The immune response in rabbits to bacterial somatic antigen administered via the oral mucosa

Archs oral BioL Vol. 14, pp. 7-17, 1969. Pergamon Press. Printed in Gt. Britain.

THE I M M U N E RESPONSE IN RABBITS TO BACTERIAL SOMATIC A N T I G E N A D M I N I S T E R E D VIA THE ORAL MUCOSA S. E. BERGLUND, A. A. R l z z o and S. E. MERGENHAGEN Immunology Section, Laboratory of Microbiology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. Summary--The immune response in rabbits stimulated by injecting different quantities of E. coli somatic antigen, either through the oral mucosa or intravenously, was studied by enumerating the number of antibody-forming cells effected in local and systemic lymphoid organs. In addition, antibody activity was detected in granulomas at the local injection site with a sensitive technique previously described by this laboratory. Results suggest that low doses of antigen administered intramucosally stimulated an immune response limited to the local lymph nodes. Larger doses stimulated an immune response not only in the local nodes but in the systemic antibody-forming organs as well. Conversely, intravenously administered antigen provoked an immune response only in the systemic antibody-forming organs. Serum antibody titres were consistent with the above findings and also suggested that relatively large amounts of antigen injected locally recruited more ceils into the immune response than did intravenous injection. However, smaller doses appeared to be more effective in such recruitment if administered intravenously. INTRODUCTION THE LARGEnumber of bacteria in contact with diseased periodontal tissues favours the passage of antigens into the body. Moreover, antibody specific for oral bacteria has been demonstrated in pooled human sera and in the sera of patients with severe periodontal disease (MERGENHAGEN, DE ARAUJO and VARAH, 1965; EVANS, SPAETH and MERGENHAGEN, 1966). Additional findings show that elevated amounts of immunoglobulins are present in inflamed gingival tissues (BRANDTZAEG and KRAUS, 1965; TnONARD, CROSBV AND DALBOW, 1966) and that these proteins may contain antibody specific for oral bacteria (SCHNEIDER, et aL, 1966). Since the interaction of antigen and antibody is known to provoke allergic inflammation in gingival tissues ( R l z z o and MITCHELL, 1966) the immune response to bacterial antigens could be of special significance in the pathogenesis of periodontal disease. Recent findings show that the immune response to somatic antigens of G r a m negative bacteria injected into the rabbit footpad can be largely localized in the popliteal lymph node (LAND¥ and BAKER, 1966). In view of these findings, and the possible relevance of local immune reactions to periodontal disease, experiments were undertaken to study the immune response to bacterial antigen deposited in the oral mucosa. For comparison, antigen was also administered intravenously. The resulting

8

S.E. BERGLUND,A. A. Rizzo ANDS. E. MERGENHAGEN

antibody was quantitated by determining serum titres and the number of antibodyforming cells in the cervical lymph nodes, spleen, bone marrow and blood leukocytes. MATERIALS AND METHODS

Antigen The lipopolysaccharide somatic antigen (SA) from Escherichia coli strain 0127:B8 (Difco Laboratories, Detroit, Michigan) was employed. Antigen doses prepared from a stock solution were injected in either 0-2 or 2.0 ml volumes.

Identification of cervical lymph nodes One-tenth microgram of SA contained in a 0-2 ml volume was injected into the palatal mucosa of female New Zealand white rabbits weighing 2.0-2.5 kg. The injection was made with a 0.25 ml syringe fitted with a 30 gauge needle which was placed as superficially as possible at a site in the right maxillary palatal mucosa centred between the midline and the distolingual corner of the last molar. Five days later the ipsilateral and contralateral submaxillary, parotid, and upper deep cervical lymph nodes were removed and assayed as described below for antibody-forming cells.

Immunization Groups of at least three rabbits were immunized either intramucosally or intravenously. Animals to receive antigen intramucosally were anaesthetized with pentobarbital sodium ("Diabutal", Sodium Pento-Barbital, Diamond Laboratories, Des Moines, Iowa). The antigen contained in a 0.2 ml volume was then injected into the palate as described above. Equal doses of antigen were administered intravenously in 2-0ml volumes. Controls received similar injections of pyrogen-free saline. In addition, several rabbits were injected intramucosally with 0.25 ml complete Freund's adjuvant (Difco Laboratories) containing 100/~g of SA. Additional rabbits were similarly injected with adjuvant not containing SA.

