Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate.

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity and depletes lymphoid cells of PIP3. Hence we propose that Cdt toxicity results from depletion of this signaling lipid and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signaling. We have now focused on the relationship between cell susceptibility to CdtB and differences in the status of baseline PIP3 levels. Our studies demonstrate that the baseline level of PIP3, and likely the dependence of cells on steady-state activity of the PI-3K signaling pathway for growth and survival, influence cell susceptibility to the toxic effects of Cdt. Jurkat cells with known defects in both PIP3 degradative enzymes, PTEN and SHIP1, not only contain high baseline levels of PIP3, pAkt, and pGSK3ß, but also exhibit high sensitivity to Cdt. In contrast, HUT78 cells, with no known defects in this pathway, contain low levels of PIP3, pAkt, and pGSK3ß and likely minimal dependence on the PI-3K signaling pathway for growth and survival, and exhibit reduced susceptibility to Cdt. These differences in susceptibility to Cdt cannot be explained by differential toxin binding or internalization of the active subunit. Indeed, we now demonstrate that Jurkat and HUT78 cells bind toxin at comparable levels and internalize relatively equal amounts of CdtB. The relevance of these observations to the mode of action of Cdt and its potential role as a virulence factor is discussed.

Variants of Porphyromonas gingivalis lipopolysaccharide alter lipidation of autophagic protein, microtubule-associated protein 1 light chain 3, LC3.

Porphyromonas gingivalis often subverts host cell autophagic processes for its own survival. Our previous studies document the association of the cargo sorting protein, melanoregulin (MREG), with its binding partner, the autophagic protein, microtubule-associated protein 1 light chain 3 (LC3) in macrophages incubated with P. gingivalis (strain 33277). Differences in the lipid A moiety of lipopolysaccharide (LPS) affect the virulence of P. gingivalis; penta-acylated LPS1690 is a weak Toll-like receptor 4 agonist compared with Escherichia coli LPS, whereas tetra-acylated LPS1435/1449 acts as an LPS1690 antagonist. To determine how P. gingivalis LPS1690 affects autophagy we assessed LC3-dependent and MREG-dependent processes in green fluorescent protein (GFP)-LC3-expressing Saos-2 cells. LPS1690 stimulated the formation of very large LC3-positive vacuoles and MREG puncta. This LPS1690 -mediated LC3 lipidation decreased in the presence of LPS1435/1449 . When Saos-2 cells were incubated with P. gingivalis the bacteria internalized but did not traffic to GFP-LC3-positive structures. Nevertheless, increases in LC3 lipidation and MREG puncta were observed. Collectively, these results suggest that P. gingivalis internalization is not necessary for LC3 lipidation. Primary human gingival epithelial cells isolated from patients with periodontitis showed both LC3II and MREG puncta whereas cells from disease-free individuals exhibited little co-localization of these two proteins. These results suggest that the prevalence of a particular LPS moiety may modulate the degradative capacity of host cells, so influencing bacterial survival.

Flow cytometric analysis of the cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human natural killer cells.

The goal of this investigation was to determine if human natural killer (NK) cells were susceptible to the cytolytic effects of the Actinobacillus actinomycetemcomitans leukotoxin (LTX). Following treatment with LTX (0-200 ng/ml), NK cell activation by interleukin-2 (IL-2) was evaluated. LTX inhibited the IL-2-induced expression of both CD69 and the IL-2 receptor. Furthermore, the up-regulation of CD56 was also impaired. To determine whether the observed functional deficits were the result of cell death, NK cell viability was evaluated by flow cytometry. Changes in forward and side light scatter patterns consistent with cell death were observed within 60 min. Direct analysis of cell viability by measuring propidium iodide exclusion, however, indicated little change in the viability of LTX-treated NK cells. Electron microscopic analysis of NK cells exposed to LTX revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation, and condensation of nucleoplasm. However, no change in membrane integrity was initially noted. Finally, LTX caused a rapid and sustained elevation in the intracellular levels of Ca2+. These morphological and biochemical changes are consistent with the notion of programmed cell death.

Role of polyclonal cell activation in the initiation of immune complex-mediated pulmonary injury following antigen inhalation.

