Serum Antibody in Actinobacillus actinomycetemcomitans-infected

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1 INFECTION AND IMMUNITY, May 1991, p /91/ $2./ Copyright X) 1991, American Society for Microbiology Vol. 59, No. 5 Serum Antibody in Actinobacillus actinomycetemcomitans-infected Patients with Periodontal Disease J. L. EBERSOLE,* M.-N. SANDOVAL, M. J. STEFFEN, AND D. CAPPELLI Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas Received 16 July 199/Accepted 21 February 1991 This study was designed to (i) delineate the characteristics of serum antibody responses to Actinobacilus actinomycetemcomitans in patients with periodontitis who are infected with A. actinomycetemcomitans; irrespective of disease classification; (ii) assess the relationship of the elevated antibody levels to colonization of the oral cavity by A. actinomycetemcomitans; and (iii) describe the serotype distribution of A. actinomycetemcomitans and antibodies to the microorganism in infected patients with various clinical classifications. To compare the levels of various isotype-specific antibodies to the different antigens, studies were performed that allowed quantitation of each isotype-specific antibody in a human reference standard. By using this reference standard, it was shown that the levels of immunoglobulin G (IgG), IgM, and IgA responses to A. actinomycetemcomitans were similar among the infected patients, irrespective of disease classification. Also, we demonstrated that the serum antibody response to serotype b was quantitatively greater in all isotypes. Our findings indicate that b was the most frequent A. actinomycetemcomitans serotype detected in the patients and appears to be capable of initiating a substantial serum IgG antibody response that may contain cross-reactive antibodies to other serotypes of A. actinomycetemcomitans. Generally, in cases in which the response to a single serotype was elevated, only that type of A. actinomycetemcomitans was detected in the plaque. Individuals exhibiting elevated antibodies to multiple serotypes were most consistently colonized by the serotype b microorganism. This study represents the first report detailing the distribution of IgG subclass antibodies to A. actinomycetemcomitans in periodontal disease. The results demonstrated that the primary responses of patients with periodontitis to A. actinomycetemcomitans were of the IgGl and IgG3 subclasses, which is consistent with elicited responses to protein antigens. In contrast, the primary subclass response in normal subjects was limited to the IgG2 subclass and may represent broader cross-reactivity to polysaccharide antigens-lipopolysaccharide from the bacteria. Actinobacillus actinomycetemcomitans is a microorganism that is the most likely candidate as an etiologic agent in periodontal disease. Numerous microbiologic studies have identified correlations of A. actinomycetemcomitans with progressing periodontitis (6, 8, 39, 43). Initial evidence concerning the association of A. actinomycetemcomitans with periodontitis was based upon extensive colonization of patients with localized juvenile periodontitis (LJP) with this microorganism (18, 27, 31, 34, 41, 44). On the basis of these studies, A. actinomycetemcomitans became intimately associated with this clinical disease classification. However, in many of these investigations, substantial proportions of patients with other clinical classifications were also shown to harbor A. actinomycetemcomitans in disease sites (6, 43, 44). Thus, while A. actinomycetemcomitans has been linked to 75 to 9% of patients with LJP, routinely 5% of rapidly progressive periodontitis (RPP) (e.g., advanced destructive periodontitis and generalized juvenile periodontitis) cases and 25 to 3% of adult periodontitis (AP) cases have been associated with A. actinomycetemcomitans infection (35, 44, 51, 55, 56). In contrast, the microorganism is present in only 5 to 1% of the clinically normal population (44, 55, 56). Similar results concerning host antibody responses to A. actinomycetemcomitans have been published. Generally, serum antibody responses to the microorganism are significantly elevated in high percentages of patients with LJP (9, 16, 17, 19-21, 24, 28, 47, 49, 53). This group of patients was * Corresponding author shown to have elevated systemic responses to A. actinomycetemcomitans compared with both normal subjects and patients with other types of periodontal diseases. These studies were related primarily to descriptions of immunoglobulin (IgG) antibody levels in the sera. However, it is well recognized that human serum IgG is composed of four subclasses that appear to be elicited by different types of antigens and have different functional capabilities (25, 26, 29, 3, 52). One purpose of this study was to delineate the characteristics of serum antibody responses to