Preparation of tissues for assay According to LANDVand BAKER(1966), maximum numbers of antibody-forming cells appear in the regional lymph nodes and the spleen 5 days after a local injection of lipopolysaccharide somatic antigen. Similar observations have been made by this laboratory (unpublished observations) and by others (LANDY et al., 1965; M6LLER, 1965). Based on these findings, assays for immune responses were conducted 5 days after either intramucosal or intravenous immunization. Blood was drawn by cardiac puncture to obtain serum and blood cells. The animal was then sacrificed with an overdose of pentobarbital sodium and the ipsilateral and contralateral upper deep cervical lymph nodes, spleen, and a pool of bone marrow from the long bones of each leg were removed. Blood cells were obtained by drawing 10 ml of blood into an equal volume of cold Alsever's solution. No attempt was made to separate leukocytes from erythrocytes

THE IMMUNE RESPONSE IN RABBITS TO BACTERIAL SOMATIC ANTIGEN

9

which were immediately centrifuged and washed three times with 6 volumes of cold phosphate-buffered saline at pH 7.2. The washed cells were then suspended at a concentration of 2 x 106 leukocytes per ml in cold Ham's F~ 2 tissue culture medium (HAM, 1965). Suspensions of lymph node, spleen, and bone marrow cells were obtained by placing each tissue in 4-0 ml of cold Ham's medium and mincing it with scissors. The minced tissues were then gently homogenized with a loose-fitting teflon pestle and the organ stroma was removed by passing the crude cell suspensions through several layers of cheese cloth. Cell concentrations were determined by haemocytometer counts and dilutions were prepared which contained either 2 x 10' or 2 x 106 cells per ml.

Quantitation of the immune response Antibody contained in serum was assayed by the photometric bactericidal assay technique of MUSCHELand TREFFERS(1956). The assay employed for the quantitation of antibody-releasing cells in lymphoid tissues and blood was that described by JERNE, NORD1N,and HENRY (1963) as modified by SCHWARTZand BRAUN(1965). This procedure was conducted as follows: E. coli was grown in l0 ml of brain heart infusion broth (Difco Laboratories) for 18-24 hr. The cells were harvested, washed three times, and suspended in 5.0 ml of Ham's medium so that the concentration of viable cells was approximately 1.2 x 108 per ml. Two-tenths ml of the bacterial suspension was pipetted into a test tube placed in a 4245°C water bath. One-half ml of a lymphoid cell suspension was then added. Two ml of 0.5 per cent agarose ("Seakem" agarose, Lawshe Instrument Co., Bethesda, Md.) suspended in Ham's medium at 50°C was added and the tube contents were mixed by gentle pipetting. The mixture was subsequently layered equally over two 15 x 100 mm petri dishes previously prepared by covering the bottoms with 4 ml of 0.5 per cent agarose. When the agar had gelled, the dishes were incubated for 1 hr at 37°C. Two and one-half ml of complement (fresh guinea pig serum, Texas Biological Laboratories, Fort Worth, Texas) diluted 1/10 with cold phosphate buffered saline was then added to each petri dish. Incubation proceeded at 37°C for an additional 3-4 hr during which time bacteria not affected by antibody and complement grew throughout the agar. Bacteria in the immediate zone of an antibody-releasing cell, however, were sensitized by specific antibody and their subsequent growth was inhibited by the action of complement. Such zones were termed plaques and the centrally located effector cell was termed the plaque-forming cell (PFC). At the termination of the 3-4 hr incubation period, those petri dishes which contained erythrocytes and leukocytes were flooded with 2 per cent acetic acid to lyse contaminating erythrocytes. Such treatment enabled plaques in the bacterial growth to be more readily perceived. The number of plaques on each dish was counted with the aid of a dissecting microscope at x 7 magnification and expressed as the number of PFC per lymph node or spleen (Fig. 1). The immune response in blood leukocytes and bone marrow cells was evaluated on a relative basis by comparing the number of PFC per l06 cells assayed in these tissues to the number of PFC per 106 cells assayed in the spleen.

