Clin. exp. Immunol. (1989) 76, 103-110

Bacteroides-specific IgG and IgA subclass antibody-secreting cellsisolated from chronically inflamed gingival tissues

T. OGAWAt, M. L. McGHEE, Z. MOLDOVEANU,* S. HAMADAt, J. MESTECKY*, J. R. McGHEE* &H. KIYONO The Departments of Oral Biology and *Microbiology, The Research Center in Oral Biology, The University ofAlabama at Birmingham, Alabama, USA and tThe Department of Oral Microbiology, Faculty of Dentistry, Osaka University,

Suita, Osaka, Japan

(Acceptedfor publication 20 December 1988)

SUMMARY

The emergence of cells that produce IgG and IgA subclass antibodies to Bacteroides gingivalis(Porphyromonas gingivalis) fimbriae and lipopolysaccharide (LPS) antigens was examined inmononuclear cells isolated from inflamed gingiva ofdifferent stages (slight, moderate or advanced) ofadult periodontitis (AP). Antigen-specific IgM, IgG (including IgG 1, IgG2, IgG3 and IgG4) and IgA(including IgA1 and IgA2) producing cells were enumerated by the ELISPOT assay and werecompared with total Ig-producing cells ofeach isotype or subclass. In advanced AP, the B. gingivalisfimbriae-specific IgG- and IgA-secreting cells represented 5% of total IgG- or IgA-secreting cells,while those from the moderate stage comprised approximately 1% of these two isotypes. Cellsproducing antibody specific for B. gingivalis LPS were observed at frequencies of 0-1% and 04% forIgG and IgA cells, respectively in the advanced stage. When IgG subclasses were analysed inmoderate AP, the anti-fimbriae subclass responses were largely IgGl (60%), followed by IgG2 (20%),IgG3 (10%) and IgG4 (10%). Fimbriae-specific IgG subclass responses were elevated in the advancedstage of AP, and IgG4 (40%) and IgGl (30%) were dominant, followed by IgG3 (20%) and IgG2(10%). IgAl cells predominated in both the moderate and advanced stages, however a relativeincrease in IgA2 cells occurred in advanced AP. Mononuclear cells isolated from gingiva of APpatients did not contain cells producing antibody to antigens such as Escherichia coli K235 LPS,cholera toxin or the hapten dinitrophenyl coupled to bovine serum albumin. These results show thatlocal IgG and IgA subclass responses occur to a protein antigen of a major periodontal disease (PD)-associated pathogen, B. gingivalis, and the increase in IgG4 and IgA2 responses may be associatedwith host protection.

Keywords Bacteroides gingivalis (Porphyromonas gingivalis) fimbriae lipopolysaccharide IgAsubclasses IgG subclasses periodontal disease adult periodontitis

INTRODUCTION

In humans the presence of IgG and IgA subclasses may beadvantageous to the host, because antibodies of various IgGand possibly IgA subclasses display different effector functions.For example, it is now established that IgGl and IgG3 areeffective bacterial opsonins and activate complement (C) via Clbinding, while IgG2 and IgG4 show little or no such function,respectively (Reid, 1983; Woof et al., 1986). Unlike IgGsubclasses the effector functions associated with IgA I and IgA2are less well defined. Generally IgA antibodies efficiently bind tobacterial antigens, however they do not activate C via Cl and

Correspondence: Dr Hiroshi Kiyono, Department of Oral Biology,University of Alabama at Birmingham, UAB Station, Birmingham,Alabama 35294, USA.

are considered to be poor opsonins (reviewed in Kilian,Mestecky & Russell, 1988).

Chronic inflammatory diseases, such as periodontitis, arecharacterized by a local accumulation of lymphocytes, macro-phages, neutrophils and abundant plasma cells, that are inducedby the penetration of bacterial products into the gingivalcrevice. Anaerobic, black pigmented Bacteroides, especially B.gingivalis (porphyromonas gingivalis), have been isolated fromthe advanced periodontitis lesion (Slots, 1986). The correlationbetween the incidence of B. gingivalis and alveolar bone loss isalso well established (Miller et al., 1986). Further, patients withperiodontitis possess elevated antibody levels to B. gingivalis inserum and in gingival crevicular fluid (Tew et al., 1985; Ebersoleet al., 1986; Naito, Okuda & Takazoe, 1987). Implantation of B.gingivalis into the gingival region ofprimates resulted in alveolarbone loss and elevated serum anti-B. gingivalis antibody levels

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(Holt et al., 1988). However, it is still not clear if the increase inplasma cells which occurs in periodontitis is due to immuneresponses to specific antigens of bacteria and other tissueproducts or to polyclonal responses induced by the bacterial cellwall components.