A. actinomycetemcomitans in patients with periodontitis who are infected with A. actinomycetemcomitans, irrespective of disease classification. This will allow more accurate assessment of differences in host responses to this microorganism related to patient age or disease severity. The relationship of the systemic antibody responses to infection has generally been supported by associating patients with disease with a particular microorganism and demonstrating elevated levels of antibody to that microorganism (12, 15, 23, 38). However, relation of individual paired sera to detection of the homologous microorganism in the subgingival plaque has not been uniformly consistent (35, 47, 53, 54). Thus, it was of interest to assess the relationship of the elevated antibody levels to colonization of the oral cavity by A. actinomycetemcomitans. Studies by Zambon and colleagues have defined three serotypes of A. actinomycetemcomitans (7, 32, 55-57). The existence of these serotypes has been confirmed, and serotype-specific antigens have been characterized (1, 58, 59). An interesting aspect of the serotype definition has been

2 1796 EBERSOLE ET AL. delineated, in that all serotype b A. actinomycetemcomitans strains have been shown to produce a leukotoxin, while serotype c strains are generally devoid of this capability (3). Also, serotype b of this species has been suggested to predominate in patients with LJP, and limited serotypebiotype distribution within families has been described (3, 5, 57). We have extended this information by describing the serotype distribution of A. actinomycetemcomitans and antibodies to the microorganism in infected patients with various clinical classifications. MATERIALS AND METHODS Patient samples. The subjects in this study were patients being monitored at the University of Texas Health Science Center at San Antonio in the Clinical Research Center. All patients received appropriate information concerning the procedures to be used in the study and signed approved consent forms. Clinical categorization of the patients included LJP (n = 23), which included subjects <3 years old with a typical localized molar-incisor bone loss with no more than two additional teeth involved and a mean age of 18.4 (range, 12 to 29) years (46); rapidly progressive periodontitis (RPP; n = 31), which included patients 535 years old with severe alveolar bone loss on 14 or more teeth with no clear localization and a mean age of 23.7 (range, 13 to 34) years (37); and adult periodontitis (AP; n = 39), which included subjects >35 years old who exhibited generalized severe alveolar bone loss with vertical defects with a mean age of 41.6 (range, 35 to 67) years (46). The normal population was defined as having no detectable bone loss and some mild gingivitis. This population represents a group of sera from normal subjects (n = 75) that was assembled at both Forsyth Dental Center and the University of Texas Health Science Center at San Antonio over a period of years. The groups ranged in age from 16 to 65 which, in general, spans the age ranges of the different populations of patients. An additional group of samples from patients representing LJP (n = 28), RPP (n = 25), and AP (n = 22) was obtained from a population at Forsyth Dental Center. Similar consent procedures were used, and the clinical classifications were identical. Microorganisms. The A. actinomycetemcomitans strains used in these studies as antigens to detect systemic antibody levels were prepared from broth cultures of microorganisms as described previously (1). Representatives of the three serotypes (57) of A. actinomycetemcomitans included A. actinomycetemcomitans ATCC (serotype a), Y4 (serotype b), and ATCC (serotype c). Formalin-killed bacteria were used as antigens on enzyme-linked immunosorbent assay (ELISA) plates to detect antibody in serum, as well as for immunization of rabbits to produce polyclonal antisera for bacterial identification. Identification of colonization. Rabbit antisera to be used for identification of A. actinomycetemcomitans in subgingival plaque was prepared as described previously (11). The gamma globulins were prepared by salt fractionation and used in a SeroELISA (1) or colony immunoblot procedure. The colony immunoblot process was essentially similar to that described by McArthur et al. (33), with some modification (9a). The subgingival plaque samples were collected from at least four sites per patient by using paper points (27) or the procedure of Newman and Socransky (36). The bacteria were dispersed and anaerobically plated on 5% sheep blood agar plates containing 5,ug of hemin per ml and.3,ug of menadione per ml. All plates were incubated at INFECT. IMMUN. 