Porphyromonas gingivalis-specific serum IgG and IgA antibodies originate from immunoglobulin-secreting cells in inflamed gingiva.

Patients with adult periodontitis (AP) exhibit elevated serum antibody levels to Porphyromonas (Bacteroides) gingivalis; however, it is not known whether these antibodies originate from plasma cells in the local disease site or from peripheral lymphoid tissues. We studied the isotype and subclass levels and origin of antibodies to P. gingivalis fimbriae, since elevated serum anti-fimbriae responses were seen when compared with sera of healthy controls. IgG anti-fibriae titres were dominant and the subclass response was IgG3 much greater than IgG1 greater than IgG2 much greater than IgG4; however, some IgA anti-fimbriae antibodies were also seen. The IgA subclass fimbriae-specific response was mainly IgA1; however, significant IgA2 anti-fimbrae antibodies were seen. We also assessed numbers of anti-fimbriae antibody producing cells from peripheral blood mononuclear cells (PMBC) and from either healthy or inflamed gingiva of AP subjects. Gingival mononuclear cells (GMC) of AP patients exhibited high numbers of immunoglobulin-producing (spot-forming) cells (SFC) including fimbriae-specific antibody secreting cells in a pattern of IgG greater than IgA greater than greater than greater than IgM. However, low numbers of SFC were seen in GMC from healthy gingiva; further, no anti-fimbriae SFC responses were noted in healthy GMC. Although no fimbriae-specific immunoglobulin-producing cells were seen in PBMC, low numbers of antigen-specific SFC were found in pokeweed mitogen-triggered PBMC from AP subjects. Treatment of AP patients for plaque and surgical removal of inflamed gingiva resulted in significant reductions in serum anti-fimbriae responses. These studies show that AP patients exhibit brisk serum IgG and IgA subclass anti-fimbriae antibodies, whose origin appear to be the plasma cells present in the localized inflamed tissues.     

Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases

The level of serum IgM, IgG and IgA antibodies including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 subclass-specific antibodies to Bacteroides (Porphyromonas) gingivalis fimbriae and to lipopolysaccharide (LPS) were analysed in patients with different forms of periodontal disease (PD) and control subjects by ELISA. Among PD subjects, sera obtained from adult periodontitis (AP), rapidly progressive periodontitis (RPP) and gingivitis contained high titres of fimbriae-specific IgG antibodies (7500-15,000 ELISA units) followed by IgA (90-700 units) and IgM (30-90 units). In contrast, sera from localized juvenile periodontitis (LJP) subjects exhibited much lower titres of fimbriae-specific IgG (89 +/- 11 units), IgA (31 +/- 5 units) and IgM (17 +/- 3 units) antibodies. A similar response pattern was also seen in sera from normal subjects aged 35-41 years who practice normal oral hygiene, while sera of younger adults (aged 18-24) with superior hygiene did not have any antigen-specific antibodies. Analysis of IgG subclass anti-fimbriae responses revealed that the major response was IgG3 followed by IgG1, IgG2 and IgG4 in AP, RPP and gingivitis. Although lower, a similar pattern of IgG subclass titre was seen in LJP and normal subjects aged 35-41 years. When IgA subclass responses were measured in AP and RPP, higher titres of the fimbriae-specific response were noted with IgA1 when compared with IgA2. However, lower but approximately equal levels of fimbriae-specific IgA1 and IgA2 titres were seen in other PD groups. When anti-B. gingivalis LPS-specific responses were measured, the sera of AP patients contained high levels of IgG antibodies (2265 +/- 224 units) followed by IgA (411 +/- 90 units) and IgM (214 +/- 56 units). Further, IgG anti-LPS responses were mainly IgG2 followed by IgG4, IgG3 and IgG1. For IgA subclass responses, higher titres of anti-LPS-specific antibodies were noted in IgA2 subclass over IgA1. These results showed that higher anti-B. gingivalis antibody responses occur in PD when compared with healthy individuals and protein and lipid-carbohydrate antigens of B. gingivalis induce distinct patterns of antigen-specific IgG and IgA subclass responses.     

Effects of IL6 on B cells in mucosal immune response and inflammation.