The lung, by virtue of its anatomic situation, provides environmental antigens with unique access to host lymphoid tissues. In order to better understand the biologic consequences of antigen inhalation, we developed in animal model in which soluble proteins are administered in aerosol form to rabbits. By labeling these proteins with fluorochrome dyes or radioactive isotopes, the uptake, distribution, and fate of such proteins can be demonstrated both morphologically and quantitatively. Prompt host-antibody responses can be demonstrated to inhaled antigen, but not to comparable amounts of ingested antigen. Repeated administrations of antigen aerosol to immune animals produced little injury; in contrast, administration of aerosols containing phytohemagglutinin or cancanavalin A (Con A), plant lectins which activate leucocytes in a polyclonal fashion, induced a diffuse interstitial pneumonitis. When immune animals inhaled antigen plus Con A, devastating pulmonary necrosis was induced, in association with localized deposits of immune complexes containing antigen, antibody and complement. Such necrotic injury healed by scarring within 4 weeks. The necrotizing injury could be prevented by either decomplementation with cobra venom factor, or through inhibition of leucocyte responsiveness to Con A by administration of cholera toxin, a cAMP agonist. These studies indicate that antigen inhalation may serve as an important means of establishing "natural" immunity to environmental agents, but also may lead to severe pulmonary injury and fibrosis where the agents inhaled act not only as antigens but as polyclonal leucocyte activators as well.

Activated human T lymphocytes exhibit reduced susceptibility to methylmercury chloride-induced apoptosis.

Mercurials have been shown to cause apoptosis in human T cells. The objective of this study was to evaluate and compare the relative susceptibility of resting versus activated T cells to methyl mercury chloride (MeHgCl)-induced cell death. Apoptosis was assessed by Hoechst 33258 and 7-AAD staining and annexin V binding. Our results show that activation of T cells by PHA, PMA, and ionomycin, or IL-2, reduces mercury-induced apoptosis by approximately 50%. We have previously shown that the underlying basis for these toxic effects involves perturbation of mitochondrial function leading to oxidative stress and the release of cytochrome c to the cytosol. Therefore, the ability of MeHgCl to alter the mitochondrial transmembrane potential (delta psi m) and to induce the generation of reactive oxygen species (ROS) was evaluated in activated T-cells. Both resting and activated cells treated with MeHgCl exhibited a decrease in delta psi m when compared to respective control cells. ROS production was elevated in resting cells following treatment with mercury; in contrast, fewer activated T cells exhibit increased levels of ROS in the presence of MeHgCl. Similarly, MeHgCl treatment resulted in the release of cytochrome c to the cytoplasm in non-activated T cells but failed to do so in the activated population. These results lead us to examine intracellular levels of bcl-2, a protein that has been shown to regulate apoptosis, presumably via its ability to associate with the mitochondrial membrane. Bcl-2 levels were found, in resting cells, to be low in the presence or absence of mercury. In comparison, activated T cells expressed elevated levels of bcl-2. The relationship between mercury-induced apoptosis in human T cells, mitochondrial dysfunction, and intracellular levels of bcl-2 are discussed.

Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species, development of mitochondrial membrane permeability transition and loss of reductive reserve.

The focus of this investigation was to examine the effects of low concentrations of organic mercuric compounds on human monocyte function and to relate these effects to apoptosis. Following exposure of monocytes to 0-5 microM MeHgCl, phagocytic function and capacity to generate a respiratory burst, following PMA activation, were determined. We found that the mercury-treated cells exhibited reduced phagocytic activity. Exposure to the same mercury concentration range, also caused a marked increase in cell death. To ascertain if monocyte death was due to apoptosis, a number of flow cytometric studies were performed. Mercury-treated cells exhibited increased Hoechst 33258 fluorescence, while maintaining their ability to exclude the vital dye 7-aminoactinomycin D. Furthermore, monocytes exhibited changes in light scatter patterns that were consistent with apoptosis; these included decreased forward light scatter and increased side scatter. The percentage of cells undergoing apoptosis was dependent upon the mercury content of the medium, regardless of whether the metal was present as methyl, ethyl or phenyl mercury. Mercury-treated cells also exhibited changes in lipid organization within the plasma membrane as evidenced by increased uptake of the fluorescent probe, merocyanine 540, and by elevated annexin V binding to phosphatidylserine. Using the fluorescent probes DiOC6(3) and rhodamine 123 we noted that within 1 h of exposure to mercury, monocytes exhibited a decrease in mitochondrial transmembrane potential (psi m). Since a decreased psi m is associated with altered mitochondrial function, the hypothesis that mercury potentiated reactive oxygen species (ROS) generation and that these species promoted apoptosis was tested. We noted that treated cells generated ROS, as evidenced by oxidation of hydroethidine and the generation of the fluorescent product, ethidium. Finally, since ROS would also lower monocyte reductive reserve, we also measured GSH levels in mercury-treated cells. Chemical measurement of GSH indicated that there was thiol depletion. We suggest that the low thiol reserve predisposes cells to ROS damage and at the same time activates death-signaling pathways.