In this study we have isolated mononuclear cells fromgingival tissue of patients with adult periodontitis, and haveanalysed the isotype and subclass of antibody produced tofimbriae and lipopolysaccharide (LPS) of B. gingivalis at thesingle cell level. Our results show that antigen-specific immuneresponses occur at the local disease site, and provide evidencethat the fimbriae antigen of B. gingivalis induce IgG- and IgA-subclass-specific responses.

MATERIALS AND METHODSMonoclonal anti-human-IgG- and subclass-specific antibodiesAnti-IgGI (HP 6012), anti-IgG2 (HP 6014), anti-IgG3 (HP6047) and anti-IgG4 (HP 6022) (kindly provided by Dr C. B.Reimer, CDC, Atlanta, GA) were used for the ELISPOT assay(see below). The specificity of these MoAb was tested by anELISA using purified human myeloma IgGl, IgG2, IgG3 andIgG4 proteins, kindly furnished by Dr F. Skvaril ofWHO, anddetails are described in another manuscript (Ogawa et al., 1989).The MoAb anti-human IgAl and IgA2 antibodies, a kind giftfrom Dr J. Radl (Institute of Experimental Gerontology, TheNetherlands) have been extensively characterized and describedelsewhere (Crago et al., 1984).

Bacteriodes fimbriae andlipopolysaccharide (LPS) preparationsBacteroidesgingivalis 381 (Porphyromonasgingivalis) (originallyisolated by Dr S. Sockransky, Forsyth Dental Center, Boston,MA) was grown anaerobically in GAM broth (Nissui, Tokyo,Japan) supplemented 5 mg/l with haemin (Wako Pure ChemicalIndustries, Ltd. (Wako), Osaka, Japan) and menadione (10 yg/l,Wako) at 37°C for 26 h in a mixed gas with 5% C02, 5% H2 and90% N2.

Fimbriae were prepared on a large scale by a modification ofa method described previously (Yoshimura et al., 1984).Bacterial cells from a 16-1 culture were harvested by centrifuga-tion at 10 000 g for 30 min and were suspended in 2 1 of 20 mmTris-hydrochloride (pH 7 4), 0-15 M NaCI, and 10 mM MgCl2 byrepeated pipetting. The bacterial suspension was gently agitatedwith a magnetic bar for 15 min to avoid cell lysis, and thesupernatant was obtained by centrifugation at 10 000 g for 30min at 25'C. Ammonium sulphate was added to the bacterialwash fluid to 40% saturation, and the precipitated proteins werecollected by centrifugation and resuspended in a small volumeof 20 mm Tris-hydrochloride (pH 8 0). This material wasdialysed against 25 1 of 20 mm Tris-hydrochloride (pH 8-0). Thedialysates were clarified by centrifugation at 10 000g for 15 min,and the sample was applied to a column of DEAE-SepharoseFAST Flow (5 by 15 cm). The column was washed with 1 5 1 of20 mm Tris-hydrochloride (pH 8 0) and eluted with a step-wisegradient at 0 and 0-15 M NaCl in 20 mm Tris-hydrochloride (pH8 0). When column fractions were analysed by SDS-PAGE, the48 kD fimbriae protein was detected in the 0 15 M NaCl fraction.The column eluate containing the 43 kD protein was concen-trated by ammonium sulphate precipitation and dialysedagainst 10 1 of 20 mm Tris-hydrochloride (pH 8 0). The 0-15 M

NaCl fraction was tested by double diffusion in an agar platewith rabbit anti-fimbriae-specific and anti-B. gingivalis wholecell antisera. No other contaminating proteins were present. Thespecific immunologic and biochemical characterization of puri-fied fimbriae proteins will be described elsewhere (manuscript inpreparation).