10

S.E. BERGLUND,A. A. RIzzo ANDS. E. MERGENHAGEN

Determination of antibody activity in oral tissues Attempts were made to demonstrate antibody activity at the site of antigen deposition in oral mucosa by using a modification of the plaque technique just described. Instead of testing cell suspensions from oral mucosa for plaque formation, frozen sections of this tissue were tested directly for the presence of antibody activity, Rabbits were injected with adjuvant and sacrificed 10 days later. The resulting granulomas were excised and bisected. The cut surfaces were wiped free of adjuvant and the tissue mass was mounted on an object disc held in dry ice so that serial sections of about 10tz in thickness could be obtained. Even-numbered sections were placed in 10 per cent buffered formalin for subsequent staining with haematoxylin and eosin. Odd-numbered sections were treated as previously described (BERGLUND, MARKEY and MERGENHAGEN, 1967) SO that the presence of specific antibody within the tissue could be demonstrated. The assay was employed as originally described; however, the following modifications were made: (1) sheep erythrocytes were replaced with an E. coli inoculum as described above in the PFC technique; (2) one-half ml Ham's medium was added to the agar instead of 0.5 ml of complement and (3) after 1 hr of incubation, complement diluted 1 to 10 was pooled on top of the agar overlay instead of incorporating it into the gel. Incubation then proceeded for an additional 3-4 hr. During the initial 1 hr incubation period, antibody within the tissue section diffuses into an overlying agar layer which contains E. coli. This results in the formation of bacteria-antibody complexes. The addition of complement and subsequent incubation effects growth inhibition of those organisms combined with antibody but it does not affect the growth of those organisms which did not react with antibody. Since the inhibition of bacterial growth is effected by the action of antibody and complement, the following controls were run to rule out other growth inhibitors which might be present in the tissue specimen: (1) sections were prepared from granulomas induced with adjuvant in the absence of SA., (2) sections from granulomas induced with adjuvant containing SA were overlayed with agar containing bacteria and incubated with heat-inactivated complement. Similar sections were overlayed with agar that contained 100/zg of SA per ml in addition to E. coli. The presence of SA in the agar would block the combining sites of homologous antibody and thereby prevent antibody-mediated growth inhibition. RESULTS Table 1 shows that afferent lymphatic vessels at the injection site drained to the ipsilateral upper deep cervical lymph node. It was therefore decided to assay only the ipsilateral and contralateral upper deep cervical nodes in an attempt to measure the immune response in local lymphoid tissues draining the oral mucosa. The immune response manifested by systemic antibody-forming organs was estimated by quantitating the number of PFC which appeared in spleen, bone marrow and blood leukocytes. A comparison of the immune response manifested by cells from these sites indicated that the spleen alone would serve as the best indicator of the systemic immune response since, with the doses of antigen employed, it always

11

THE IMMUNE RESPONSE I N RABBITS TO BACTERIAL SOMATIC ANTIGEN

TABLE 1. T O T A L NUMBER OF PLAQUE-FORMING CELLS FOUND IN REGIONAL LYMPH NODES AFTER AN INTRAMUCOSAL IMMUNIZATION W I T H 0 " 1 Fg SOMATIC ANTIGEN FROM E. coil*

Ipsilateral lymph nodes

Contralateral lymph nodes

Animal Upper deep cervical 1

Submaxillary plus parotid

Upper deep cervical

Submaxillary plus parotid

88

29 67 40

117 5 14

1011

2 3

851 792

59 62

* The numbers shown in the "ipsilateral, submaxillary plus parotid" column, as well as those in the two columns of "contralateral lymph nodes", fall within the control range. All numbers in the "ipsilateral upper deep cervical node" column are elevated above controls. s h o w e d the largest n u m b e r o f P F C . F u r t h e r m o r e , only spleen cells d e m o n s t r a t e d increased n u m b e r s o f P F C after i n t r a v e n o u s i m m u n i z a t i o n with low doses o f antigen ( 1 0 - 3 a n d 10-4/~g). The n u m b e r o f P F C f o u n d in local a n d systemic l y m p h o i d tissues after either i n t r a m u c o s a l o r i n t r a v e n o u s i m m u n i z a t i o n is shown in Fig. 2. These d a t a clearly show the d i s t r i b u t i o n o f the i m m u n e response between the cervical l y m p h nodes a n d the INTRAMUCOSA/

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FIG. 2. The numbers of antibody-forming cells found in the ipsilateral and contralateral upper deep cervical lymph nodes and the spleen 5 days after either an intramucosal or an intravenous immunization with various doses of somatic antigen. Each bar represents the mean 4- standard error of groups composed of at least three rabbits.