Freshly isolated surface IgA+ (sIgA+) B cells from human gut-associated lymphoreticular tissues (GALT), e.g. the appendix, express high levels of IL6 receptor (IL6R) and respond to IL6 with significant increases in the number of IgA-secreting cells. On the other hand, neither sIgM+ nor sIgG+ B cells from appendix express IL6R. When the effect of IL6 on IgA subclass antibody synthesis was examined, the numbers of both IgA1- and IgA2-producing cells were increased upon incubation of GALT B cells with IL6; however, 60-70% of IgA-secreting cells were IgA2 subclass. Aberrant local production of IL6 can contribute to increased B-cell responses that occur in mucosal inflammation such as gingiva of patients with adult periodontitis (AP). When gingival mononuclear cells (GMC) isolated from AP patients were cultured without any stimulus, GMC spontaneously produced biologically active IL6 which induced peripheral blood mononuclear cells (PBMC) from the same patients to become IgG- and IgA-producing cells. Further, mRNA extracted from GMC possessed high message for IL6. When the expression of IL6R was compared between GMC and PBMC isolated from AP patients, IL6R-bearing cells were only seen in the former population. Thus, a high production of IL6, which have the ability to regulate later stages of IL6R+ B-cell development and to induce them to become Ig-secreting plasma cells and to support plasmacytoma growth, are important immunopathological elements for the induction of the increased B-cell response region in inflamed mucosal tissues.     

Lipopolysaccharide- and cholera toxin-specific subclass distribution of B-cell responses in cholera.

The immunoglobulin subclass responses to homologous lipopolysaccharide (LPS) and to cholera toxin (CT) in adult patients infected with Vibrio cholerae O1 and V. cholerae O139 were studied. LPS-specific antibody-secreting cells (ASC) of both the immunoglobulin A1 (IgA1) and IgA2 subclasses were seen, with the IgA1 ASC response predominating in both V. cholerae O1- and O139-infected patients. For antibodies in plasma, by day 11 after onset of disease, all V. cholerae O1- infected patients responded to homologous LPS with the IgA1 subclass (P = 0.001), whereas fewer (68%) responded with the IgA2 subclass (P = 0.007). About 89% of V. cholerae O139-infected patients responded with the IgA1 subclass (P = 0.003), and only 21% responded with the IgA2 subclass (not significant [NS]). Both groups of cholera patients showed significant increases in LPS-specific IgG1, IgG2, and IgG3 antibodies in plasma. In feces, the response to homologous LPS occurred in both groups of patients with the IgA1 and IgA2 subclasses, with 55 to 67% of patients showing a positive response. V. cholerae O1- and O139-infected patients showed CT-specific ASC responses of the different IgG and IgA subclasses in the circulation, and the pattern followed the order IgG1 > IgA1 > IgG2 > IgA2, with low levels of IgG3 and IgG4 ASC. Plasma anti-CT antibody responses in all subclasses were seen by day 11 after onset of disease. Although there were no increases in CT-specific ASC of the IgG3 (NS) and IgG4 (NS) subtypes, there were significant increases of these two subclasses in plasma (P 相似文献   

The early cellular and humoral immune response to primary and booster oral immunization with cholera toxin B subunit

The immune response to cholera toxin B subunit given orally was studied in 13 human volunteers. A serum IgG and IgA antitoxin response was observed, which was boosted by a second immunization. Using an immunospot assay, cells spontaneously secreting anti-toxin IgG and IgA, but not IgM appeared transiently in the blood after immunization. There were 105 IgG- and 87 IgA-secreting cells per 2 x 10(6) mononuclear cells 7 days after the first immunization, and 282 IgG- and 413 IgA-secreting cells 5 days after the second immunization. A polyclonal increase in total IgM-secreting cells was observed. Few anti-toxin-secreting cells were observed in the bone marrow at the peak of the circulating cell response, which could be accounted for by contamination of the sample with peripheral blood, suggesting that the bone marrow is not a significant site of anti-toxin-secreting cells after oral immunization.     