Lipopolysaccharide (LPS) was prepared from B. gingivalisby the standard hot phenol water method as previouslydescribed (Koga et al., 1984). Briefly, lyophilized B. gingivaliscells were suspended in 350 ml ofpyrogen-free water and 350 mlof90% phenol. The mixture was stirred vigorously at 65°C for 5min and then centrifuged at 5000 g for 30 min. The aqueousphase was removed, and the phenol phase and insolubleprecipitate were re-extracted with 350 ml of water. The twoaqueous phases were combined, dialysed extensively againstdistilled water, lyophilized, and stored at - 20°C until used. Thebiochemical and biologic properties of phenol-water-extractedLPS (LPS (Ph)) from B. gingivalis are described elsewhere(Koga et al., 1984; Hamada et al., 1988).

Isolation ofmononuclear cellsfrom gingival tissuesSurgically removed inflamed tissues (range of 100-900 mg, wetweight) were placed in Petri plates containing RPMI 1640(Mediatech, Washington, DC) supplemented with L-glutamine,HEPES, gentamycin, penicillin and streptomycin (incompletemedium) and were then dissected into small fragments (1-2mm3). The tissues were washed twice with incomplete mediumfor 10 min at 37°C with stirring, and were then transferred to aPetri plate containing 1 5 mg/ml of the neutral protease enzymeDispase (Boehringer-Mannheim Biochemicals, Indianapolis,IN) in Joklik's minimal essential medium (Gibco Laboratories,Grand Island, NY). The plates were incubated at 37°C for 30min with continuous stirring. Non-adherent cells were removedand washed with incomplete medium. Cells were pelleted bycentrifugation, resuspended in incomplete medium containing2% fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT)and stored on ice. This enzymatic dissociation procedure wasrepeated at least four additional times. The cells from individualfractions were pooled and placed on a mini gradient consistingof 1 ml of Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO)in order to obtain the mononuclear cell fraction. Cell yieldsobtained by this procedure averaged 22 + 0-5 x 106 cells andwere >98% viable as determined by trypan blue exclusion.Further, this preparation consisted of macrophages (40-68%)and lymphocytes (30-50%) followed by plasma cells (1-25%)and granulocytes (0-3%). It should be noted that the number ofplasma cells increased according to the disease severity (e.g.,slight: 3 1+1 2; moderate: 6 9+1-3; advanced: 18-0+2 0).Extensive characterization of Dispase isolated gingival mono-nuclear cells has been described elsewhere (McGhee et al., 1989,Ogawa et al., 1989).

Enumeration ofantigen-specific Ig-producing cells by ELISPOTTo determine numbers of antigen-specific IgM, IgG (and IgGI,IgG2, IgG3, and IgG4 subclasses) and IgA (including IgAI andIgA2 subclasses) secreting cells present in the inflamed gingivaltissues, the enzyme linked immunospot (ELISPOT) assay wasused as described previously (Czerkinsky et al., 1987; Ogawa etal., 1989). Lids of polystyrene plates (Costar 24-well clusterplates, Mark II 3424, Cambridge, MA) were coated with eitherB. gingivalis fimbriae (10 pg/well) or LPS (Ph) (100 pg/well). For

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Antigen-specific IgG and IgA subclass antibody-secreting cells in periodontal disease

polysaccharide antigen, LPS preparations were coupled withpoly-L-lysine as described by others (Gray, 1979). To ensure

optimal coating conditions for polysaccharide antigen, periph-eral blood mononuclear cells (PBMC) isolated from subjectsimmunized with pneumococcal vaccine were tested on pneumo-coccal-polysaccharide-coated plates. PBMc from subjectsimmunized with pneumococcal polysaccharide vaccine were

tested on pneumococcal-polysaccharide-coated plates. PBMCfrom subjects immunized with pneumococcal vaccine supportedgood antigen-specific spot forming cell responses (e.g., IgM180 + 89; IgG 210+ 72; IgA 390+ 96/106 cells). Optimal coatingconcentrations were also determined previously by checker-board titrations using spleen cells from mice which hadpreviously been immunized with B. gingivalis fimbriae or LPS(Ph). All wells were then blocked with 10% FCS. Enzymatically-isolated gingival mononuclear cells were incubated in antigen-coated plates for 4 h at 370C. The lids were washed and thenincubated with biotinylated goat anti-human-p, -y or -a chain-specific antisera (Tago, Burlingame, CA). After overnightincubation the lids were washed and treated with peroxidase-conjugated avidin (Zymed, San Francisco, CA) for 2 h. Antigen-specific spots were developed with agar-substrate containing 1%Noble agar (Difco Laboratories, Detroit, MI), para-phenylene-diamine (Sigma), methanol and H202. In some experiments, 100pg/well of LPS (Ph) from Escherichia coli K235, 5 pg/well ofcholera toxin and 2 pg/well of dinitrophenyl-coupled bovineserum albumin (DNP-BSA) were also used as coating antigen.Control wells were coated with 10% FCS. Active secretion ofantigen-specific antibodies in the ELISPOT assay was revealedby the addition of cycloheximide (25 pg/ml), which reduced the