37 C under anaerobic conditions (5% H2, 1% C2, 85% N2) for approximately 5 days. The presence and serotype of A. actinomycetemcomitans in the plaque sample was then assessed by using the serological procedures described above. ELISA for isotype and subclass antibody. IgG, IgM, and IgA antibodies to the A. actinomycetemcomitans serotypes in sera were determined by using an ELISA as described previously (1). The only modification to this assay was the use of affinity-purified goat anti-human isotype-specific antibodies (Calbiochem) and affinity-purified rabbit anti-goat IgG conjugated to alkaline phosphatase (Sigma) as the developing reagent. To quantitate the levels of the immunoglobulin isotypes in the sera and allow comparison among the antibody isotypes, we developed a method for approximation of the antibodies in each serum. For this procedure, we isolated IgG, IgM, and IgA antibodies to A. actinomycetemcomitans by affinity chromatography using Formalin-killed A. actinomycetemcomitans as the insoluble matrix (14a). A pool of highly reactive human serum was used as the antibody source and mixed with the A. actinomycetemcomitans. The bound antibody was eluted from the bacteria by using 1 M NH4H, ph 11.. Contaminating bacterial antigens that had eluted from the microorganisms were removed by an affinity column prepared from rabbit anti-a. actinomycetemcomitans globulins attached to Affi-Gel 1 (Bio-Rad). The IgG was isolated by using a protein G matrix (Pierce). IgM and IgA were obtained from the eluted antibody by binding and elution from anti-igm or anti-iga affinity columns composed of goat anti-igm or -IgA bound to Affi-Gel 1. The purity of the isolated anti-a. actinomycetemcomitans immunoglobulins was assessed by gel diffusion, immunoelectrophoresis, and ELISA. Protein levels in these preparations were determined by using BCA analysis (Pierce) with bovine gamma globulin as the standard. To define the characteristics of the purified antibody preparations in an ELISA, the purified immunoglobulins were attached to plates and the wells were probed with monospecific anti-isotype serum. The resulting reactions showed that only a single isotype was present in each preparation. These purified antibody preparations were then used in an isotype-specific ELISA to quantitate the amount of anti-a. actinomycetemcomitans antibody of each isotype in a reference human serum pool. The characteristics of the binding curves for each isotype compared between the purified antibody and the antibody in the human serum were similar. Thus, we determined that the reference standard contained 6.75,ug of IgG per ml, 2.6,ug of IgM per ml, and.22 jig of IgA per ml for serotype a; 13.89,Ig of IgG per ml, 2.73,ug of IgM per ml, and.36,ug of IgA per ml for serotype b; and 8.11 jig of IgG per ml, 2.25,ig of IgM per ml, and.27,ug of IgA per ml for serotype c. This reference standard was subsequently included on each ELISA plate for comparison of the experimental sera. Within the IgG isotype responses to various antigens, one can identify a distribution of subclass composition that is often related to the type of antigen, the genetics of the host, or the phase of antibody response development. A similar procedure was used to quantitate the level of IgG subclass antibodies to A. actinomycetemcomitans in the human sera. In this case, human myeloma proteins representing each of the four subclasses were obtained (Calbiochem) and used to coat ELISA plates at various concentrations. The wells were developed with mouse monoclonal antibodies (MAbs) specific for human IgGl (HP612; Calbiochem), IgG2 (HP614; Calbiochem), IgG3 (HP647; Calbiochem), IgG4 (HP622;

3 VOL. 59, 1991 Calbiochem), and affinity-purified goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma). The reactivities of known amounts of the IgG subclass were then compared with reactions under similar conditions with human serum antibodies to A. actinomycetemcomitans. In this manner, the amount of each IgG subclass antibody in the reference serum standard could be compared with the myeloma standards. Initial experiments were devised that would examine the specificity of the mouse MAbs to human IgG subclasses, as well as assess the similarity of binding dynamics of the MAb binding to human myeloma proteins and to human subclass antibody bound to A. actinomycetemcomitans. In these studies, myeloma proteins of each subclass were coated individually onto wells of ELISA plates covering a concentration range of 5 jig to 1 pg. The wells were developed with the subclass-specific MAbs, and the results showed that the murine MAbs were specific for the different subclasses. Additional wells were coated with A. actinomycetemcomitans, and the human serum reference standard was analyzed by using the same developing conditions as those used for the myeloma proteins. The results showed similar curves with detection of each subclass reaction in the human serum versus those defined by interaction of the MAb with the purified myeloma