Immunologic dysfunction in the pathogenesis of periodontal diseases.

Despite significant progress in our understanding of the pathogenesis and etiology of periodontal diseases, the nature and contribution of the immune system to this disorder remains unclear. Several studies provide evidence for either a protective or destructive rôle. These conflicting findings are difficult to reconcile, since most interpretations tend to argue for a static contributory rôle (i.e., either protective or destructive) of the immune system. Current theories on the rôle of the immune response do not address these conflicting findings as well as the contradictory observation of a detectable immune response in the face of persistent infection in these patients. In this article, we present a model, based on available data, for the contribution of the immune system to the pathogenesis of periodontal disease. This model ascribes a dynamic rôle for the immune response. As documented in other infectious diseases, it is entirely possible, for example, that a state of immunologic dysfunction may occur in the earliest stages of periodontal disease progression; this may then be followed by a period of active immune reactivity (humoral and/or cellular) that would represent either a delayed or depressed response. This model is discussed in conjunction with recent findings that several suspected periodontal pathogens are capable of producing immunosuppressive agents. Many of the apparently contradictory clinical observations concerning the host immune response to oral pathogens and its correlation (or lack of) with both the progression and severity of periodontal disease may be accounted for in this model.

Suppression of human lymphocyte responsiveness by forskolin: reversal by 12-O-tetradecanoyl phorbol 13-acetate, diacylglycerol and ionomycin.

Forskolin, a potent activator of adenylate cyclase, was examined for its ability to alter human peripheral blood lymphocyte (HPBL) activation by both mitogens and antigens. We found that forskolin, at concentrations ranging from 0.04 to 25 micrograms/ml, caused a dose-dependent inhibition of HPBL responses to mitogens (concanavalin A, phytohemagglutinin, pokeweed mitogen and Staphylococcus aureus) and to recall antigens (tetanus toxoid and streptokinase/streptodornase). Inhibition was reflected in altered DNA, RNA and protein synthesis, including immunoglobulin production, and was not due to altered cell viability. Forskolin also induced a 19-fold increase in HPBL cyclic AMP levels at the same concentrations that suppressed HPBL function. To further define the mechanism(s) by which these elevations in cyclic AMP suppressed HPBL function, we tried to reverse these inhibitory effects with several agents; ascorbic acid, carbachol and levamisole had no effect. However, the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, as well as L-alpha-1,2-dioleoyl diacylglycerol were able to completely reverse the inhibition. Furthermore, the Ca2+ ionophore, ionomycin, was also able to act synergistically with lower and less effective concentrations of 12-O-tetradecanoyl phorbol 13-acetate to reverse the inhibitory effects of forskolin. The data suggest that forskolin-induced elevations in cyclic AMP may lead to inhibition (or, more correctly, prevents the activation) of protein kinase C, presumably by inhibiting phospholipid turnover. Our studies suggest a linkage between these two opposing membrane-signal transduction systems with protein kinase C representing a pivotal point for various regulatory signals that ultimately control lymphocyte activation and function.

Suppression of human peripheral blood lymphocytes by Fusobacterium nucleatum.

Fusobacterium nucleatum has been implicated in the pathogenesis of several diseases, including urinary tract infections, bacteremia, pericarditis, otitis media, and disorders of the oral cavity such as pulpal infections, alveolar bone abscesses, and periodontal disease. In this study, we examined sonic extracts of F. nucleatum strain FDC 364 for its ability to alter human lymphocyte function. We found that the soluble cytoplasmic fraction (CF) of the sonic extract was able to cause a dose-dependent inhibition of human lymphocyte responsiveness to Con A, PHA, PWM, and the recall antigen SKSD. Suppression involved altered DNA, RNA, and protein synthesis; there was no effect on cell viability. The suppressive activity is nondialyzable and heat labile. To achieve maximal suppression in 96-hr cell cultures, the CF had to be added to cells during the first 24 hr of incubation. Inhibition was reduced when the CF was added at 48 hr, and no suppression was observed when addition was at 72 or 96 hr (along with [3H]TdR). Furthermore, cells could be protected from the suppressive effects of the CF by washing within 24 hr of exposure. Suppression did not involve nonspecific effects on thymidine utilization. Although the mechanism of action of the F. nucleatum immunosuppressive activity has not yet been determined, we can rule out a requirement for monocytes/macrophages and activation of T suppressor cells. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many diseases. The data presented in this paper suggest that local and/or systemic immunosuppression could be initiated by F. nucleatum. This immunosuppression may alter the nature and consequences of host-parasite interactions, thereby enhancing the pathogenicity of F. nucleatum itself or that of some other opportunistic organism.