Isotype of Diagnosis ofresponse periodontitis B.gingivoais fimbriae-specific

Slight

IgG Moderate

Advanced

Slight

IgA Moderate

Advanced

Slight

IgM Moderate

Advanced

number ofSFC by 75-96%. The addition of rabbit IgG anti-B.gingivalis fimbriae antibody to the fimbriae-coated plate alsoresulted in the inhibition of spot formation by 72-95%, whereasnormal rabbit IgG was without effect.

Antigen-specific IgG and IgA subclass antibody-secretingcells were similarly enumerated on lids of polystyrene plates.After appropriate incubation of mononuclear cells on coatedplates, the cells were removed and MoAb to IgG subclasses(anti-IgGl (HP 6012), anti-IgG2 (HP 6014), anti-IgG3 (HP6047), or anti-IgG4 (HP 6022)) (Ogawa et al., 1989) or to IgAsubclasses (anti-IgA1 or -IgA2; Crago et al., 1984) (- I pg/I00p1/well) were added to individual wells. After overnight incuba-tion, biotin-labelled F(ab')2 fraction of goat IgG anti-mouse Igantibodies (absorbed with human serum) (Southern Biotechno-logy Associates, Birmingham, AL) were added and incubatedfor 4 h at 25°C followed by avidin-peroxidase. Spots were

developed with agar-substrate as described above. The numbersof spots present were counted macroscopically on a whitebackground under direct illumination.

Determination of total spontaneous spot-forming cellsIn order to enumerate total numbers of IgM-, IgG or IgA-secreting cells in the mononuclear cell fraction isolated frominflamed gingiva, plates were coated with mouse monoclonalanti-human-p chain (DA 44) or with goat F(ab')2 anti-humanIgG (Jackson ImmunoResearch, West Grove, PA) or with goatF(ab')2 anti-human IgA (Pel Freez, Rogers, AR). Dissociatedgingival cells were added to the antibody-coated plates, incu-bated for 4 h at 37°C and then removed as described above.

Total

0I ~H:H1HH1 H 20 30000

0 500 1000 0 1000 2000 3000

50 100 0 1000 2000 3000

0 50 100 0 100 200

Number of spot forming cells (SFC)/106 Mononuclear cells

Fig. 1. Bacteroides gingivalis fimbriae-specific IgM, IgG and IgA SFC in mononuclear cells isolated from inflamed gingival tissues. The

number of fimbriae-specific or spontaneous IgM, IgG and IgA SFC were determined from different stages ofAP (slight (4 patients);

moderate (8 patients); advanced (8 patients)) by ELISPOT assay.

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Individual wells were further incubated with appropriate biotin-labelled goat anti-human-si, -y or -a chain-specific antibodies(Tago, Burlingame, CA) followed by avidin-peroxidase andagar substrate for development of spots.

Patients and gingival tissue collectionChronically inflamed gingival tissues were obtained frompatients with adult periodontitis (AP) during surgery at threeprivate periodontal clinics headed by Drs James E. Roberts,Alvin W. Stevens and Debra A. Woods in the Birmingham area.

The details of the study were discussed with individual patientsand they signed a Consent Form allowing their tissues to be usedin this study. The excised tissue was placed in sterile tubescontaining RPMI 1640 medium supplemented with 5% FCS.Less than 1-2 h after surgery tissues were transferred to UABand isolation of mononuclear cells was performed as describedabove. The gingival tissues were diagnosed (e.g., slight, moder-ate or advanced from AP) according to criteria described by theAmerican Academy of Periodontology (Current ProceduralTerminology for Periodontics, 5th edition, November, 1987).Inflamed gingival tissues were obtained from a total of 140patients with AP (slight: 9 cases; moderate: 45 cases; advanced:86 cases), and this represents the total number of gingival tissueswhich were used in this study. The number of samples is alsolisted in the individual figure legends.