protein. These findings indicated that the development system detected individual molecules of the IgG subclasses in a similar fashion, whether bound to plates or to the microorganism. Therefore, we could reliably calculate the subclass IgG levels in the reference standard by comparison of the curves. Consequently, the reference serum standard was estimated to contain 7.53,ug of IgGl per ml, 1.9 jig of IgG2 per ml, 4.37,ug of IgG3 per ml, and.48 jg of IgG4 per ml. Within the limits of error of the methods, the summation of these levels was close to the total IgG anti-a. actinomycetemcomitans antibody level determined previously (13.89,ug/ml). Levels of antibodies to each of the A. actinomycetemcomitans serotypes were assessed in the population of normal subjects. Mean levels and their standard deviations was determined. Significantly elevated levels in the test patient group were defined as activities that were >2 standard deviations above the mean of the normal group (46). Statistical analyses. Statistical differences in the data among the groups were assessed by Kruskal-Wallis analysis of variance and a Wilcoxon-Mann-Whitney test. The differences in frequency of elevated responses in the patients were determined by Chi-square analysis. Correlation analyses of the data were assessed by using a Spearman correlation coefficient. All of the tests were performed by using Statgraphics (STSC) on an IBM PC-based system. RESULTS Serotype distribution ofa. acfinomycetemcomitans. Primary cultivation plates of subgingival plaque were obtained from 177 patients. The cultures were probed by using monospecific rabbit polyclonal antiglobulins for each of the A. actinomycetemcomitans serotypes. The results in Table 1 demonstrate that the predominant serotype of A. actinomycetemcomitans in each of the patient classifications was b. However, it did appear that the LJP group was specifically enriched for colonization by this serotype. Sera from patients from whom A. actinomycetemcomitans could be cultivated were then examined for the level and isotype distribution of antibody to A. actinomycetemcomitans. In this population of A. actinomycetemcomitans-infected subjects, 1, 91, 87, and 4% of the LJP, AP, RPP, and normal ANTIBODY TO A. ACTINOMYCETEMCOMITANS 1797 TABLE 1. Distribution of A. actinomycetemcomitans serotypes in infected patients Disease Distribution (%) in pa- % (range) of classificationa Serotype tients plaque with sample" positive positive samplesc plaque LiP a (12-41) b (15-79) c (13-75) RPP a (21-75) b (25-82) c (33-8) AP a (8-5) b (19-4) c (14-32) Normal a (4-25) b (4-25) c (4-25) a The numbers of subjects in each disease classification with positive plaque samples were as follows: LJP, 35; RPP, 46; AP, 31; normal, 5. b Mean percentage of each serotype detected in plaque samples from the subjects. c Mean percentage (range) of plaque samples from subjects with detectable A. actinomycetemcomitans. Plaque samples collected from individual teeth in the patients ranged from 6 to 28. groups, respectively, exhibited elevated levels of IgG antibody to the microorganism. Thus, it appeared that A. actinomycetemcomitans infections are generally associated with elevated systemic antibody levels. Serum isotype antibody responses to A. actinomycetemcomitans in periodontitis. Groups of patients with periodontitis were identified on the basis of colonization of at least one subgingival plaque sample with A. actinomycetemcomitans. The results shown in Fig. 1 depict the levels of antibody to each serotype of A. actinomycetemcomitans in the LJP, Disease Classification Jp AP N RP AP Nn JrP AP? _ I ug/mi * IgG D 19M 3 IgA FIG. 1. Levels of IgG, IgM, and IgA antibodies in serum from A. actinomycetemcomitans-infected patients with LJP (JP; n = 51), RPP (n = 56), or AP (n = 61) patients and from normal (N) subjects (n = 75). Panels: A, serotype a; B, serotype b; C, serotype c. The bars show mean values for the populations. Variances from the means were no greater than 27 (IgG), 12 (IgM), and 23% (IgA). Responses to serotype b were significantly greater (P <.1) than those to the other serotypes in all groups of patients. Additionally, all antibody levels in patients were significantly greater (P <.25) than the corresponding levels in normal subjects. A B c