12

S.E. BERGLUND,A. A. PdZZOANDS. E. MERGENHAGEN

spleen after intramucosal injections of different antigen doses. An intramucosal injection of a 1 "0t~g dose provoked increased numbers of PFC in the local cervical lymph nodes as well as the spleen, whereas 10- or 100-fold reductions of the antigen dose (0.1 or 0.01/zg) stimulated the appearance of PFC only in the ipsilateral lymph node. In contrast, intravenous immunization with the same antigen doses effected a different distribution of the immune response as indicated by the appearance of PFC in the spleen alone (Fig. 2). The injection of 1.0/zg SA into the mucosa resulted in elevated serum antibody titres (Fig. 3). Doses of 0.1/zg or less administered by this route, however, failed to

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Fro. 3. The titres of bactericidal antibody in the serum of rabbits 5 days after either an intramucosal or an intravenous immunization with various doses of somatic antigen. Titres represent the greatest serum dilution which effected 50 per cent kill of a standard E. con inoculum. Each bar represents the mean ~ standard error of groups containing at least three rabbits. provoke elevated titres. On the other hand, immunization via the intravenous route with either the 1.0 or 0.1/~g dose did effect increased titres. The presence of specific antibody activity at a site of chronic inflammation induced with adjuvant containing SA is illustrated in Fig. 4. Figure 4(A) shows areas of bacterial growth inhibition over and around the tissue which contained antibody. Histological examination revealed a heavy cellular infiltrate which included large numbers of macrophages, lymphocytes, and plasma cells. The infiltrate distribution generally corresponded with the areas of antibody activity. This contrasts to the

THE IMMUNE RESPONSE IN RABBITS TO BACTERIAL SOMATIC ANTIGEN

]3

picture seen in Fig. 4(B) which illustrates a control section that shows no bacterial growth inhibition. Such tissue sections did not demonstrate growth inhibition because either antibody was absent, heat-inactivated complement was employed, or antibody present in the tissue was blocked by the presence of SA in the agar (see Methods). DISCUSSION

The results of this study clearly show that small quantities of lipopolysaccharide somatic antigen are capable of provoking a remarkable antibody response in regional lymphoid tissues which drain the oral mucosa. By comparing the distribution of the immune response in local and systemic lymphoid organs after an intramucosal immunization, the importance of the regional lymph nodes in the immune response elicited by locally-deposited antigen becomes apparent. This is emphasized by the observation that stimulation with minimal antigenic doses (0.1-0-01 ~g) resulted in detectable antibody production only by cells in the local ipsilateral lymph node. Larger doses (1.0/~g) similarly administered provoked splenic antibody-forming cells in addition to stimulating such cells in the local lymph node. In contrast, an intravenous injection of the same antigen dose stimulated antibody-forming cells in the spleen but not in local lymph nodes. These findings correlate with serum antibody titres which reflect composite antibody synthesis of all antibody-forming cells in the animal. The serum antibody titres also suggest that more antibody-forming cells were recruited into the immune response by microgram doses when administered intramucosally, whereas smaller doses ( ~ 0.1/~g) were more effective in such recruitment if an intravenous injection was employed. The immune response to bacterial antigens entering the body through oral tissues in patients with periodontal disease could be analogous to the immune response described in this study, since the number of bacteria in human periodontal pockets can account for the doses of somatic antigen employed (RIzzo and MERGEN,AGEN, 1964) and antibody titres against oral bacteria have been demonstrated in human sera (EvANs et al., 1966; MERGENHAGENet al., 1965). The presence of antibody specific for oral bacteria in serum would presumably result in antibody localized at a site in the oral tissues containing bacterial antigens (GmsoN and SHANNON, 1964; COURANTand BADER, 1966). Such localization could be effected through the formation of antigen-antibody complexes within the tissue. The increased amount of immunoglobulins demonstrated in inflamed periodontal tissues (BRANDTZAE6 et al., 1965; TI-IONARD et al., 1966) is consistent with this reasoning. Such antigens need not be intact bacterial cells, but could be cell-free antigens resulting from bacterial degradation. On the other hand, the origin of immunoglobulins in diseased gingival tissue may not be entirely derived from serum. Such antibody may result from de novo synthesis at the inflammatory site itself, since plasma cells are characteristic features of the inflammatory infiltrate (RIZzOand MERGENHAGEN,1965), antibody-forming cells have been demonstrated in the gingiva of rats after repeated administration of antigen (TnoNARD and DALBOW, 1965), and antibody synthesis has been demonstrated by cells in chronically inflamed tissue (granuloma) (AsKONASand