RESULTS

Analysis of antigen-specific spot forming cells (SFC) in themononuclear cellfractions isolatedfrom inflamed gingivaThe isotype of B. gingivalis fimbriae or LPS-specific SFC were

assessed in mononuclear cell fractions derived from gingiva ofpatients with different stages of adult periodontitis (AP) (e.g.,sight, moderate and advanced) by the ELISPOT assay.

Although the total number of plasma cells increased in relationto the severity of disease (McGhee et al., 1989; Ogawa et al.,1989), the major isotype detected in all stages was IgG followedby IgA (Fig. 1). Small numbers of IgM SFC were seen.

Significant numbers of fimbriae-specific IgG SFC were found inmononuclear cells isolated from both moderate and advancedstages of AP (Fig. 1). In the moderate stage, -1% of the totalIgG SFC population were fimbriae-specific, however up to 5%of IgG SFC in the advanced stage were specific for this antigen.Fimbriae-specific IgA SFC also increased as the disease pro-gressed in severity. Approximately 1% of IgA-secreting cellspossessed antigen-specificity to fimbriae in moderate AP, whilecells isolated from the advanced stage of disease containedfimbriae-specific IgA SFC at frequencies of 4-6% (Fig. 1).Essentially no fimbriae-specific IgM SFC were seen in any stageof disease.

When LPS-specific SFC were enumerated in a similarmanner, lower frequencies of LPS-specific antibody-producing

Isotype of Diagnosis ofresponse periodontitis

Slight

IgG Moderate

Advanced

Slight

IgA Moderate

Advanced

Slight

IgM Moderate

Advanced

B. gingivolis LPS-Specific Total

o 25 50 0 10 000 20 000 30 000

0 25 50 0 1000 2000 3000

0 25 50 0 100 200Number of spot forming cells (SFC)/106 Mononuclear cells

Fig. 2. B. gingivalis LPS-specific IgM, IgG and IgA antibody-secreting cells in mononuclear cells isolated from inflamed gingival tissues.

The number of LPS-specific or spontaneous IgM, IgG and IgA SFC were determined at different stages of AP (slight (3 patients);moderate (7 patients); advanced 8 patients) by ELISPOT assay.

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Antigen-specific IgG and IgA subclass antibody-secreting cells in periodontal disease

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Fig. 3. Comparison of B. gingivalis fimbriae-specific IgG subclassantibody-secreting cells in gingival mononuclear cells isolated fromdifferent stages of AP. The number of fimbriae-specific IgGl, IgG2,IgG3, IgG4 and total IgG were determined in moderate (6 patients) andadvanced (7 patients) AP by ELISPOT assay.

cells were found in all isotypes tested. LPS-specific SFC werenoted at a frequency of 0-1% and 04% for IgG- and IgA-producing cells, respectively, regardless of the stage of diseaseanalysed (Fig. 2). Bacteroides LPS-specific IgM SFC were notdetected in mononuclear cells isolated from different stages ofdisease.

Since the IgG and IgA SFC responses were higher tofimbriae when compared with LPS, the next experiments wereperformed to ensure that antigen-specific SFC responses werebeing detected in enzymatically isolated gingival mononuclearcells. For these studies we selected E. coli K235 LPS, choleratoxin, or DNP-BSA for analyses of antigen-specificity. Cellsfrom different stages of diseased gingiva did not contain SFCspecific for any of these antigens (Table 1). On the other hand,the same mononuclear cell preparations possessed B. gingivalisfimbriae- and LPS-specific antibody-secreting cells. Theseresults suggest that the detection of high numbers of IgG andIgA SFC which occur in the gingival tissues are antigen-specificand not polyclonal responses.

