPX~N, M. J., DAMES, D. A. and H urctn~so~, A. M. 1958. The serological specificities of Pasteurelk pseudoruberculosis somatic antigens. J. gen. Microbial. 18, 129-139. DRAPER, L. R. and HIRATA,A. A. 1968. Antibody responses in rabbits to soluble particulate forms of bovine serum albumin. Imrmrnology 15 23-30. FREEMAN,M. J. 1968. Heterogeneity of antibody response of rabbits immunized with acrylic particle BSA complexes. Immwwlogy 15.481492. KIM,Y. B., BRADLJX,S. G. and WATSON,D. W. 1968. Ontogeny of the immune response-V. Further characterization of 19s $3 and 7s $ immunoglobulins in the true primary immune response in germ-free colostrum deprived piglets. J. Immunol. 101, 224-236. LANDY, M., SANDERSON, R. P. and JACKSON,A. L. 1965. Humoral and cellular aspects of the immune response to the somatic antigen of Salmonella enteritidis. J. exp. Med. 122,483-504. MOLLBR,G. 1965. 19s antibody production against soluble lipopolysaccharide antigens by individual lymphoid cells in vitro. Nature, Loti. 207, 1164-l 168. MULHOLLAND,J. H., WOLFF, S. M., JACKSON.A. L. and LANDY M. 1965. Quantitative studies of febrile tolerance and levels of specific antibody evoked by bacterial endotoxin. J. clin. Invest. 44, 920-928.

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NOSSAL,G. and ABBOT,A. 1968. Bases for the recognition of antigenicity. Cancer Res. 28.1423-1429. RANNBY,R. R. 1970. Specific antibody in gingiva and submandibular nodes of monkeys with allergic periodontal disease. J. periodont. Res. 5, 1-17. RIZZO, A. A. and MITCHELL,C. T. 1966. Chronic allergic intlammation induced by repeated deposition of antigen in rabbit gingival pockets. Periodontics 4, 5-10. ROBBINS,J. B., KENNY, K. and SIITBR,E. 1965. The isolation and biological activities of rabbit YM and yG anti-Salmonella typhimurium antibodies. J. exp. Med. 122, 385-402. SCIIBLLING,M. and SILVERMAN,P. 1968. The effect of route of injection upon the development of circulating antibody in response to a variety of antigens. Immunology 14, 781-785. WEI, M., and STAVITSKY,A. B. 1967. Molecular forms of rabbit antibody synthesized during the primary response to human albumin. Immunology 12, 341444. YOUNG, W. G., DICK, H. M. and TROIT, J. R. 1969. The primary immune response of mice to two submucosal antigens. J. periodont. Res. 4,268-273.

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1 OVERLEAP

A. T. BALL and J. R. TROT-I

FIG. 2. Immuno-electrophoretic

pattern of IgG (top) and IgM (bottom) developed with goat anti-rabbit globulin.

PLATE

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