14

S.E. BERGLUND,A. A. RIzzo Aha) S. E. MERGENHAGEN

HtrMrrmEY, 1958). Although the data presented herein do not allow the conclusion that de nero antibody synthesis has occurred in oral tissues, the findings are suggestive of, and consistent with, such antibody production. The potential of somatic antigens from gram-negative organisms and reacting antibody to serve as activators of the terminal components in the complement system has been recently shown (BLADEN,GEWURZ,and MERGENHAGEN,1967; GEWURZ, et al., 1968). Such activated complement components are known to generate pharmacologically active effector substances which contract smooth muscle and increase vascular permeability (anaphylatoxin) (JENSEN, 1967; D~AS OA SILVA, EISELE and LEPOW, 1968 ; COCHRAN~and MOLLER-EBERHARD, 1968 ; LICHTENSTEINet al., 1968) and induce polymorphonuclear chemotaxis (WARD, COCHRhNE and MOLLER-EaERHARD, 1966; SNYDERMAN, GEWURZ and MERGENHAGEN, 1968) both of which are hallmarks of the inflammatory response. Thus, it appears as though the immune response to oral bacterial antigens m a y represent a two-edged sword. One edge of this sword represents the protection afforded by antibody against active bacterial infection while the other edge represents the features of immune reactions which mediate the inflammatory process and may, therefore, adversely affect periodontal health. Although in general both features aid the host in its attempt to combat infection and noxious agents, the periodontium represents an area of the body which is generally thought to be deleteriously affected by events which occur during the inflammatory response. Since increased vascular permeability and leukocytic migration do occur in inflamed h u m a n gingival tissues, immune reactions provoked by bacterial antigens must be considered as a significant factor in mediation of the inflammatory response characteristic of periodontal disease. Acknowledgements Acknowledgement is made to Mr. JOHN B. KENNEDY for his superior technical assistance. Appreciation is also expressed to Drs. SAMUEL KAKErmsm, HENRY GEWURZ, and JENS WAERI-tAUG for their helpful suggestions in the preparation of this manuscript. Rrsumr--La rrponse immunitaire de lapins, obtenue en injectant diverses quantit~s, d'anti#ne somatique d'E. cell, soit dans la muqueuse buccale, soit par voie intraveineuse, est 6tudire en comptant le hombre de cellules formant des anticorps au niveau des organes lymphoides rrgionaux et grnrraux. En outre, l'activit6 en anti-corps est drterminre h l'aide d'une technique originale, au niveau de granulomes apparaissant au point d'injection. I1 semble que des doses faibles d'antigrnes, injectrs dans la muqueuse, provoquent une rrponse immunitaire, lirnit6e aux ganglions lymphatiques rrgionaux. Des doses plus 61evresprovoquent, non seulement, une rrponse rrgionale, mais intrressent 6galement les organes grnrraux, forrnant des anticorps. Des antigrnes, injectrs par voie intra-veineuse, ne provoquent une rrponse qu'au niveau des organes grnrraux producteurs d'anticorps. La drtermination des anticorps srriques confirme les rrsultats prrcrdents et suggrre que des quantitrs relativement plus importantes d'antigrnes, injectrs localement, intrressent plus de cellules, au cours de la rrponse immunitaire que les injections intraveineuses: cependant, des doses moindres paraissent plus effectives par voie intra-veineuse. Zusammenfassung--Durch Ausz/ihlung der AntikOrper-bildenden Zellen in lokalen und allgemeinen Lymphorganen wurde die Immunreaktion bei Kaninchen nach Injektion verschiedener Mengen yon E. coil-Antigen untersucht. Das Antigen wurde entweder in die