IgG and IgA subclass mRNA-bearing plasma cells in periodontitis gingival tissue and immunoglobulin levels in the gingival crevicular fluid

The humoral immune response, especially the production of IgG and IgA, is considered to have a protective role in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. In order to determine local IgG and IgA production, we investigated the presence of human IgG and IgA subclass mRNA-bearing plasma cells within periodontal tissue by in situhybridization using digoxigenin-labelled oligonucleotide probes in 24 gingival biopsy samples (pocket depth>5 mm) which were obtained from eight patients with adult periodontitis. Furthermore, we examined IgG and IgA subclass proteins and digested IgA1 Fab portions in the gingival crevicular fluid (GCF), corresponding to the sites from which the tissues were taken, by ELISA. IgG and IgA subclass mRNA-expressing cells were detected in all serial formalin-fixed/paraffin-embedded gingival tissue sections sampled. Plasma cells showed strong cytoplasmic staining with a high contrast and a good retention of morphology with these probes. IgG1 mRNA-expressing cells were predominant (mean 63%) and IgG2 mRNA-expressing cells were present at around 23% of total IgG plasma cells, while IgG3 and IgG4 mRNA-expressing cells were present to a much lesser extent (3% and 10%, respectively). Similar proportions of IgG subclass proteins in GCF were detected, which were also consistent with ‘normal’ serum levels. In terms of IgA subclass, IgA1 mRNA-positive cells were predominant (mean 65.1%, P<0.001). In contrast, IgA2 protein in the GCF samples were detected at higher concentrations than IgA1 (P<0.001). The ratio of total IgG to IgA mRNA-positive plasma cells was ≈7.5:1. There was a good correlation between the amounts of IgG subclass proteins in GCF and the number of IgG subclass mRNA-positive cells in the same sites, but not between IgA subclass proteins and the number of IgA subclass mRNA-positive cells. These data suggest that IgG and IgA subclass proteins can be locally produced in the periodontitis gingiva. In addition, as we detected IgA1 Fab fragments in GCF, this is further confirmation that secreted IgA1 protein in GCF may be digested by periodontal bacteria.     

Quantitative assessment of IgG and IgA subclass producing cells in rectal mucosa during shigellosis.

AIMS: To assess quantitatively both the morphological changes in the rectal mucosa and the changes in the relative frequency of IgA and IgG subclass producing cells found in the rectal mucosa during the acute phase of shigellosis and at convalescence. METHODS: Rectal biopsies from 25 Shigella dysenteriae 1 infected patients, 10 Shigella flexneri infected patients, and 40 uninfected controls were studied. Morphological changes in the mucosa were graded. The frequency of IgA and IgG subclass producing cells was assessed. In addition, immunostaining for secretory component in epithelial cells was analysed. RESULTS: Using morphological grading, 20% of the 35 patients studied had advanced inflammation (grade 3) in the acute phase of the disease. At convalescence, grade 1 inflammation was seen in 37% of the patients and in 10% of the controls. In the acute phase, as well as at convalescence, the number of IgA1, IgA2, and IgG2 positive cells was significantly higher than in the controls. The results were related to the histopathological degree of inflammation. CONCLUSIONS: In shigellosis, there is evidence for a prolonged humoral response residing in the mucosa long after the clinical symptoms have resolved, suggesting that shigellosis induces persisting mucosal humoral immune and inflammatory responses, remaining at least until 30 days after the infection.     

Ontogenesis of the secretory immune system and innate defence factors in human parotid glands

Immunoglobulin-producing cells and epithelial expression of secretory component (SC), amylase, lysozyme (Ly) and lactoferrin (Lf) were studied by immunohistochemistry to obtain information about the development of mucosal immunity. Tissue specimens were obtained from 20 fetal and 40 postnatal parotid glands. (1) Fetal specimens. Occasional IgM- and IgA- but no IgD-, IgG- or IgE- producing cells were seen (ratios, IgM:IgA:IgD:IgG:IgE approximately 4:1:0:0:0). The IgAl subclass dominated (median 90%, range 50-95%) and these cells were mostly J-chain-positive (median 97%, range 94-98%). Only few IgA2-producing cells were seen (median 10%, range 5-50%) and they were also mostly J-chain-positive (median 99%, range 98-100%). Amylase, Ly and Lf were most prominent in early fetal life, while only small amounts of SC were present. (2) Postnatal specimens. Secretory component increased markedly along with a growing number of IgA- and IgD-producing cells (IgA:IgM:IgD:IgG:IgE approximately 4:2:1:1:0). The IgAl subclass remained predominant (median 65%, range 50-90%) although the proportion of IgA2-positive cells tended to be raised (median 35%, range 10-50%). Most IgAl (median 97%, range 67-100%) and IgA2 (median 94%, range 75-100%) cells were J-chain-positive. These features probably reflected local activation of the immune system in response to environmental factors. The amount of amylase, Ly and Lf decreased shortly after delivery, perhaps because the cellular stores were emptied by postnatal increase in secretory activity.     

Analysis of IgG subclass production in cell cultures from IgA deficient patients and in normal controls as a function of age

IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age. Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures. Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency. When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal. The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced for IgG2; IgG2 plasma cell differentiation was particularly depressed in LPS cultures. In contrast to normal adult cells, blood MNC from the nine children with IgA deficiency and age-matched controls (2-17 years) yielded more IgG1 than IgG2 plasma cells in both control and LPS cultures, while the pattern of response to PWM was similar in all groups (gamma 1 greater than gamma 2). A good concordance was found between the level of secreted Ig in the culture supernatants and the relative number of IgM or, IgG and IgA plasma cells identified by immunofluorescence staining of cytoplasmic immunoglobulins.     

Humoral response to Porphyromonas (Bacteroides) gingivalis in rats: time course and T-cell dependence.

In this study, we describe the time course and T-cell dependence of the serum antibody response to the periodontopathogen Porphyromonas (Bacteroides) gingivalis in an experimental rat model. Normal Fischer rats were challenged by a local injection of P. gingivalis (2 x 10(8) bacteria) into gingival tissue or by the administration of a similar number of bacteria by the intravenous (i.v.) route on days 0, 2, and 4. Serum antibody activity was detected within 1 week and peaked at 8 weeks after gingival challenge. A similar but lower response was seen for rats challenged by the i.v. route. The response in both groups of rats was mainly of the immunoglobulin G (IgG) isotype; some IgM but no IgA antibody activity was detected. Analysis of the IgG subclass revealed mainly IgG2c in animals challenged locally in the gingiva with P. gingivalis, whereas IgG2b predominated in rats challenged by the i.v. route. The importance of T cells in the response was established by demonstrating the absence of serum IgG antibodies in nude rats after a local challenge of gingival tissue with P. gingivalis. Nude rats given purified splenic T cells from normal rats immunized systemically with P. gingivalis prior to a local gingival challenge showed a rapid appearance of serum antibody activity that peaked between 4 and 6 weeks. This initial peak occurred 2 to 4 weeks earlier than that seen in normal animals. Fluorescence-activated cell sorter analysis of splenic lymphoid cells from these nude rats revealed a helper T-cell population. The levels of serum IgG antibodies in nude rats given nonimmune T cells rose slowly, and the antibodies were mainly of the IgG2a and IgG2b subclasses. Nude rats given immune T cells showed a rapid increase primarily in IgG2b antibody levels following a local gingival challenge. These findings suggest that the immune helper T-cells contributed to the rapid development of the response to P. gingivalis. Furthermore, it is likely that the IgG subclass response to P. gingivalis in these nude rats was related to the splenic origin of the T cells used for adoptive transfer.     

Physiology of IgD. IX. Effect of IgD on immunoglobulin production in young and old mice

Weekly i.p. injections of IgD from birth in (SJL X BALB/c)F1 mice were found to accelerate the development of IgG- and IgA-secreting cells and to increase the numbers of Ig-secreting cells of all isotypes in 17-28-day-old mice, but not in 7-10-day-old mice. Similarly, repeated weekly injections of IgD in normal adult BALB/c mice increased the numbers of reverse plaque-forming cells/spleen for all isotypes studied, including IgM, IgG1, IgG2, and IgA, but not for IgD itself. No such effect was observed in IgD-treated aged (20 months old) BALB/c mice. The absence of an effect of IgD on Ig secretion appeared to correlate with a lack of induction of receptors for IgD on T cells of the host, both in 7-10-day-old and in aged mice. In 7-10-day-old mice this lack of induction appeared due to their very low numbers of L3T4+ T cells. A comparison was made between the effect of a single injection of IgD or lipopolysaccharide (LPS) on numbers of Ig-secreting cells in the spleen determined 1-7 days after injection. Both agents caused increases, but the increase in IgM-producing cells was much greater after LPS (day 4), while IgD caused a relatively greater increase in IgG2 and IgA (days 4-7). Increases in IgG1 and IgG3-producing cells induced by LPS and IgD were of similar magnitude (days 6-7). IgD production, however, was not increased. The number of cells producing antibody of anti-trinitrophenyl (TNP) specificity was enhanced by LPS (day 4), but not by a single injection of IgD, although more than one injection of IgD caused a significant increase in anti-TNP-producing cells above background. LPS, but not IgD, caused B cell proliferation in vitro in the presence or absence of gamma-irradiated T delta cells. However, in vivo, IgD injections caused a significant increase in the percentage of lymphoid follicles with germinal centers in lymph nodes from 17-21-day-old and normal adult mice, but not in 7-10-day-old or aged mice. Such an effect was also absent in 24-28-day-old mice, where germinal center development, even in untreated mice, was very high.     

Human nasopharyngeal-associated lymphoreticular tissues. Functional analysis of subepithelial and intraepithelial B and T cells from adenoids and tonsils

Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells ( approximately 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset ( approximately 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.     

Delayed and reduced adaptive humoral immune responses in children with shigellosis compared with in adults

We hypothesized that increased susceptibility to Shigella infection, increased severity of disease and high mortality in children compared with adults were consequences of insufficient adaptive immune responses. Antigen-specific immune responses were studied in paediatric patients (n = 38, 2-10 years) with shigellosis and compared with those of adult patients (n = 30, 18-45 years). Peak frequencies of antigen (invasion plasmid coded antigen B, Ipa-B; lipopolysaccharide, LPS)-specific immunoglobulin (IgM)-antibody secreting cells (ASC) were seen within 3-5 days after the onset of diarrhoea in children, while peak IgA- and IgG-ASCs were obtained 8-10 days later in line with adults. Antigen-specific ASC responses in children ranged between 2 and 4% of the total ASC responses, in contrast to 8-15% in adults. The kinetics of LPS-specific IgG subclass titres was different in younger children (2.5-5 years) (IgG1 > IgG2 > IgG4 > IgG3) compared with in older children (6-8 years) (IgG2 > IgG1 >IgG3 > IgG4) and adults. Secretory IgA levels in stool peaked 8-10 days after onset in both adults and children. However, a rapid induction of stool LPS-specific IgA, IgA1 and IgA2 occurred in adult patients within 3-5 days of onset, while in children, this was delayed by 8-10 days. Similarly, higher number of tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma expressing cells in vitro were seen in adult patients in response to antigens (LPS and Ipa-B) in the acute stage in contrast to paediatric patients. Thus, paediatric patients with shigellosis have reduced and delayed adaptive immune responses compared with adult patients.     

Serodiagnosis of Porphyromonas gingivalis infection by immunoblot analysis with recombinant collagenase.

The Porphyromonas gingivalis collagenase-specific serum immunoglobulin A (IgA), IgM, and IgG responses from 20 patients with early-onset periodontitis (EOP), 20 patients with adult periodontitis, (AP), and 20 age- and sex-matched healthy controls were examined by immunoblot analysis. A recombinant collagenase antigen used for the immunoblot analysis was produced by using the plasmid pGEX-2T, which allows the fusion between the collagenase and glutathione S-transferase. There was no significant difference in collagenase-specific IgG antibody detection between samples from the EOP, AP, and control groups. In contrast, 85% of AP and EOP sera had collagenase-specific IgA antibodies, whereas only 20% of control sera showed collagenase-specific IgA reactivity. Plaque samples from all groups were assessed by PCR with primers complementary to the collagenase-encoding gene prtC. The results indicated that 90% of AP and EOP plaque samples and 10% of control samples were positive for P. gingivalis. All patients with collagenase-specific IgA antibodies were PCR positive. The results of the study indicate a nearly complete concordance (k = 0.856) between the presence of collagenase-specific IgA antibodies and PCR detection of P. gingivalis. By using PCR as the "gold standard," the sensitivity and specificity of the IgA immunoblot test were 94.7 and 90.9%, respectively. Therefore, the recombinant collagenase is a potential candidate for use in the serodiagnosis of periodontitis.     

Humoral immune responses in periodontal disease may have mucosal and systemic immune features

The humoral immune response, especially IgG and IgA, is considered to be protective in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. Immunoglobulins arriving at the periodontal lesion are from both systemic and local tissue sources. In order to understand better the local immunoglobulin production, we examined biopsy tissue from periodontitis lesions for the expression of IgM, IgG, IgA, IgE and in addition the IgG and IgA subclasses and J-chain by in situ hybridization. Tissues examined were superficial inflamed gingiva and the deeper granulation tissue from periodontal sites. These data confirm that IgM, and IgG and IgA subclass proteins and J-chain can be locally produced in the periodontitis tissues. IgG1 mRNA-expressing cells were predominant in the granulation tissues and in the gingiva, constituting approx. 65% of the total IgG-expressing plasma cells. There was a significantly increased proportion of IgA-expressing plasma cells in the gingiva compared with the granulation tissue (P < 0.01). Most of the IgA-expressing plasma cells were IgA1, but a greater proportion expressed IgA2 mRNA and J-chain mRNA in the gingival tissues (30.5% and 7.5%, respectively) than in the periodontal granulation tissues (19% and 0-4%, respectively). The J-chain or dimeric IgA2-expressing plasma cells were located adjacent to the epithelial cells, suggesting that this tissue demonstrates features consistent with a mucosal immune response. Furthermore, we were able to detect the secretory component in gingival and junctional epithelial cells, demonstrating that the periodontal epithelium shares features with mucosal epithelium. In contrast, deeper tissues had more plasma cells that expressed IgM, and less expressing IgA, a response which appears more akin to the systemic immune response. In conclusion, this study suggests that immune mechanisms involved in the pathogenesis of periodontitis may involve features of both the mucosal and systemic immune systems, dependent on tissue location.     

Mechanisms of monophosphoryl lipid A augmentation of host responses to recombinant HagB from Porphyromonas gingivalis

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 microg) alone or with MPL (25 microg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was approximately 1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.     

Delineation of producing ability of IgG and IgA subclasses by naive B cells in newborn infants and adult individuals

Neonatal B cells with the naive (sIgD⁺) phenotype are able to generate IgG- and IgA-producing cells as well as IgM production in the presence of memory CD4⁺ T cells expressing L-selectin (CD62L) in pokeweed mitogen-stimulated cultures. We used this system to examine comparatively the ability of naive B cells to produce IgG and IgA subclasses in newborn infants and adult individuals. Naive B cells were enriched from both donors on the basis of sIgD positivity, and memory (CD45RO⁺) CD4⁺ T cells with CD62L expression were isolated from adults. We here demonstrate some differences in profiles of IgG and IgA subclass production between neonatal and adult naive B cells. In neonatal B cells, IgG1 and IgG3 were predominantly produced, but IgG2 and IgG4 production was virtually absent. Similar to neonatal B cells, adult naive B cells produced mainly IgGq and IgG3, although memory (sIgD’) B cells from adults secreted all of the IgG subclasses. It should be noted that low but detectable levels of IgG2 and IgG4 were found in adults’naive B cell cultures. Although IgA produced by neonatal B cells was exclusively IgA1, IgA2-secreting cells were identifiable in adult naive B cells. The results suggest that further class switch of naive B cells to IgG2, IgG4 and IgA2 in addition to IgG1 and IgG3 may be controlled by their own age-dependent maturation process.     

Serum immunoglobulin G antibody to Porphyromonas gingivalis in rapidly progressive periodontitis: titer, avidity, and subclass distribution.

Porphyromonas gingivalis is a suspected pathogen in rapidly progressive periodontitis (RPP). We have determined the anti-P. gingivalis serum immunoglobulin G (IgG) isotype response and avidity and the subclass titer distributions for 30 RPP patients and 30 age-, sex-, and race-matched healthy subjects by using enzyme-linked immunosorbent assay technology. Patients and control subjects were classified as seropositive if their total IgG response to P. gingivalis was twofold or more than the median response in healthy subjects. The predominant antibody responses for both patients and healthy subjects were IgG2 and IgG3, with a subclass order of IgG2 greater than IgG3 greater than IgG1 greater than IgG4. The avidity of the IgG response was highest for the seropositive healthy subjects and was no different between seronegative and seropositive RPP patients. The subclass antibody responses did not depend on gender, and there were no correlations between titer, avidity, or subclass with disease severity in the RPP patients as measured by pocket depth or bone loss on dental X rays. The seronegative RPP patients exhibited antibody responses that were greater than the responses of seronegative healthy subjects for all four subclasses, while the seropositive RPP patients had higher IgG1 and IgG4 levels than seropositive healthy subjects. These findings are consistent with the hypothesis that both carbohydrate and protein antigens are important in the IgG response to P. gingivalis. The relative predominance of IgG2, a subclass which lacks strong complement fixation and opsonic properties, and the low avidity of patient anti-P. gingivalis IgG antibodies suggest that humoral responsiveness to infection with P. gingivalis may be ineffective in clearing this organism.     

Immunoglobulin subclass distribution of synovial plasma cells in rheumatoid arthritis determined by use of monoclonal anti-subclass antibodies

The distribution of IgG and IgA subclass plasma cells among dissociated synovial cells from 14 rheumatoid arthritis (RA) synovia was examined by immunofluorescence using mouse monoclonal anti-human subclass antibodies. Of the IgG plasma cells 81 +/- 9% were IgG1, 4 +/- 2% IgG2, 14 +/- 9% IgG3, and 0.9 +/- 0.6% IgG4. While IgG1 predominated in all 14 synovia (which is similar to what is seen in normal tissues), in 5/14 20% or greater of IgG plasma cells were IgG3, suggesting a selective increase in IgG3 production in the synovia of certain RA patients. Among IgA plasma cells 89 +/- 5% were IgA1 and 8 +/- 3% were IgA2. This distribution is similar to the distribution in normal serum.     

Kinetics and Isotype Profile of Rheumatoid Factor Production During Viral Infection: Organ Distribution of Antibody Secreting Cells

The kinetics and isotype profile of influenza virus-specific IgG antibodies were studied in correlation with the serum titre of IgG-reactive autoantibodies. An increased level of IgG isotype-specific, rheumatoid factor-type autoantibody secretion was observed in the late phase of the virus-specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti-viral antibody response. As revealed by a preparative immunosorbent technique combined with isotype quantitation the majority of IgG2a- or IgG1-bound immunoglobulins isolated from the serum of virus-infected mice belonged to the same subclass as the target antibody. Comparison of the kinetics of appearance and the number of IgM-, IgG- and IgA-type IgG2a-reactive autoantibody secreting cells during the primary and memory anti-viral antibody responses showed isotype switch of IgM rheumatoid factor secreting cells predominantly to IgA. Localization of IgM and IgA antibody secreting cells demonstrated the wide organ distribution of IgM-type rheumatoid factor secreting cells. On the contrary, IgA rheumatoid factor production was observed only in Peyer's patches and at the site of the local virus-specific immune response, i.e. in mediastinal lymph nodes and in the lung. These results demonstrate that B cells specific for self IgG are activated and differentiated in concert with the virus-specific antibody response in similar microenvironments. The predominant involvement of the mediastinal lymph nodes and the spleen in the production of IgG2a-specific IgM-type autoantibodies suggest a regulatory function of this type of autoantibodies in modulating IgG2a production in both systemic and local anti-viral immune responses. The results also suggest a strictly regulated rheumatoid factor production which, however, can be unbalanced by repeated viral infections resulting in the escape of high affinity, isotype-switched autoantibodies.