IgG subclass distribution offimbriae-specific SFCSince fimbriae-specific SFC were predominantly of the IgGisotype, the frequency of antigen-specific IgG subclass SFC wasexamined in mononuclear cells isolated from gingival tissues ofpatients with the moderate or advanced stage of AP (Fig. 3).Fewer IgG SFC were seen in the moderate stage (- 100

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IgAFig. 4. Distribution of B. gingivalis fimbriae-specific IgAl and IgA2antibody-secreting cells in gingival mononuclear cells isolated fromdifferent stages of AP. The number of fimbriae-specific IgAl, IgA2 andtotal IgA were determined in moderate (7 patients) and advanced (7patients) stages of AP by ELISPOT assay.

fimbriae-specific SFC/106 cells), and most were of the IgGIsubclass, followed by IgG2, and small IgG3 and IgG4 responses(Fig. 3). Higher numbers of antigen-specific IgG SFC wereobserved in the advanced stage of AP, and ranged from 900 to1300 SFC/106 mononuclear cells (Figs 1 and 3). Increasednumbers of fimbriae-specific SFC were seen in each IgGsubclass. For example, 15-20-fold higher fimbriae-specific spotswere found in the IgG3 and IgG4 subclasses, respectively, whencompared with the moderate stage (Fig. 3). Furthermore, IgGland IgG2 antigen-specific SFC increased five-fold when com-pared with the moderate stage of disease. Of interest was thedistinct pattern of IgG subclass distribution found in cellsisolated from advanced AP when compared with the moderatestage. IgG4 fimbriae-specific SFC were higher (approximately40% of antigen-specific IgG SFC) followed by IgGi (-30%),IgG3 (--20%) and IgG2 (- 10%) (Fig. 3). These results indicatethat selective increases in both IgG4 and IgG3 fimbriae-specificSFC occur in the advanced stage of disease.

Fimbriae-specific SFC in the IgA I and IgA2 subclassesWhen IgA fimbriae-specific antibody-secreting cells wereassessed for their subclass distribution, IgAl SFC were mostprevalent in both moderate and advanced stages ofAP (Fig. 4).Although the frequency ofIgA fimbriae-specific SFC was 1% ofthe total IgA-secreting cells present in mononuclear cellsisolated from gingiva in moderate AP, 95% were of the IgAl

107

nir

T. Ogawa et al.

Table 1. Mononuclear cells isolated from inflamed gingival tissues contain B. gingivalis antigen-specific antibody secreting cells*

Antigen used in ELISPOT (specific SFC/106 cells)

B. gingivalisDiagnosis of Isotype of Total SFC/ E. coli Choleraperiodontitis response 106 cells Fimbriae LPS (Ph) LPS(Ph) toxin DNP

Moderate IgM 77 + 24t 1+1 0 0 0 0IgG 15 675+2401 153+38 13+6 0 0 0IgA 1082+ 130 16+5 4+2 0 0 0

Advanced IgM 84+ 15 4+4 0 0 0 0IgG 24 675+2740 1178+235 29+9 0 0 0IgA 2210+376 121 +29 10+4 0 0 0

* Mononuclear cells were prepared by dissociation of inflamed gingival tissue with Dispasefollowed by enrichment on a Ficoll-Hypaque gradient. Both spontaneous and antigen-specificIgM, IgG and IgA SFC responses were determined by the ELISPOT assay.

t The values are expressed as number of SFC+s.e.m./106 cells.

subclass. Significant increases offimbriae-specific IgA SFC wereseen in the advanced stage where one of 20 IgA SFC werespecific for B. gingivalis fimbriae (Figs I and 4). Although IgAsubclass analysis showed that 80-90% of the fimbriae-specificSFC were of the IgAl subclass, the numbers of IgA2 fimbriae-specific SFC increased almost 20-fold when compared to themoderate stage (Fig. 4). These results suggest that the distribu-tion of fimbriae-specific IgA-secreting cells are predominantlyof the IgA 1 subclass, however an increase in IgA2 cells occurs asthe disease develops in severity.

DISCUSSION

The nature of antigen, e.g., protein or carbohydrate, as well asother properties such as their display in particulate or solubleform, or innate mitogenicity, will influence the isotype andsubclass of antibody response seen in the host. Microbialinfections can lead to an increase in a particular isotype orsubclass of antibody response to a particular bacterial compo-nent. However, most studies of this type have analysed antibodyresponses in serum (reviewed in Hammarstr6m & Smith, 1986;Mestecky & Russell, 1986). Since the analysis of antigen-specificantibody levels in serum may not reflect the local response, it isimportant to assess the specific response which occurs in thetissue site where the actual infection persists. In the presentstudy, antibody responses to relevant antigens, e.g., fimbriaeand lipopolysaccharide (LPS) of Bacteroides gingivalis (Por-phyromonas gingivalis) were assessed in mononuclear cellsisolated from clinically different disease stages of adult perio-dontitis (AP).