4 1798 EBERSOLE ET AL. RPP, AP and normal groups. The level of IgG to serotype b in serum was significantly increased versus each of the other serotypes; however, these levels were not significantly different among the three disease classifications in A. actinomycetemcomitans-infected patients. Likewise, there were no significant differences in IgG antibody to either serotype a or c, although the levels of antibody to serotype c appeared to be somewhat increased in the AP group versus the other disease classifications. IgM levels in serum were comparable among the groups of patients and with respect to each of the A. actinomycetemcomitans serotypes, and in each case these were significantly higher than those of the normal subjects (Fig. 1). Although levels of antibody to serotype b were generally higher than those of antibodies to the other serotypes, this difference reached statistical significance only in the LJP group. The level of IgA to serotype b A. actinomycetemcomitans in serum was significantly higher than that of IgA to either serotype a or c bacteria in the disease groups (Fig. 1). Interestingly, substantial elevations in this isotype antibody to serotype b bacteria were detected in the LJP and RPP groups compared with the AP group. Levels of IgG subclass antibodies to A. actinomycetemcomitans in serum in periodontitis. On the basis of the predominance of antibodies to serotype b in serum in this population, we concentrated our efforts on determination of the IgG subclass responses to this serotype. Figure 2 shows that the principal distribution of antibody to A. actinomycetemcomitans is IgGl and IgG3 in the three groups of patients. These findings were expressed in all patients with LJP, RPP, and AP (Fig. 2A). As has been noted previously, patients with LJP exhibited antibody levels significantly higher than those of all other groups. When the patients were categorized on the basis of colonization by A. actinomycetemcomitans (Fig. 2B), their IgG subclass responses were generally similar. It was of interest that only the IgGl response was specifically elevated in the LJP and RPP groups versus the AP group. The IgG response in normal subjects appeared to consist exclusively of IgG2 antibody. The sequence of subclass responses was estimated by assessing the frequency of each subclass that was significantly elevated in the patient population. These significant elevations were determined by comparison with the range of antibody values in the normal population. The results in Table 2 indicate that the sequence of responses to A. actinomycetemcomitans in these infected patients was IgGl, followed by IgG3. This is indicated by the frequency of elevated IgGl-IgG3, together and a consistently higher frequency of elevated IgGl alone versus elevated IgG3 alone. Also, these were the only two subclasses that demonstrated unique elevations in the absence of other subclasses. The IgG2-IgG4 response appeared less frequently in the group and was typically accompanied by elevated IgGl and/or IgG3 responses. In contrast, IgG4 antibodies were never elevated in the absence of elevations of other subclasses. This interpretation is based on the fact that frequencies of elevation of subclasses of IgGl, IgG2, and IgG3 were greater than those of IgGl, IgG3, and IgG4. Serotype distribution of Antibody to A. actinomycetemcomitans in serum. Previous results were presented as means of the populations within disease categories. Of interest was the relationship of responses of individual patients to each of the A. actinomycetemcomitans serotypes. The distribution of serotype responses in the patients is shown in Table 3. The results indicate that while a substantial proportion of the c ow -.c JP RPP AP N U) JP j i RPP AP &ORM ul 4 g/ml 5 * IgGl IgG2 193 IgG FIG. 2. Levels of IgG subclass antibodies to A. actinomycetemcomitans serotype b in all subjects (A) and in subjects with positive plaque samples for A. actinomycetemcomitans. The bars show the means of the populations. Variances from the means ranged from 13 to 32%. In panel A, the levels of IgGl and IgG3 in the LJP group are significantly greater (P <.1) than those of all other groups. IgGl and IgG3 levels were significantly greater (P <.25) in the RPP group than in the AP and normal (N) groups. IgG4 (P <.1) and IgG2 (P <.1) levels were significantly greater in all groups of patients than in normal subjects. In panel B, the IgG3 level in patients with LJP is significantly greater (P <.1) than that in the AP group. patients exhibited elevated antibodies to only one serotype (a, 3.9%; b, 52.1%; c, 14.3%), very frequently high levels of IgG antibodies to two or even all three serotypes were found in serum. These findings are consistent with the increased prevalence of serotype b A. actinomycetemcomitans in this population, which elicited a significant antibody response of multiple isotypes. However, the antibody patterns also indicate an array of antibody responses that are directed toward common antigens among the three serotypes of this species. Table 4 depicts the relationship between elevations of IgG antibody and the serotype distribution of A. actinomycetemcomitans in subgingival plaque. Generally, in cases in which the response to a single serotype was elevated, only that type of A. actinomycetemcomitans was detected in the plaque. Additionally, individuals exhibiting elevated levels of antibodies to each of the serotypes were most consistently colonized by the serotype b microorganism. DISCUSSION Various studies have investigated the relationship of A. actinomycetemcomitans to human periodontal disease. Both I m m -1 INFECT. IMMUN. A --.J. B

