Immunology (1991) 74 362-364 BRIEF COMMUNICATION

Immune response gene regulation of the humoral immune response to Porphyromonas gingivalis fimbriae in mice H. SHIMAUCHI, T. OGAWA & S. HAMADA Department of Oral Microbiology, Osaka UniversitY Faculty of DentistrY, Yamadaoka, Suita-Osaka, Japan

Acceptedfor publication 3 June 1991

SUMMARY Among various strains of mice immunized orally with Porphyromonas gingihalis fimbriae and adjuvant GM-53 in liposomes, BALB/c and DBA/2 mice (H-2d) were found to be high responders to the fimbriae, CBA/J and C3H mice (H-2k) were intermediate, while C57BL/6 mice were low responders in terms of serum IgG and salivary IgA responses. Furthermore, humoral immune responses were examined using congeneic mice of B 10 background showing different H-2 haplotypes, and it was revealed that B10.D2 mice (H-2d), followed by B10.BR (H-2k), responded well to antigenic stimulation of the fimbriae, while C57BL/10 mice (B 10, H-26) were low responders to the fimbriae. Hybrids between BALB/c and C57BL/6 mice were found to reflect a phenotype of low responders. Thus, the humoral immune responses to P. gingivalis in mice are restricted by H-2 haplotype.

Porphjyromonas gingivalis has been recognized as a major pathogen of adult periodontitis,' and the periodontal infections by P. gingivalis are associated with elevated levels of serum antibodies specific for the fimbriae of this organism.2 Local immune responses to the fimbrial protein of P. gingivalis were enhanced in the gingival tissues of adult periodontitis patients.3 4 Furthermore, we have also shown that P. gingicalis fimbriae can induce serum anti-fimbriae immunoglobulin G (IgG) and salivary IgA responses when administered orally with an acyl derivative of muramylpeptide in liposomes to BALB/c mice.5 6 It was revealed that the major histocompatibility complex (MHC) could influence the immune responses to some bacterial antigens and susceptibility to infection.7 " In this study, we describe the genetic control of serum and salivary antibody responses to P. gingivalis fimbriae administered orally with an adjuvant in liposomes in various strains of mice possessing different genetic backgrounds. Six inbred mouse strains, BALB/c, DBA/2, CBA/J, C3H/ HeN, C3H/HeJ and C57BL/6 mice, were obtained from Charles River Japan (Atsugi City, Japan). Three H-2 congenic strains,

B10.D2, Bl0.BR and C57BL/10 mice, were purchased from Japan SLC (Hamamatsu City, Japan). (BALB/c x C57BL/6) F, and (C57BL/6 x BALB/c) F, hybrids were bred at our facility. All mice used were 6 weeks old and male, except for F, hybrid mice.

P. gingivalis strain 381 was grown anaerobically in GAM broth (Nissui, Tokyo, Japan) supplemented with hemin and menadione at 370 for 26 hr, and the fimbriae were prepared as described previously.5 The basic structure of fimbriae (fimbrilin) was identified as a single band of the 41,000 molecular weight (MW) protein by sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE). Five-hundred micrograms of P. gingivalis fimbriae and the same amount of adjuvant GM-53 (Dainippon Pharmaceutical Co., Osaka, Japan) incorporated into liposomes consisting of lecithin (DL-a-phosphatidyl choline, dipalmitoyl, Grade I; Sigma Chemical Co., St Louis, MO) and cholesterol (Sigma) were used as immunogen. With the aid of an intubation needle, the fimbriae with GM-53 in liposomes, or liposomes alone as a control (0 25 ml per mouse), were orally administered to groups of 8-12 mice on Days 0 and 1. The mice received the secondary (booster) oral administration of the immunogen on Days 27 and 28 in the same way as the primary immunization, and then were bled from the inferior ophthalmic vein on Day 33 after the primary immunization. Saliva samples were then collected from the mice after treatment with pirocarpine hydrochloride (Wako Pure Chemical Industries, Osaka, Japan) under anaesthesia with pentobarbital (Nembutal; Dainippon Pharmaceutical Co., Osaka, Japan). These serum and saliva specimens were divided into small aliquots and stored at -80° until use. The isotypes and the levels of anti-fimbriae antibodies in serum and saliva of mice were determined by the enzyme-linked

Abbreviations: BSA, bovine serum albumin; CT, cholera toxin; ELISA, enzyme-linked immunosorbent assay; GM-53, sodium fl-Nacetylglucosaminyl-(l-14)-N-acetylmuramyl-L-alanyl-D-isoglutami-nyl(L)-stearoyl-(D)-meso-2,6-diaminopimelic acid-(D)-amide-D-alanine; Ir, immune response; LPS, lipopolysaccharides; MDP, muramyl dipeptide; MHC, major histocompatibility complex; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TD, thymic-dependent. Correspondence: Dr S. Hamada, Dept. of Oral Microbiology, Osaka University Faculty of Dentistry, Yamadaoka, suita-Osaka, 565 Japan.

362

363

Immune response to P. gingivalis Fimbrilae-speciflc antibodies ( Mean ± SE) Serum (jg / ml )

Strain

H-2

0

BALB/c

d

a

DBA12

d

c

CBA/J

k

I

C3H/HeN

k

I

C3H/HeJ

k

I

20

bgA

igG

IgM

igh

0

20

40

60

10

I

100

120

1111,11,11w* .M

D N|

1440

0 _ 20

IgG 0

100

Saliva(ng/ml) 1gA 0

100

200

300

400

I

I

_*

b b C57BU6 _______ -_-

B1IO.D2

d

b

B1O.BR

k

b

C57BL/10

b

b

Figure 1. Serum and salivary antibody responses to P. gingivalis fimbriae administered orally with GM-53 in liposomes in six inbred and three congenic strains of mice. Two groups each of 8-12 strains of mice (male, 6 weeks old) were immunized on Days 0, 1, 27 and 28 by oral administration of 500 gg fimbriae with 500 yg ofGM-53 in liposomes or liposomes alone (control, data not shown). * ** Statistical differences from the value for the control group, P < 0-05 and P < 0 01, respectively.

immunosorbent assay (ELISA) with a minor modification, as described previously.5 The titres of anti-fimbriae antibodies in serum and saliva were expressed as jug/ml and ng/ml, respectively. Comparisons between groups were made by the Student's t-test for independent samples. Serum and salivary immune responses to P. gingitalis fimbrial antigen were examined using the six inbred strains of mice (Fig. 1). Ranking of the six tested strains on the basis of serum antibody levels showed good resolution into three groups; BALB/c and DBA/2 mice (H-2d) were found to be high responders to fimbrial antigen, C57BL/6 mice (H-2b) were low responders, while C3H/HeN, C3H/HeJ and CBA/J mice (H-2k) made intermediate responses. Furthermore, the salivary IgA anti-fimbriae response of the BALB/c and DBA/2 mice was markedly higher than in other strains of mice. The results also showed a positive correlation between the serum IgG and salivary IgA anti-fimbriae levels in these strains of mice. The immunoglobulin heavy chain (Igh) haplotype did not affect either serum or salivary antibody response to fimbrial antigen. Evidence for the controlling role of H-2 haplotypes corresponding to the six strains of mice was investigated in H-2 recombinants on BlO background (Fig. 1). B1O.D2 (H-2d), followed by B10.BR (H-2k), were found to be high responders, while C57BL/ 10 (BlO, H-2b) were low responders to fimbrial antigen. These results were consistent with the results shown in the six inbred strains, and strongly suggested that the antibody responses to the orally administered fimbrial antigen of P. gingivalis were restricted by the H-2 haplotype. The response of (C57BL/ 6 x BALB/c) F. and (BALB/c x C57BL/6) F. mice, hybrids between high and low responder strains, when compared with parental strains generally reflected a phenotype of the low responder; the low responsiveness of F. mice to the fimbrial antigen was dominant in both strains of reciprocal F. mice (Fig. 2). There were no differences between the response of male and female in both F. hybrids. Our previous studies demonstrated that P. gingivalis fimbriae administered orally together with GM-53 in liposomes led to specific antibody production in serum as well as in saliva of BALB/c mice (H-2d), while oral administration of fimbriae

without the adjuvant in liposomes could induce the antibody response in serum only.5'6 In this study, we chose oral immunization which could induce both serum and salivary immune responses to fimbriae. H-2b mice failed to produce significant anti-fimbriae antibodies not only in saliva but also in serum under the same experimental conditions. These results strongly suggested that the responsiveness to P. gingivalis fimbrial antigen in mice was genetically controlled by H-2-linked genes. In this regard, genetic control of the serum antibody responses to Actinomyces viscosus T14V type 1 and type 2 fimbriae was suggested previously by Haber & Grinnel. 12 However, they only showed the difference among three inbred strains of mice which had diverse genetic backgrounds, including H-2 and Igh loci, and the gene affecting the immune response to the A. viscosus fimbrial antigen was not clarified. The MHC immune response (Ir) gene control of secretory IgA has been reported previously.'3 The report indicated that the mice of H-2b and H-2q haplotypes generated high levels of specific antibodies to cholera toxin (CT) in the intestinal secretion, while H-2k and H-2d mice developed only low levels of intestinal anti-CT IgA in the intestinal fluid. It also revealed that the same pattern occurred in plasma IgG anti-CT levels after CT feeding. Except for the kind of haplotypes stimulated, these results agree with our data with regard to the MHC gene control of both mucosal and systemic immune responses to orally administered antigen. Staruch & Wood'4 showed that the adjuvanticity of muramyldipeptide (MDP) was genetically influenced in mice with the C57BL background, and/or the H-2b haplotype responded weakly to the immunopotentiating action of MDP, whereas those with the BALB/c and C3H background strains and/or mice with H-2d or H-2k haplotypes responded strongly. Since we used GM-53, an acyl derivative of MDP, as an adjuvant in this study, the genetic influence of the adjuvanticity of MDP may affect the humoral immune response to fimbrial antigen of P. gingivalis. Of interest is that our results shown in Fig. 1 are consistent with the degree of the adjuvanticity of MDP: BALB/c and DBA/2 (H-2d) were high responders, while C57BL/6 (H-2b) were low responders. Furthermore, a congenic strain with high

364

H. Shimauchi, T. Ogawa & S. Hamada Fimbriae-specific antibodies ( Mean ± SE)

1gM

Fi mice

Sex

0

IgG 20 0

lgG

IgA 20 0

REFERENCES

Saliva(ng/mI)

Serum(gg/ml) 20

0

IgA 100 0

100

(BALB/c x C57BL/6) M (BALB/c x C57BL/6) F

(C57BL/6 x BALB/c) M (C57BL/6 x BALB/c) F

Figure 2. Serum and salivary antibody responses of(BALB/c x C57BL/6) F1 and (C57BL/6 x BALB/c) F, hybrid mice following oral immunization with P. gingivalis fimbriae. The experimental protocols are the same as described in the legend to Fig. 1. M, male; and F, female. **Statistical differences from the value for the control group, P < 00 1.

responder H-2d on B10 background (B10.D2) exhibited lower responses in serum and saliva compared with the strains with other backgrounds, such as BALB/c and DBA/2 (Fig. 1). These results suggest that non-MHC background genes may affect the expression of H-2-encoded genes. The humoral immune responses of F, (BALB/c x C57BL/6) and the reciprocal F, (C57BL/6 x BALB/c) were found to be low (Fig. 2). These results indicate that the low responsive trait is dominant in both F, hybrid mice. In this regard, the dominant inheritance of low antibody in high x low responder F, hybrid progeny has been demonstrated in mice with Mycobacterium leprae phenolic glycolipid I antigen. Furthermore, this dominant inheritance of low antibody response to this antigen might be attributable to an active suppressor effect.'5 It has been widely assumed that not only Ir genes but also immune suppression (Is) genes exist in the I-region of the murine H-2 complex.16 From these findings, it may be speculated that unresponsiveness to P. gingivalis fimbrial antigen may be influenced by Is gene control. The existence of Ir genes not linked to the H-2 complex has been proposed. One suggestion is immunoglobulin heavy chain (Igh)-linked Ir genes,'7 and the other is X-linked Ir genes.'8 It has been suggested that neither Igh-linked nor X-linked genes influence the antibody responses to P. gingivalis fimbriae, as shown in Fig. 1. It was demonstrated that IgA responses to orally administered thymic-dependent (TD) antigens were regulated by the lipopolysaccharide (LPS) gene; the LPS nonresponsive C3H/HeJ mice developed higher IgA immune responses to orally administered TD antigen compared with the LPS-responsive mice.'9 In this study, C3H/HeJ mice could not induce higher salivary IgA antibodies compared with other LPS-responsive strains of mice (Fig. 1). Thus, it appears that the LPS genes failed to affect the immune response to P. gingivalis fimbriae. Taken together, the results shown above are the first demonstration of H-2 Ir gene control of antibody responses to the fimbrial antigen from P. gingivalis.

1. SLOTS J. & LISTGARTEN M.A. (1988) Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinonmycetemcornitans in human periodontal diseases. J. clin. Periodontol. 15, 85. 2. OGAWA T., KUSUMOTO Y., HAMADA S., MCGHEE J.R. & KIYONo H. (1990) Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases. Clin. exp. Immunol. 82, 318. 3. OGAWA T., MCGHEE M.L., MOLDOVEANU Z., HAMADA S., MESTECKY J., MCGHEE J.R. & KIYONo H. (1989) Bacteroides-specific IgG and IgA subclass antibody-secreting cells isolated from chronically inflamed gingival tissues. Clin. exp. Immunol. 76, 103. 4. OGAWA T., KONO Y., MCGHEE M.L., MCGHEE J.R., ROBERT J.E., HAMADA S. & KIYONo H. (199 1) Porphvromonas gingivalis-specific serum IgG and IgA antibodies originate from immunoglobulinsecreting cells in inflamed gingiva. Clin. exp. Immunol. 83, 237. 5. OGAWA T., SHIMAUCHI H. & HAMADA S. (1989) Mucosal and systemic immune responses in BALB/c mice to Bacteroides gingivalis fimbriae administered orally. Infect. Immun. 57, 3446. 6. OGAWA T., SHIMAUCHI H., KUSUMOTO Y. & HAMADA S. (1990) Humoral immune responses to Bacteroides gingivalis fimbrial antigen in mice. Immunology,. 69, 8. 7. DE VRIES R.R.P. (1989) regulation of T cell responsiveness against Mycobacterial antigens by HLA class 2 immune response genes. Rev. Inlect. Dis. II, S400. 8. NIIYAMA T., KOJIMA H., MIZUNo K., MATSUNO Y., Fuiii H., MISONOU J., NATORI T., AIZAWA M. & OIKAWA K. (1987) Genetic control of the immune responsiveness to Streptococcus mutans by the major histocompatibility complex of the rat (RTI). Infect. Immun. 55, 3137. 9. HORMAEAECHE C.E., HARRINGTON K.A. & JOYSEY H.S. (1985) Natural resistance to Salnmonerae in mice: control by genes within the major histocompatibility complex. J. Infect. Dis. 152, 1050. 10. SASAZUKI T. (1982) HLA: genetic control of immune response and disease susceptibility. Jap. J. Human Genet. 27, 81. 11. DOUGLAS-JONES A.G. & WATSON J.D. (1985) Immunity to leprosy. II. Genetic control of murine T cell proliferative response to MYcobacteriumn leprae. J. Immunol. 135, 2824. 12. HABER J. & GRINNELL C. (1989) Analysis of the serum antibody response to type I and type 2 fimbriae in mice immunized with Actinomyi~ces zisucosus TI 4V. J. Period. Res. 24, 81. 13. ELSON C.O. & EALDING, W. (1987) Ir gene control of murine secretory IgA response to cholera toxin. Eur. J. Immunol. 17, 425. 14. STARUCH M.J. & WOOD D.D. (1982) Genetic influences on the adjuvanticity of muramyl dipeptide in vitvo. J. Immunol. 128, 155. 15. TEUSCHER C., YANAGIHARA D., BRENNAN P.J., KOSTER F.T. & TUNG K.S.K. (1985) Antibody response to phenolic glycolipid I in inbred mice immunized with Mycobacterium leprae. Infect. Immun. 48,474. 16. GERMAIN R.N., THEZE J., WALTENBAUGH C., DORF M.E. & BENACERAAF B. (1978) Antigen-specific T cell-mediated suppression. I. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine5") (GT)-specific T cell suppressor factor (GT-TsF) of suppressor T cells (Ts2) bearing distinct I-J determinants. J. Inmmunol. 121, 602. 17. BLOMBERG, B., GECKELER W.R. & WEIGERT M. (1972) Genetics of the antibody response to dextran in mice. Science, 177, 178. 18. AMSBAUGH D.F., HANSEN, C.T., PRESCOTT B., STASHAK P.W., BARTHOLD D.R. & BAKER P.J. (1972) Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. J. exp. Med. 136, 931. 19. COLWELL D.E., MICHALEK S.M. & MCGEE J.R. (1986) LPS gene regulation of mucosal immunity and susceptibility to Salmonella infection in mice. Curr. Top. Microbiol. Immunol. 124, 121.'