Past studies have shown that high levels ofIgG antibodies towhole cells or to crude extracts of B. gingivalis were detected inserum of patients with AP (Naito et al., 1984; Tew et al., 1985;Ebersole et al., 1986). Antibodies to purified capsule, haemag-glutinin and LPS of B. gingivalis were all shown to be increasedin the serum of patients with this disease (Naito et al., 1987).Furthermore, antibodies to B. gingivalis have also been detectedin crevicular fluid of subjects with periodontitis (Naito et al.,1984; Tew et al., 1985). These findings raise two important and

related questions, namely: are these responses antigen-specific,and does the antibody present in serum result from localsynthesis at the diseased gingival site? The findings ofthe presentstudy indicate that up to 5% of the total IgG cells wereproducing fimbriae-specific antibodies (Fig. 1 and Table 1).These antibodies were newly synthesized by immunoglobulin-secreting cells isolated from inflamed gingiva, and cyclohexi-mide treatment inhibited formation of antigen-specific SFC,clearly indicating that de novo synthesis had occurred. Further,this response was antigen-specific, since the addition of rabbitanti-fimbriae antibodies to the fimbriae-coated plate blockedantigen-specific SFC. Definitive support for the specific natureof this response was provided by the finding that mononuclearcells isolated from different stages of inflamed gingiva did notexhibit SFC to antigens not ordinarily encountered in humans,e.g., cholera toxin. LPS from E. coli K235 or dinitrophenylhapten (Table 1). These results clearly suggest that IgG SFCspecific for B. gingivalis fimbriae are produced by B lineage cellsin localized inflammatory tissues. Although the present studyprovides direct evidence that PD-associated bacterial antigen-specific immune responses are induced in the local disease site, itis still possible that the B. gingivalis fimbriae-specific responsesnoted were caused by the dual effects of antigen-specifictriggering and polyclonal activation. In this regard, dentalplaque-associated bacteria possess several biologically activemolecules including LPS, peptidoglycan and muramyl dipep-tide. These macromolecules could act as adjuvants to enhancethe antigen-specific immune response which occurred in thedisease lesion. These cell wall components could also act aspolyclonal B cell activators (PBA) where these molecules wouldclonally expand total numbers of specific subpopulations of Bcells and could further induce their differentiation into plasmacells. Therefore, it is possible that specific-antigen and PBA actsynergistically on a lineage of fimbriae-specific B cells to inducethem to proliferate and differentiate into plasma cells whichresulted in the high frequency of fimbriae-specific antibodysecreting cells seen. In addition, it is possible that this locally-produced, antigen-specific IgG contributes to the serum anti-bodies to B. gingivalis detected by others (Naito et al., 1984 and

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Antigen-specific IgG and IgA subclass antibody-secreting cells in periodontal disease 109

1987; Tew et al., 1985; Ebersole et al., 1986). Further support fo]this contention was provided by our studies with mitogentriggered peripheral blood mononuclear cells (PBMC) from Alpatients. In this study no fimbriae-specific antibody or SFC werdetected in these cultures (manuscript in preparation).