5 VOL. 59, 1991 TABLE 2. Distribution of IgG subclass responses to A. actinomycetemcomitans b in infected patients with periodontitis IgG subclass(es) LJP RPP AP Frequency (%) of elevated antibody response' , , ,4.6 3, , 2, , 3, a Elevated antibody responses were defined as levels >2 standard deviations above the mean of the normal population. microbiologic (6, 8, 18, 27, 31, 34, 39, 41, 43, 44) and immunologic (9, 16, 17, 19-21, 24, 28, 47, 49, 53) studies have supported the probable role of this microorganism in disease processes. In this study, we extended and expanded these findings by delineating the relationship of A. actinomycetemcomitans infection to the characteristics of the systemic antibody response to the microorganism. As in previous studies, we determined that in general, A. actinomycetemcomitans serotype b predominates in LJP (28, 55-57). We also showed that this serotype predominates in both RPP and AP, albeit not at the same frequency as in LJP. Previous studies of antibody responses to A. actinomycetemcomitans showed a significant elevation in IgG antibody levels in LJP compared with other disease classifications or normal subjects (9, 16, 17, 19-21, 24, 28, 47, 49, 53). However, because of the nature of this disease and the current concept that a finite group of bacteria is directly involved in disease, a heterogeneous disease etiology would be expected within the AP group (42, 45). In this study, patients identified as being colonized by A. actinomycetemcomitans were stratified and antibody levels were compared. In this design, it was shown that the levels of IgG, IgM, and IgA responses to A. actinomycetemcomitans were similar among the infected patients, irrespective of disease classification. To compare the levels of various isotype antibodies to the different antigens, studies were performed that allowed quantitation of each isotype antibody in a human TABLE 3. Distribution of elevated levels of IgG antibody to A. actinomycetemcomitans serotypes in infected patients with periodontitis Disease classification a b c a-b a-c b-c a-b-c Serotype specificity of elevated response' LiP RPP AP Avg a Percentage of A. actinomycetemcomitans-infected patients with antibody levels >2 standard deviations above the mean of the normal subjects. A x2 test showed that the distribution of responses in the AP group was significantly different from that of responses in the other groups at P =.3. ANTIBODY TO A. ACTINOMYCETEMCOMITANS 1799 TABLE 4. Relationship of antibody specificity to distribution of A. actinomycetemcomitans serotypes Serotype of bacteria' Serotype specificity of elevated response a b c a b c a-b a-c b-c a-b-c a Percentage of subjects with plaque samples positive for a serotype of A. actinomycetemcomitans accompanying elevated IgG antibody to one or more serotypes in serum. Percentages not included represent subjects in which more than one serotype of A. actinomycetemcomitans was detected (.9 to 3.4%). reference standard. By using this reference standard, it was demonstrated that for all isotypes the antibody response to serotype b in serum was quantitatively greater than responses to serotype a or c. Analyses were also performed to determine the relationship between serotype colonization of the subgingival plaque and elevations of antibody responses in serum. The findings were similar to those of Gmur et al. (22), in which the elevated responses of many subjects were not limited to one serotype. This is in contrast to the report of Zambon et al. (57), in which there was a more direct correlation between the colonizing A. actinomycetemcomitans serotype and the systemic antibody response. Our findings indicate that b was the most frequent A. actinomycetemcomitans serotype detected and appears to be capable of initiating a substantial IgG antibody response in serum, along with cross-reactive antibodies to other serotypes of A. actinomycetemcomitans. Previous findings by Asikainen (2) indicated that while patients with LJP retain elevated anti-a. actinomycetemcomitans antibody levels, older subjects with LJP showed a lower incidence of recovery of the microorganism. In the present study, there was no evidence of initiation of disease in the RPP and AP groups as a localized disease at a younger age. Consequently, by sampling all interproximal sites, we were not able to demonstrate this age-related decline in A. actinomycetemcomitans infection. Previous studies from our laboratory have indicated that IgG antibody levels in serum can remain elevated for prolonged periods following treatment (13), although these studies did not provide substantive data