THE IMMUNE RESPONSE IN RABBITS TO BACTERIAL SOMATIC ANTIGEN

15

Mundschleimhaut oder intraven/Ss appliziert. Weiterhin wurde die Antik6rperaktivitiit in Granulomen am lnjektionsort mit einer yon uns frtiher beschriebenen empfindlichen Methode bestirnmt. Die Ergebnisse deuten darauf hin, dab kleine, intramuk~Ssapplizierte Antigen-Mengen eine auf die 6rtlichen Lymphknoten begrenzte Immunreaktion hervorrufen. Gr/SBereDosen verursachten eine Immunreaktionnicht nur in den lokalen Lymphknoten sondern auch in den allgemeinen Antik6rper-bildenden Organen. Umgekehrt fiihrte das intraven6s angewandte Antigen lediglich zu einer Imrnunreaktion in den allgemeinen Antik6rper-bildenden Organen. Die Antik6rpertiter stimmten mit diesen Befunden iiberein und legten auch nahe, dab relativ groBe, lokal irjizierte Antik6rpermengen mehr Zellen in die lmmunreaktion einbezogen, als dies bei intravent~ser Injektion der Fall war. Kleinere Dosen schienen jedoch in dieser Hinsicht wirkungsroller zu sein, wenn sie intravenSs angewandt wurden. REFERENCES ASKONAS,B. A. and HUMPHREY,J. H. 1958. Formation of specific antibodies and gamma globulin in vitro; a study of the synthetic ability of various tissues from rabbits immunized by different methods. Biochem. J. 68, 252-261. BERGLUND,S. E., MARKEY,P. A. and MERGENHAGEN,S. E. 1967. Observations on the kinetics of the hemolytic antibody response by localized hemolysis in gel over frozen sections of mouse spleen. Proc. Soc. exp. Biol. Med. 126, 84-88. BLAOEN, H. A., GEWURZ,H. and MERGENHAGEN,S. E. 1967. Interactions of the complement system with the surface and endotoxic lipopolysaccharide of Veillonella alcaleseens. J. exp. Med. 125, 767-786. BRANDTZAEG,P. and KRAUS,F. W. 1965. Autoimmunity and periodontal disease. Odont. Tidsk. 73, 285-381. COCHRANE, C. G. and M~3LLER-EBERHARD,H. J. 1968. The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. exp. Med. 127, 371-386. COURANT,e. R. and BADER,H. 1966. Bacteroides melaninogenicus and its products in the gingivae of man. Periodontics 4, 131-136. DIAS, DASILVA,W., EISELE,J. W. and LEPOW,I. W. 1968. Complement as a mediator of inflammation. III. Purification of the activity with anaphylatoxin properties generated by interaction of the first four components of complement and its identification as a cleavage product of C'3. J. exp. Med. 126, 1027-1048. EVANS,R. T., SPAETH, S. and MERGENHAGEN,S. E. 1966. Bactericidal antibody in mammalian serum to obligatorily anaerobic gram-negative bacteria. J. Immunol. 97, 112-119. GEWURZ, H., SHIN, H. S., MAYER, M. M. and MERGENHAGEN,S. E. 1968. Further studies of the interactions between the complement system and endotoxic lipopolysaccharides. (Abs.). Fed. Proc. 27, 479. GIBSON, W. A. and SHANNON,I. L. 1964. Microorganisms in human gingival tissues. Periodontics 2, 119-121. HAM, R. G. 1965. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proe. natn. Acad. Sei. U.S.A. 53, 288-293. JENSEN, J. 1967. Anaphylatoxin in its relation to the complement system. Science 155, 1122-1123. JERNE,N. K., NORDIN,A. A. and HENRY,C. 1963. The agar plaque technique for recognizing antibodyproducing cells: In Cell-bound Antibodies. Wistar Institute Press, Philadelphia. pp. 109-125. LANDY, M. SANDERSON,R. P., BERNSTEIN,M. R. and LERNER, E. M. 1965. Involvement of thymus in immune responses of rabbits to somatic polysaccharides of gram-negative bacteria. Science 147, 1591-1592. LANDY, M. and BAKER,P. J. 1966. Cytodynamics of the distinctive immune response produced in regional lymph nodes by Salmonella somatic polysaccharide, d. lmmunol. 97, 670-679. LICHTENSTEIN,L. M., GEWURZ,H., ADKINSON,N. F., SHIN, H. S. and MERGENHAGEN,S. E. 1968. Interactions of the complement system with endotopic lipopolysaccharides: The generation of an anaphylatoxin. Immunol. in press. MERGENHAGEN,S. E., DE ARAUJO,W. C. and VARAH,E. 1965. Antibody to Leptotrichia buccalis in human sera. Archs oral Biol. 10, 29-33. M6LLER, G. 1965.19S antibody production against soluble lipopolysaccharide antigens by individual lymphoid cells in vitro. Nature, Lond. 207, 1166--1168.

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S.E. BERGLUND,A. A. RIZZO AND S. E. MERGENHAGEN

MUSCHEL,L. H. and TREFFERS,H. P. 1956. Quantitative studies on the bactericidal actions of serum and complement. I. A rapid photometric growth assay for bactericidal activity. 3". lmmunoL 76, 1-I0. Rizzo, A. A. and MERGENnAGVN,S. E. 1964. Histopathologic effects of endotoxin injected into the rabbit oral mucosa. Archs oral Biol. 9, 659-670. Rtzzo, A. A. and MERGENHAGEN,S. E. 1965. Studies on the significance of local hypersensitivity in periodontal disease. Periodontics 3, 271-274. Rlzzo, A. A. and MITCHELL,C. T. 1966. Chronic allergic inflammationinduced by repeated deposition of antigen in rabbit gingival pockets. Periodontics 4, 5-10. SCHNEmER, T. F., TOTO, P. D., GARGIULO,A. W. and POLLOCK,R. J. 1966. Specific bacterial antibodies in the inflamed human gingivae. Periodontics 4, 53-57. SCrnVARTZ, S. A. and BRAUN, W. 1965, Bacteria as an indicator of antibody formation by spleen cells in agar. Science 149, 200--201. SNYDERMAN,R., GEWURZ,H. and MERGENr~AGEN,S. E. 1968. Interactions of the complement with endotopic lipopolysaccharide: Generation of a factor chemotactic for polymorphonuclearleukocytes. J. exp. Med. 128, 259-275. TnONARD, J. C. and DALBOW,M. H. 1965. Local cellular antibodies. I. Plaque formation by sensitized oral mucosal cells from conventional animals. J. ImmunoL 95, 209-213. TFIONARD,J. C., CROSBY, R. C. and DALBOW, M. H. 1966. Detection of IgM and IgA immunoglobulins in diseased human periodontal tissue. Internat. Ass. for Dent. Res. preprinted abstracts, 44th General meeting Abstract 328. WARD, P. A., COCFmANE,C. G. and MOLLER-EBEm-IARD,H. J. 1966. Further studies on the chemotactic factors of complement and its formation in vivo. Immunol. 11, 141-153.

THE IMMUNE RESPONSE IN RABBITS TO BACTERIALSOMATIC ANTIGEN

FIG. 1. A field p h o t o g r a p h e d f r o m a petri dish d e m o n s t r a t i n g the typical a p p e a r a n c e o f plaques (circular d a r k spots) o n a grainy m a t o f bacterial growth. E a c h plaque contains a single centrally-located cell which released specific a n t i b o d y into the s u r r o u n d i n g agar. x 5"6.

PLATE 1 A . O . B . f . p . 16

S. E. BERGLUND, A. A. RIZZO AND S. E. MERGENHAGEN

PLATE 2

THE IMMUNE

RESPONSE

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PLATE

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FIG. 4~. A section of palatal tissue containing a granuloma induced with complete Freund’s adjuvant and E. coli somatic antigen. The section is covered with agar supporting a grainy mat of E. coli growth. The dark zone immediately around much of the tissue periphery (arrows) results from antibody-mediated bacterial growth inhibition. Such growth inhibition also appears directly over much of the tissue, but it can not be seen in the picture because of poor contrast.

FIG. 4b. A similar section incubated in the presence of heat-inactivated complement which demonstrates no inhibition of bacterial growth. x 10.

A.O.B. 14/1-B

17