Different IgG subclass response patterns are often seen t'different forms of bacterial antigens, e.g., those induced biprotein or polysaccharide antigens. Proteins mainly induceIgGi with some contribution from the IgG3 and IgG4 sub.classes (Mortimer & Widdowson, 1979; Hammarstrom &Smith, 1986). On the other hand, IgG2 antibody is selectivelyinduced by polysaccharide antigens (Matter et al., 1985Hammarstrom & Smith, 1986; Persson et al., 1986). The anti-fimbriae SFC responses in this study were mainly of the IgGIisotype in mononuclear cell fractions obtained from subjectswith the moderate stage ofAP (Fig. 3). However, in the adancecstage of AP the response profile was mainly IgG4 and IgG1followed by IgG3 (Fig. 3). This selective increase in the IgG&subclass is significant and merits additional discussion. Th(IgG4 response in humans is low and may be related to itsposition in the Igh genome. The Cy4 gene is the most 3' amongthe y subclasses, and only Ce and Ca2 are more 3' than this geneFurthermore, it has been shown that prolonged exposure tcprotein antigen, e.g. bee venom in beekeepers, results in theenhancement of antigen-specific IgG4 responses (Aalberse, varder Gaag & van Leeuwen, 1983). Higher levels of IgG4antibodies to food antigens, e.g. milk proteins and egg albumin.occur when compared with other IgG subclasses, suggestingthat chronic exposure to ingested food antigens enhancesresponses in this subclass (Merrett et al., 1983; Shakib, Brown &Stanworth, 1984). Thus, more chronic exposure of B cells ingingival tissues to B. gingivalis fimbriae antigen may induceswitches from a predominant IgG I to a downstream IgG4response. This tendency of sustained antigenic exposure tcinduce a shift towards production of IgG4, a subclass notassociated with C activation or opsonization, could suggest thaiIgG4 anti-fimbriae antibodies may protect the host from tissuedamage induced by IgG I and IgG3 antibodies, which effectivelyactivate C and augment phagocytosis. A relationship may alsoexist between IgG4 and IgE responses, since patients with atopicdisease or with chronic parasite infections often exhibit eleva-tions in these two isotypes (Iskander, Das & Aalbere, 1981;Merrett et al., 1984). Further, patients with advanced periodon-titis may also exhibit increased IgE responses in local tissues(Hyyppa, 1984; Hara et al., 1987). The precise role of IgEantibody in the development ofAP still needs further investiga-tion.

The finding that IgA-specific anti-fimbriae SFC are seen ingingival mononuclear cells obtained from disease sites alsomerits discussion. Interestingly, the predominant fimbriae-specific SFC were of IgAI subclass, regardless of the diseasestage. In this regard, it has been shown that differences in theIgA-subclass, antigen-specific antibodies may also be related tothe nature of the inducing antigen (e.g., protein vs carbo-hydrate), and to the source of IgA assayed (e.g., serum orexternal secretion). A predominance of IgA I antibodies tobacterial protein and to LPS and IgA2 antibodies to dextran wasfound in serum (Brown & Mestecky, 1985). Although anti-bodies to most protein and carbohydrate antigens are of theIgA 1 subclass in secretions, secretory IgA antibodies to LPS andto lipoteichoic acid were found to be higher in the IgA2 subclass

when compared with IgAl (Brown & Mestecky, 1985).The lower frequency of SFC to LPS of B. gingivalis was also

of interest (Fig. 2), since it is often assumed that LPS in gingivalplaque induces both polyclonal and antigen-specific responses.Several possible explanations may be given for this finding. Thelow anti-LPS SFC may be related to the B. gingivalis LPS itself,since it has been shown to differ in chemical structure from LPSderived from other Gram-negative micro-organisms (Man-sheim et al., 1979; Koga et al., 1984). Further, biologicalresponses to LPS from B. gingivalis are lower than to LPSisolated from enterobacteria when assessed by lymphocytemitogenicity, the Schwartzman reaction, the chicken embryolethality test and pyrogenicity (Mansheim et al., 1978; Nair etal., 1983; Hamada et al., 1988). Although B. gingivalis LPSexhibits a strong bone resorption activity (Miller et al., 1985),this molecule may be poorly immunogenic. Further investiga-tions will be required to elucidate why this LPS exhibits certainpathological functions for PD, but induces lower immuneresponses in the host.

ACKNOWLEDGMENTS

The authors wish to thank Drs James E. Roberts, Alvin W. Stevens andDebra A. Woods for providing gingival tissues from their periodontalsurgery. We thank Dr Hidetoshi Shimauchi for assistance in thepreparation of antigens, and Drs Maureen G. Bruce and Noel K.Childers for critical review ofthis manuscript. We also thank Ms SandraRoberts and Mr Masahiko Amano for typing the paper and forcomputer graphic work, respectively. This work was supported by USPublic Health Service grants DE 08228, DE 04217, Al 21032, Al 18958,DE 08182 and Al 19674, and by grant-in aid 63771486 and 63870072 forspecific research from the Ministry of Education, Science and Culture ofJapan. HK is the recipient of a Research Career Development Award,DE 00237.

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