concerning elimination of the microorganism (antigen) from the oral cavity associated with treatment. Therefore, the question of the persistence of elevated systemic antibody levels in the absence of oral colonization-infection with a specific microorganism remains to be elucidated. It was also noted that while A. actinomycetemcomitans serotype b was the predominant isolate from the patient population examined in this investigation, there did appear to be a real disease classification-dependent difference in the distribution of other serotypes. Specifically, patients with AP showed a higher frequency of serotype c than did patients with LJP or RPP. This was accompanied by an increased frequency of elevated levels of IgG antibody to the serotype in the sera of patients with AP. A previous report by Tsai and Taichman (5) described the dynamics of leukotoxic A. actinomycetemcomitans in juvenile periodontitis. The relationship of that study to ours is that in general, serotype b strains of A. actinomycetemcomitans have been

6 18 EBERSOLE ET AL. described as leukotoxin positive, while serotype c strains have been uniformly negative (3). The results from this study suggested that younger patients with juvenile periodontitis were colonized by leukotoxic A. actinomycetemcomitans, while the older individuals tended to be infected with nonleukotoxic strains. Thus, it may be implied that either leukotoxin production in the younger patients is eventually selected against by host mechanisms and these strains lose the ability to produce the leukotoxin or the leukotoxic strains (i.e., serotype b) are eventually eliminated and replaced by nonleukotoxic strains (i.e., serotype c) in the plaque ecology. While neither of these options has been clearly defined, it has been unequivocally demonstrated that patients with LJP produce IgG antibody in serum that is effective in inhibiting the function of the leukotoxin (48). Thus, this host response may be critical in the selection process for the A. actinomycetemcomitans type in the subgingival plaque. Previous studies have routinely identified elevated levels of IgG antibody to A. actinomycetemcomitans in serum in various type of periodontitis (9, 16, 17, 19-21, 24, 28, 47, 49, 53). Human IgG antibody responses have been shown to be composed of four subclasses. The distribution of these subclasses in an immune response has been shown to be controlled by antigen characteristics, extent of stimulation, and host genetic composition (25, 26). The significance of the variances in response patterns resides in the functional differences among the subclass molecules (25). Some previous studies have examined IgG subclass levels or specificities and their relationship to periodontal disease (29, 3, 4, 52). However, this study represents the first report detailing the distribution of IgG subclass antibodies to A. actinomycetemcomitans. We have developed an ELISA that provides an estimation of absolute levels of each of the subclasses. The results demonstrated that the primary responses to A. actinomycetemcomitans were of the IgGl and IgG3 subclasses. On the basis of other experimental systems, these responses suggest that protein antigens from the microorganism are involved in the primary elicitation of host antibody responses. These findings are also consistent with studies in which Western immunoblotting analyses were performed to delineate the characteristics of antigens from A. actinomycetemcomitans to which humans respond. In some of these cases, the antibodies in serum appeared to be directed towards outer membrane protein antigens from the microorganism (7, 14, 53). In contrast, the primary subclass response in normal subjects is limited to the IgG2 subclass and may represent a broader cross-reactivity to polysaccharide antigens-lipopolysaccharide from the bacteria. A recent report by Bardardottir et al. (5) showed that immunization of humans with pneumococcal polysaccharide vaccine resulted in elevated antibody levels in serum in each of the IgG subclasses. Thus, the exact antigenic determinants that elicit the elevated antibody to this microorganism in serum, as well as the dominant antigens across populations, have yet to be determined. Additionally, we derived an apparent order of IgG antibody responses to A. actinomycetemcomitans and showed that IgGl appears to be elevated initially in the response, followed sequentially by IgG3, IgG2, and IgG4. A critical step in our understanding of the mechanisms of disease associated with this microorganism will be a precise insight into the functional capabilities of these antibody molecules (4, 13a, 48). INFECT. IMMUN. ACKNOWLEDGMENTS This work was supported by U.S.P.H.S. grant DE-789 from the National Institute of Dental Research. We thank S. S. Socransky and A. C. R. 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