Immunomodulatory effects of Bacteroides products on in vitro human lymphocyte functions.

Bacteroides spp. have been implicated in the pathogenesis of several diseases, including periodontal diseases. In this study sonic extracts of 6 Bacteroides spp. were examined for their abilities to alter human lymphocyte function. We found that soluble extracts from Bacteroides intermedius, Bacteroides endodontalis, Bacteroides asaccharolyticus, Bacteroides melaninogenicus, and to a lesser degree Bacteroides loescheii, caused dose-dependent inhibition of human lymphocyte responsiveness to both mitogens and antigens. Suppression involved altered DNA, RNA and protein synthesis as well as immunoglobulin production. In contrast, Bacteroides gingivalis did not suppress these responses; instead, it stimulated lymphocyte proliferation and enhanced immunoglobulin production. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many infections. The data presented in this paper suggest that microbial mediated immunosuppression may conceivably alter the nature and consequences of host-parasite interactions in periodontal disease.

Fusobacterium nucleatum inhibits human T-cell activation by arresting cells in the mid-G1 phase of the cell cycle.

Fusobacterium nucleatum has been implicated in the pathogenesis of several diseases, including urinary tract infections, bacteremia, pericarditis, otitis media, and disorders of the oral cavity such as pulpal infections, alveolar bone abscesses, and periodontal disease. We have previously demonstrated that sonic extracts of F. nucleatum FDC 364 were capable of inhibiting human T-cell responses to mitogens and antigens. In this study, we have further characterized this immunosuppressive protein (FIP) and initiated experiments to determine its mode of action. The purified FIP has an apparent molecular mass of 90 to 100 kDa; sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the FIP is actually composed of two subunits with molecular masses of 48 and 44 kDa. Purified FIP retained its biological activity and was capable of inhibiting mitogen-induced proliferation of human T cells. Inhibition was dose dependent, and the FIP exhibited a specific activity approximately 250-fold greater than that of the crude extract. Cell cycle analysis indicates that FIP-treated cells were prevented from exiting the G0/G1 phase of the cell cycle. However, FIP did not alter the expression of activation markers (CD69, CD25, and CD71) or interleukin-2 secretion. The latter observations suggest that the T cells did indeed become activated and had entered the G1 phase of the cell cycle. Analysis of the expression of cyclins indicates that the phase of the cell cycle that is FIP sensitive resides somewhere beyond the restriction point of cyclin D2 (early to mid-G1) but prior to that of cyclins D3 and E (mid- to late G1). Finally, analysis of the expression of the proliferating cell nuclear antigen indicates that this is the earliest detectable defect in T cells exposed to FIP. We propose that if a block in the G1 phase of the cell cycle occurs in vivo in lymphocytes, it may result in a state of local and/or systemic immunosuppression. These suppressive effects could alter the nature and consequences of host-parasite interactions, thereby enhancing the pathogenicity of F. nucleatum itself or that of some other opportunistic organisms.

Immunosuppressive properties of Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans produces a leukotoxin that kills human polymorphonuclear cells (PMNs) and monocytes but not lymphocytes. In this study, we examined A. actinomycetemcomitans leukotoxin for its ability to alter human peripheral blood lymphocyte (HPBL) responsiveness. After a 90-min exposure to the leukotoxin, all monocytes were killed and HPBL responsiveness to mitogens and antigens was significantly inhibited. The ability of the leukotoxin to inhibit HPBL responses was not surprising, since monocytes and macrophages are required for many lymphocyte functions. However, we were unable to totally restore HPBL responsiveness when adherent autologous monocytes were added back to cultures of leukotoxin-treated lymphocytes. These studies demonstrate that A. actinomycetemcomitans leukotoxin may also exert nonlethal effects directly on lymphocytes. Furthermore, impaired lymphocyte function did not appear to be the result of indirect effects of products released by dying monocytes. Although it is not clear how A. actinomycetemcomitans acts to cause disease, several investigators have proposed that impaired host defenses may play a pivotal role. Several studies have demonstrated defects in PMN, monocyte, and lymphocyte function in patients with periodontal disease. These findings, along with the data presented in this paper, support the hypothesis that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. The effects of this suppression may be to enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.

