Vol. 59, No. 7

INFECTION AND IMMUNITY, JUlY 1991, p. 2364-2369 0019-9567/91/072364-06$02.00/0 Copyright C) 1991, American Society for Microbiology

Genetic Control of Serum Antibody Responses of Inbred Mice to Type 1 and Type 2 Fimbriae from Actinomyces viscosus T14V JEROME HABER,l.2* CHRISTINE M. GRINNELL,2 J. ELAINE BEEM,3



Department of Oral Pathology, School of Dental Medicine,' and Department of Pathology, School of Medicine,2 Tufts University, Boston, Massachusetts 02111, and Periodontal Disease Research Center, Department of Oral Biology, College of Dentistry,3 and Department of Immunology and Medical Microbiology, College of Medicine,4 University of Florida, Gainesville, Florida 32610 Received 21 September 1990/Accepted 15 April 1991

Antibodies reactive with type 1 and type 2 fimbriae from Actinomyces viscosus T14V specifically inhibit the adherence of A. viscosus T14V to salivary pellicle-coated tooth surfaces and other bacteria, and these antibodies are thought to modulate colonization by this microorganism. These studies were done to determine whether previously noted differences in the antibody responses of inbred mice to type 1 and type 2 fimbriae might be under genetic control. The serum immunoglobulin G (IgG) and IgM antibody responses of inbred, F1 hybrid, and H-2 congenic mice, immunized with A. viscosus T14V cells, were analyzed by enzyme-linked immunosorbent assays for antibodies reactive with A. viscosus T14V whole-cell type 1 and type 2 fimbriae. The results confirmed earlier findings and indicated striking variations in the amounts of IgG anti-type 1 (23-fold) and anti-type 2 (48-fold) fimbria antibodies elicited. The responses of the 17 inbred strains tested showed a relatively continuous distribution from high to low, as well as marked differences in the responses of H-2 and Igh-C identical strain pairs. An analysis of the responses of F1 hybrid and H-2 congenic mice indicated dominance of the low-responder gene(s) and control by H-2-linked genes. Antisera from two high-responder strains inhibited in vitro bacterial adherence to a much greater degree than antisera from a low-responding strain. These data suggest polygenic control of the magnitude of the IgG anti-type 1 and anti-type 2 fimbria antibody responses by H-2-linked genes as well as background genes not associated with H-2 or Igh-C loci.

Antibody responses to oral microorganisms play a major role in modulating bacterial colonization of the oral cavity (7, 8). The ability of oral bacteria to colonize depends upon complex interactions, including adsorption to salivary pellicle-coated tooth surfaces and host epithelial cells, as well as interbacterial aggregation (21). In many bacterial species, these interactions are mediated by antigenically distinct fimbriae (4, 21) and are inhibited by fimbria-specific antibody (14, 30, 32). Because adsorption is a prerequisite for bacterial colonization, variations in naturally induced antifimbria antibodies may, in part, account for differences among individuals in susceptibility to colonization by oral microorganisms such as those associated with periodontal infections. Periodontal pathogens colonize the host in supragingival and subgingival environments, where specific immunoglobulin A (IgA) and IgG antibodies are available to inhibit adherence. The immune response is genetically regulated (5, 28), and genetic diversity is a likely source of variation in host antibody responses to bacterial antigens. Genetically defined inbred mice have proven invaluable in studies of genetic regulation of the immune response. Various patterns of genetic control of the antibody response have been described, including control by H-2 major histocompatibility complex (MHC)-linked immune response (Ir) genes (5, 28), immunoglobulin allotype locus (Igh-C)-linked genes (1, 23, 33), and non-H-2-, non-Igh-C-linked background genes (3, 15, 23, 27, 31). Most of the complex protein antigens studied so far have no inherent biological function (43), and consequently little is known about the genetic regulation of responses to the complex bacterial antigens


Corresponding author.