Treponema denticola immunoinhibitory protein induces irreversible G1 arrest in activated human lymphocytes.

Oral spirochetes may contribute to the pathogenesis of a number of disorders including periodontal and periradicular diseases; however, the mechanism (s) by which these organisms act to cause disease is unknown. We have previously shown that extracts of the oral spirochete, Treponema denticola, contain an immunosuppressive protein (Sip) which impairs human lymphocyte proliferation. The objective of this study was to determine the mechanism by which Sip alters the proliferative response of lymphocytes. Human T-cells were activated by PHA in the presence or absence of Sip and cell cycle progression was assessed by flow cytometry. Cell cycle distribution was based upon DNA, RNA and protein content as well as expression of the activation markers; CD69 and IL-2R. Seventy-two hours following activation with PHA, cells were found in the G0, G1, S and G2/M phases of the cell cycle. In contrast, pretreatment with Sip resulted in a significant reduction of cells in the S and G2/M phases and a concomitant increase in the G1 phase. Sip did not alter the expression of the early activation markers CD69 and CD25R. To determine if G1 arrest resulted in activation of the checkpoint and cell death, we also monitored Sip-treated cells for apoptosis. Indeed, treatment with Sip resulted in both DNA fragmentation and caspase activation after 96 h. Our results indicate that Sip induces G1 arrest in human T-cells and, furthermore, that the arrest is irreversible, culminating in activation of the apoptotic cascade. We propose that if cell cycle arrest occurs in vivo, it may result in local and/or systemic immunosuppression and thereby enhance the pathogenicity of spirochetes and/or that of other opportunistic organisms.

Immune suppression induced by Actinobacillus actinomycetemcomitans: effects on immunoglobulin production by human B cells.

Actinobacillus actinomycetemcomitans produces an immunosuppressive factor (ISF) which has been shown to suppress mitogen- and antigen-induced DNA, RNA, and protein synthesis in human T lymphocytes. In this study, we examined purified A. actinomycetemcomitans ISF for its ability to alter immunoglobulin production by human B cells. The ISF caused a dose-dependent inhibition of pokeweed mitogen (PWM)-induced immunoglobulin G (IgG) and IgM production. Preexposure to ISF was not required to achieve maximal inhibition of immunoglobulin synthesis, as previously observed for its effect on T-cell activation. Nevertheless, the ISF appeared to act by irreversibly affecting the early stages of cell activation. While PWM-induced immunoglobulin production is under the influence of T-regulatory circuits, it appears that the ISF interacts directly with B cells. First, ISF failed to alter either the synthesis of interleukin-2 (IL-2) or the expression of IL-2 receptors on T cells. Second, experiments in which individual purified populations of cells were exposed to ISF, washed, and placed back into tissue culture indicated that when all cells (i.e., T cells, B cells, and monocytes) were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was observed only when the B-cell-enriched population was exposed to ISF. These results in conjunction with our earlier findings suggest that the ISF functions via the activation of a regulatory subpopulation of B lymphocytes, which in turn either directly or indirectly (via suppressor T cells) downregulate both B- and T-cell responsiveness. Furthermore, it is hypothesized that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. This suppression may enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.

Immunosuppressive effects of Prevotella intermedia on in vitro human lymphocyte activation.

In this study, we have assessed four strains of Prevotella intermedia, isolated from periodontally involved lesions, for their ability to inhibit lymphocyte functions. All four strains were found to cause a dose-dependent inhibition of B- and T-cell proliferation in response to mitogens and antigens. This was reflected in altered DNA, RNA, and protein syntheses. Furthermore, P. intermedia appeared to affect the early stages of cell activation. This was ascertained by kinetic analysis in which it was determined that the extract had to be present during the first 24 h of incubation to cause suppression. Moreover, direct assessment of the early stages of cell activation indicated that release of cytokines and expression of the interleukin 2 receptor and CD69 on T cells were inhibited by P. intermedia sonic extracts. Finally, preliminary characterization of the immunosuppressive agent indicates that it has a molecular mass of approximately 50 kDa and is heat labile. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many infections. The data presented in this paper suggest that microbially mediated immunosuppression may contribute to the pathogenesis of periodontal disease by altering the nature and consequences of host-parasite interactions.

Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent.