which enable infectious microorganisms to colonize their host. On this basis, we recently developed a model for analysis of variations and genetic control of the mouse antibody responses to fimbrial proteins from A. viscosus T14V (22). A. viscosus T14V was selected as a model organism because it is an early plaque colonizer associated with gingivitis in humans (29, 36), and its adherence mechanisms have been well characterized by using in vitro adsorption inhibition assays. These studies have shown that its adherence to salivary pellicle is mediated by type 1 fimbriae and inhibited by anti-type 1 fimbria antibodies (39, 40), whereas its aggregation with streptococci is mediated by type 2 fimbriae and inhibited by anti-type 2 fimbria antibodies (11-13, 32). In addition, A. viscosus T14V is well suited for study for two practical reasons. First, the availability of purified type 1 and type 2 fimbriae from A. viscosus T14V allowed the development of enzyme-linked immunosorbent assays (ELISAs) for measurement of specific anti-type 1 and anti-type 2 fimbria antibodies (22). Second, the availability of functional tests for A. viscosus T14V adsorption inhibition activity (14, 32) enabled us to assess the effects of variations in antifimbria antibody responses on bacterial adherence. Our previous analysis of the responses of three inbred mouse strains indicated marked variations in the magnitude of the serum antibody responses to type 1 and type 2 fimbriae (22). The purpose of the present study was to expand our preliminary strain survey and determine the influence of the genetic background on the magnitude of the anti-type 1 and anti-type 2 fimbria antibody responses. The present data indicate dominant low responsiveness and genetic control by an H-2-linked gene(s) and a background gene(s) independent of H-2 and Igh-C. 2364



VOL. 59, 1991

MATERIALS AND METHODS Bacteria. A. viscosus T14V was obtained from the culture collection at the University of Florida Periodontal Disease Research Center. A. viscosus T14V was maintained as multiple frozen stocks at -80°C in tryptic soy broth (Difco Laboratories, Detroit, Mich.) containing 20% glycerol (39). Cells were harvested from 15- to 18-h tryptic soy broth cultures, washed twice in 0.01 M phosphate-buffered saline, pH 7.0, and lyophilized. A. viscosus T14V was resuspended in normal saline for use as an immunogen and an immunosorbent. A stock cell suspension was obtained by passing the resuspended cells through a 30-gauge needle and adjusting the concentration of this suspension to approximately 1010 cells per ml. Animals. The following strains of female inbred F1 hybrid and congenic mice were obtained from the Jackson Laboratory, Bar Harbor, Me.: A/J, A/HeJ, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL/6J, C57BL/1OJ, C57BL/lOSnJ, C57L/J, DBA/1J, DBA/2J, LP/J, NZB/BINJ, SJL/J, SWR/J, 129/J, A.BY. A.SW, B1O.A, B10.AKM, B1O.BR, B1O.D2, (BALB/cJ x A/J)F1, (BALB/cJ x SWR/J)F1, and (C57L x A/HeJ)Fl. Mice were immunized when 6 to 8 weeks old. Immunizations. In all experiments, mice were injected intraperitoneally with 108 A. viscosus T14V cells in 0.1 ml of normal saline on day 0 and day 14. Mice were bled prior to immunization and on day 26. This immunization and bleed schedule was based on r esponse kinetics and dose response experiments, by which the optimal conditions for stimulating and detecting secondary anti-A. viscosus T14V responses were determined (22). A small volume of blood was obtained by retro-orbital bleeding, and the serum was stored at -200C. Preparation of bacterial fimbriae from A. viscosus T14V. Purified type 1 and type 2 fimbriae were prepared as previously described (13, 39). These preparations reacted appropriately with anti-type 1- or anti-type 2-specific antisera when used as antigens in an ELISA. These preparations were further examined by electron microscopy to ensure that fimbrial structures were indeed present and morphologically pure. Measurement of antibody. Levels of serum IgG and IgM antibodies to type 1 and type 2 fimbriae were measured by ELISA as previously described for IgG (22). The assays for IgM antibodies were done similarly, except a biotin-conjugated goat anti-mouse IgM reagent was used instead of a goat anti-mouse IgG reagent. Briefly, the method is as follows: ascites standards containing high levels of IgG or IgM antibody reactive with type 1 or type 2 fimbriae were prepared and characterized. Dilutions of the anti-type 1 or anti-type 2 fimbria ascites standard (in duplicate at 5 dilutions) and mouse antisera (in duplicate at 3 dilutions) were added to microtiter plates which had been previously coated with purified type 1 fimbriae or purified type 2 fimbriae. Following cycles of incubation and washing, biotin-conjugated goat anti-mouse IgG (heavy and light chain reactive; Sigma Chemical Co.) or biotin-conjugated goat anti-mouse IgM (heavy chain specific; Sigma), peroxidase-conjugated avidin, and 4-aminoantipyrine were added. After 45 min, the optical densities at 490 nm were determined in a spectrophotometer (Dynatech MR600 Microplate Reader). The reference ascites were used to construct standard curves for each assay. The antibody content of each serum sample was determined from the linear portion of each standard curve, corrected for dilution, and expressed as units of antibody per milliliter.

TABLE 1. Strain distribution of the serum IgG antibody responses to type 1 and type 2 fimbriae from A. viscosus T14V Mouse strain

Haplotype H-2 Igh-C Ly-17"


b b b d d k

b b a n a j


b d k q



a a b

e e a

k q s s





d c


b e

No. of mice

Antibody units/ml, 103 (mean + SD)' Anti-type 1 Anti-type 2












2 1 2 2

22 6 23 21 24


6 5 3

1 4

1 10

3 1

2 11 8 2


1 2 2 2

21 36 24 24

11 12 14 14

7 8 15 7

14 10 4 14

18 5 2 10

1 1 1 1 1 1 1

22 22 23 20 23 19 21

20 19 25 13 25 26 31 32 47 51 29 ± 25 21 ± 10


29 16 60 37 23 15 22 19 68 55 36 ± 30 82 ± 65

" A one-way analysis of variance indicated significant differences among inbred strains in levels of antibodies reactive with type 1 and type 2 fimbriae (F = 5.8 and 13.1, respectively; 16 df; P < 0.001). IgG antibodies were undetectable in sera from unimmunized mice. b Ly-17 haplotypes (26).

The anti-immunoglobulin reagent used in the IgG antibody ELISAs was made by using goats immunized with mouse IgG and therefore is presumed to be reactive with both IgG light and heavy chains. Although this reagent is potentially reactive with other immunoglobulin isotypes, we interpret the results of these ELISAs to reflect predominantly IgG levels for several reasons. These antibody measurements do not reflect serum IgA antibody, as we have been unable to detect IgA antibody in sera from A. viscosus T14V-immunized mice by using an IgA-specific anti-immunoglobulin reagent. Whereas IgG is the major immunoglobulin isotype in serum, IgE is present in trace amounts. Even if detected on the basis of anti-light-chain reactivity, IgE levels would be so low as to not influence the measurement of IgG antibodies. Finally, these antibody measurements do not reflect IgM responses, since the IgG responses exhibit secondary response kinetics (22) and are independent of the IgM-specific responses (Tables 1 and 2). On this basis, we refer to antibodies measured with the IgG heavy- and light-chain reagent as IgG. Assay to measure in vitro adsorption inhibition activity in mouse sera. The assay for measuring the capacity of antisera to inhibit the adherence of A. viscosus T14V to a tooth surface in vitro (adsorption inhibition activity) has been described previously (14, 39). Briefly, A. viscosus T14V was cultured in tryptic soy broth supplemented with 10 ,uCi of [3H]thymidine per ml (Schwartz/Mann, Orangeburg, N.Y.). Washed cells were suspended in adsorption buffer (pH 7.2). The efficiency of cell labeling with [3H]thymidine (the number of cells per counts per minute) in these experiments was approximately 500. Saliva-treated hydroxyapatite (SHA) was prepared by incubating 5 mg of washed hydroxyapatite (HA) beads (BDH Biochemicals Ltd., Poole, England) overnight in whole saliva. Paraffin-stimulated whole saliva was collected as previously described, clarified by centrifugation



INFECT. IMMUN. TABLE 3. Serum IgG anti-type 1 and anti-type 2 fimbria responses of (LR x HR)F1 hybrid mice

TABLE 2. Strain distribution of the serum IgM antibody responses to type 1 and type 2 fimbriae from A. viscosus T14V Haplotype

Mouse strain


No. of mice




b b d d k b d k q a b k q s

b b n a j b c d c e a j c b

2 2 1 2 2 1 2 2 2 1 1 1 1 1

Anti-type 1 24 22 23 21 24 21 36 24 24 22 23 20 23 19

Antibody units/ml,

Antibody units/ml, 103 (mean ± SD)'

3.9 3.0 2.3 1.3 1.1 1.8 4.0 1.9 2.3 2.1 2.1 1.1 2.5 2.1

+ t


+ ±


3.2 2.9 0.9 0.6 0.3 1.0 3.2 0.5 1.9 1.1 1.4 0.4 1.4 1.0

Mouse strain

Anti-type 2

2.3 3.1 1.8 2.0 0.9 1.2 2.3 1.8 1.4 2.8 1.7 1.4 1.8 3.2

± ± ± ± ± ± ± ± ± ± ± ±

1.1 3.3 0.6 1.2 0.4 0.5 2.2 0.7 1.0 1.8 1.3 0.4 0.9 1.3

a A one-way analysis of variance indicated statistically significant differences among inbred strains in levels of antibodies reactive with type 1 and type 2 fimbriae (F = 5.3 and 6.6, respectively; 13 df; P < 0.001). Background levels of IgM antibodies in sera from unimmunized mice were

Genetic control of serum antibody responses of inbred mice to type 1 and type 2 fimbriae from Actinomyces viscosus T14V.

Antibodies reactive with type 1 and type 2 fimbriae from Actinomyces viscosus T14V specifically inhibit the adherence of A. viscosus T14V to salivary ...
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