There is growing evidence that heavy metals, in general, and mercurial compounds, in particular, are toxic to the human immune system. In this regard, we have previously shown that both inorganic and organic mercurials are potent human T-cell apoptogens; moreover, mitochondria appear to be a target organelle for the induction of cell death. To ascertain whether both mercury species utilize the same molecular pathway to trigger the apoptotic cascade, cells were treated with MeHgCl or HgCl2 and mitochondrial activity was examined. We show that both mercury species affect mitochondrial activity by inducing the development of a membrane permeability transition. This state is characterized by a decline in both the transmembrane potential and the intracellular pH, as well as the generation of reactive oxygen species. We also determined that mercury exposure results in a decline in the T-cell GSH content. Since mitochondrial dysfunction and the development of a permeability transition may result in the release of cytochrome c, a factor that promotes apoptosis, we assessed the abilities of both species of mercury to induce the translocation of cytochrome c from mitochondria to the cytosol. We noted that MeHgCl caused a significant increase in cytosolic cytochrome c. Surprisingly, however, HgCl2 did not alter the level of cytosolic cytochrome c. We next determined whether the mercurials could alter the level of the anti-apoptotic protein Bcl-2. Our results demonstrate that HgCl2 induces a significant elevation in the Bcl-2 content of T-cells; in contrast, T-cells treated with MeHgCl did not exhibit altered levels of this anti-apoptotic protein. Regardless of whether cytochrome c is released from the mitochondria, both mercurial species were capable of activating the caspase cascade, as evident by cleavage of poly (ADP-ribose) polymerase. Thus, our study shows that, whereas each of the mercury species shares common features in the apoptotic process, profound differences exist in a number of key steps in the pathway. The significance of these differences is discussed.

Induction of human T cells that coexpress CD4 and CD8 by an immunomodulatory protein produced by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a gram-negative, capnophilic bacterium, is associated with several human diseases and is the suspected etiologic agent in certain forms of periodontal disease. We have previously shown that this organism produces an immunosuppressive factor (ISF) which is capable of inhibiting both T- and B-cell activation. Furthermore, these effects appear to be associated with the activation of a population of suppressor cells. We now report that the ISF induces a unique population of CD4+CD8+ dual-positive T-cells. By utilizing multiparameter flow cytometric analysis, we were able to detect the presence of dual-positive cells in cultures of human T-cells treated with PHA and ISF. The cells appeared within 48 hr and their induction was dependent upon the presence of both CD4 and CD8 cells in the culture. Dual expression of CD4 and CD8 was stable in that the cells continued to express both surface proteins after being sorted and cultured for an additional 24 hr. Phenotypic analysis indicates that these cells are also CD3+, CD2+, CD5+, TCR alpha beta+, CD45RA+ (and RO+), and CD29+. The dual-positive cells express surface markers associated with T-cell activation: CD25+, CD69+, CD71+, and HLA-DR+. In contrast, the cells were negative for CD34, CD57, CD56, and CD16. Cell cycle analysis indicates that > 80% of the dual-positive cells were in the S phase. Finally, functional analysis of these cells indicates that they are capable of suppressing the proliferative response of autologous T-cells to PHA.

Suppression of fibroblast proliferation by oral spirochetes.

Soluble sonic extracts of several strains of Treponema denticola and Treponema vincentii were examined for their abilities to alter proliferation of both murine and human fibroblasts. We found that sonic extracts of all tested strains of T. denticola caused a dose-dependent inhibition of murine and human fibroblast proliferation when assessed by both DNA synthesis ([3H]thymidine incorporation) and direct cell counts. T. vincentii had only a minimal inhibitory effect at comparable doses. No inhibition was observed when sonic extracts were added simultaneously with [3H]thymidine, indicating that suppression was not due to the presence of excessive amounts of cold thymidine in the extract, nonspecific effects on thymidine utilization by the cells (transport and incorporation), or degradation of label. RNA ([3H]uridine incorporation) and protein ([3H]leucine incorporation) synthesis were similarly altered after exposure to the T. denticola sonic extracts. There was no effect on cell viability as measured by trypan blue exclusion. Inhibition could be reversed by extensive washing of the cells within the first few hours of exposure to sonic extracts. Preliminary characterization and purification indicated that the inhibitory factor(s) is not endotoxin since it is heat labile, and elutes in a single, well-defined peak on a Sephadex G-150 chromatography column corresponding to a molecular weight of approximately 50,000. Since oral spirochetes have been implicated in the pathogenesis of periodontal disorders, it is possible that they contribute to the disease process by inhibition of fibroblast growth and therefore may, at least in part, account for the loss of collagen seen in diseased tissue.

Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis.