Production of interleukin-1 and tumor necrosis factor by human peripheral monocytes activated by periodontal bacteria and extracted lipopolysaccharides.

Whole Gram-negative bacteria associated with juvenile and adult periodontitis, and their respective extracted lipopolysaccharides (LPS), were tested for the ability to activate quiescent human peripheral blood monocytes. All pathogenic Gram-negative bacteria and all LPS tested were able to induce the production of significant amounts of IL-1 and TNF, monokines known to induce osteoclastic bone resorption. Haemophilus segnis, which has not been associated with any form of periodontal disease, did not activate monocytes. Purified LPS from Actinobacillus actinomycetemcomitans Y4 was able to elicit IL-1 and TNF release at a threshold concentration of 1-10 ng/mL. To examine the mechanism whereby whole bacteria activated monocytes, we added polymixin B in culture with glutaraldehyde-fixed bacteria to bind LPS. This resulted in the abrogation of IL-1 and TNF production. To compare the effects of Gram-positive oral bacteria on monocytes, we also tested Staphylococcus epidermidis and the Gram-positive amphipathic equivalent of LPS, lipoteichoic acid (LTA) extracted from Staphylococcus aureus bacteria. Whereas whole Gram-positive bacteria had no stimulatory effect on monocytes, LTA induced IL-1 and TNF production at a concentration range equivalent to that of the LPS. These results indicate that monocytes are activated by free LPS or LPS bound to Gram-negative pathogenic periodontal bacteria to produce monokines which may contribute to the destruction of periodontal bone.

Induction of activated lymphocyte killing by bacteria associated with periodontal disease.

Complex interactions occur among host defense cells during bacterial infection. Bacteria and bacterial products may enhance or inhibit the effector and regulatory activity of human lymphocytes. Accordingly, we tested the ability of human periodontal pathogens to activate peripheral blood lymphocytes using standard chromium-release assays to measure lymphocyte-mediated cytolysis. Human adherent-cell depleted peripheral blood lymphocytes (PBL) with the addition of glutaraldehyde-fixed bacteria at a 5:1 bacteria:lymphocyte ratio were incubated at 37 degrees C for 24 hr in RPMI 1640 medium. Six of eight bacteria tested significantly augmented lymphocyte killing of the natural killer (NK) cell-sensitive human erythroleukemia cell line K562. E. corrodens, representing activating bacteria, was also able to induce the killing of NK-resistant targets (M14, Raji), comparable with induction by interleukin-2. Lipopolysaccharides extracted from A. actinomycetemcomitans strains, when incubated with PBL, were able to enhance cytotoxicity without the presence of whole bacteria. A majority of cytotoxicity was mediated by NK cells bearing Leu-11 and NKH-1 markers.

HSV-1-infected oral epithelial cells are targets for natural killer cells.

A mechanism of cell-mediated immunity was investigated to determine whether natural killer (NK) cells were able to lyse antigenically-altered epithelial cell targets. Using standard four-hour chromium-release assays, we tested human peripheral blood lymphocytes against autologous untreated epithelial cells and autologous HSV-1-infected epithelial cells and calculated the percentage of lysis. With adherent cell-depleted peripheral blood, only epithelial cells infected with virus were lysed (p = 0.009). Evidence that NK cells were responsible for the lysis exists because: (1) peripheral blood lymphocytes were able to lyse allogeneic as well as autologous virus-infected cells; (2) when NK cells were depleted with the lysosomotropic drug L-leucine methyl ester, cytotoxicity against infected targets was abrogated; and (3) depletion of NK cells by the monoclonal antibody Leu-11b, plus complement, also eliminated cytotoxicity against virus-infected targets. Additional evidence suggests that lysis of targets does not involve antibody-dependent cellular cytotoxicity. These findings indicate that NK cells have the potential to perform a similar in vivo immunologic role in the oral cavity and initiate cell-mediated lysis of virus-infected epithelial cells.

Oral colonization and susceptibility testing of Pseudomonas aeruginosa oral isolates from cystic fibrosis patients.

Microbial samples from the oral cavities of cystic fibrosis (C.F.) patients and 20 age-matched normal control subjects were characterized. Mucoid variant Pseudomonas aeruginosa was isolated from the tongue, buccal mucosa, and saliva of C.F. patients only. Analysis of the data suggests that the oral cavity is a potential reservoir for this organism. Aspiration and cross-contamination from this reservoir may be important in perpetuating chronic pulmonary infection in C.F. patients. Susceptibility testing was performed on 20 mucoid variant P. aeruginosa oral isolates obtained from the patients according to standardized broth dilution procedures. The in vitro antimicrobial effects of sodium fluoride, stannous fluoride, and chlorhexidine were measured. Analysis of the data suggests that clinically safe and achievable levels of chlorhexidine and stannous fluoride may be antimicrobial.

Actinobacillus actinomycetemcomitans and Bacteroides gingivalis activate human peripheral monocytes to produce interleukin-1 and tumor necrosis factor.

The effects of gram-negative bacteria clearly associated with juvenile and adult periodontitis on monokine production were assessed using standard in vitro assay techniques. Actinobacillus actinomycetemcomitans and Bacteroides gingivalis were able to activate human peripheral blood monocytes to produce significant amounts of interleukin-1 (IL-1) and tumor necrosis factor (TNF). These monokines are known to induce osteoclastic bone resorption. An oral gram-positive organism, Staphylococcus epidermidis, was able to induce only modest amounts of IL-1 and TNF, slightly above unstimulated monocyte levels.

Fusobacterium nucleatum T18 aggregates human mononuclear cells and inhibits their PHA-stimulated proliferation.

In previous studies Fusobacterium nucleatum has been shown to induce either stimulatory or inhibitory effects on human mononuclear cells. We examined the interaction of human mononuclear cells with human and cynomolgus monkey strains of F. nucleatum. Peripheral blood mononuclear cells (PBMCs) isolated from normal donors were aggregated in the presence of cells of F. nucleatum but not control bacteria. The aggregation of PBMCs and F. nucleatum T18 was inhibited by either L-arginine, L-lysine, or pretreatment of the bacterial cells with heat, but was unaffected by the presence of sugars or normal human serum. Strain T18 aggregated purified T-cells and monocytes at approximately equal concentrations. When F. nucleatum T18 was incubated with PHA-stimulated PBMCs, DNA synthesis in the PBMCs was significantly inhibited and detection of IL-2R alpha on the PBMCs was reduced. These studies indicate that F. nucleatum aggregates PBMCs, and that this interaction is associated with both an inhibition of PBMC proliferation and a decrease in IL-2 receptor expression. The ability of F. nucleatum to inhibit mononuclear cell proliferation may be significant in the pathogenesis of periodontal diseases.

Bacterial-stimulated cytokine production of peripheral mononuclear cells from patients of various periodontitis categories.

Periodontitis is a general term for disease categories, including juvenile periodontitis (JP), rapidly progressive periodontitis (RPP), and adult periodontitis (AP), which may or may not share a common etiology and pathogenesis. These disease categories are characterized by differences in progression of tissue destruction and differences in age group susceptibility, but not, to our knowledge, by differences in cytokine responses of inflammatory cells. The present study examined blood cell counts and interindividual variation in the ability of PBMC of patients in three different categories of periodontitis to produce cytokines after stimulation with different oral bacterial species in vitro. The AP group had a significantly lower production of IL-1ra when stimulated with Porphyromonas gingivalis (P.g.) and Actinobacillus actinomycetemcomitans (A.a.) (P < 0.05). Streptococcus sanguis (S.s.), which is associated with normal periodontal conditions, induced extremely high levels of IL-1 alpha and TNF alpha production in all groups. The RPP group had a significantly higher number of monocytes (MC) than the AP group (P < 0.05). Additionally, JP patients had a significantly higher concentration of polymorphonuclear granulocytes compared to juvenile controls (P < 0.05). In conclusion, IL-1 alpha, TNF alpha, or IL-6 production by peripheral blood MC after in vitro stimulation with oral bacterial type stains may not distinguish different categories of periodontitis. The results support the hypothesis that the cytokine IL-1ra is produced in different concentrations in the two groups: RPP and AP. Furthermore, elevated MC concentration in the RPP group compared to the AP group may be an important pathogenic feature in RPP.

Flow cytometric analysis of human dental pulp tissue.

Existing knowledge regarding the cellular components of the dental pulp has been derived primarily from classical methods of histology and biochemistry. Since observations made from prepared tissue sections are static, it is not clear whether this accurately reflects the cellular dynamics of living pulp tissue. Therefore, we developed a method to analyze vital human pulpal tissue by flow cytometry. To test this method, two analyses of the prepared pulpal tissue were performed. First, the prepared tissue was stained with monoclonal antibodies to detect lymphocyte subpopulations. Second, the tissue was processed for DNA analysis of individual cells. Results demonstrated that lymphocytes bearing CD4 and CD8 antigens were clearly detected in pulpal tissue by this method. No B cells were found in any sample. DNA analysis revealed two distinct cell populations. Approximately 88% were small and 12% were large. According to DNA content, 90% of all cells were noncycling and 10% were cycling. These results demonstrate the feasibility of using flow cytometric analysis to examine, at a quantitative level, the cellular heterogeneity of the human dental pulp.

The effects of benzo(a)pyrene, nicotine, and tobacco-specific N-nitrosamines on the generation of human lymphokine-activated killer cells.

The effects of four major components of snuff (fine-cut smokeless tobacco) on the development of lymphokine-activated killer cells (LAK) were measured in vitro. Of the components tested: nicotine, N'-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo(a)pyrene (BaP), only BaP suppressed LAK cytotoxicity against tumour targets and LAK DNA synthesis during 3- and 7-day incubations. BaP concentrations of 0.1-1.0 micrograms/ml suppressed lymphocyte proliferation only; there was no effect on tumour cell proliferation at these concentrations. BaP had no effect on tumour target killing when incubated during 4 h natural killer (NK) or LAK cytotoxicity assays. There was no effect on LAK binding of tumour targets after 3 days culture with BaP concentration of 0.1-1.0 micrograms/ml. These data confirm that a water-soluble extract of snuff has anti-cytolytic and anti-proliferative effects on peripheral blood lymphocytes. As NK and LAK cells are important in preventing tumourigenesis and metastasis, suppression of these cells may favour neoplastic growth associated with snuff-dipping.

Activation of human natural killer cells by lipopolysaccharide from Actinobacillus actinomycetemcomitans.

Lipopolysaccharide (LPS) from the Y4 strain of this bacterium, which is implicated in the pathogenesis of juvenile periodontitis, was incubated with human peripheral blood lymphocytes (PBL) and its action compared to that of LPS from Escherichia coli. Both LPS augmented cytotoxicity measured against natural killer (NK) cell-resistant tumour targets within 24 h of incubation. Cytotoxicity was exclusively found in NK-enriched low-density large granular lymphocyte fractions, as separated by Percoll gradient. LPS activated NK cells without stimulating high levels of proliferation. The minimum concentration of A. actinomycetemcomitans LPS required to activate NK cells was 1 microgram/ml; higher concentrations did not significantly increase this activation. LPS had no synergistic effect on the induction of PBL cytotoxicity by interleukin-2. In contrast, LPS pre-activated monocytes inhibited the induction of lymphocyte cytotoxicity by either interleukin-2 or LPS.

Inhibition of human lymphokine-activated killer activity by smokeless tobacco (snuff) extract.

Chronic snuff dipping has been associated with oral cancer in man and experimental animals. Here, the effects of a water-extract of snuff on the in-vitro development of human lymphokine-activated killer (LAK) activity were examined. The snuff extract inhibited both LAK cytotoxicity and DNA synthesis in a dose-dependent fashion at concentrations of 0.125 to 2.0 per cent; above 2.0 per cent, cell viability decreased significantly. In contrast, the snuff extract had no effect on natural killer-cell cytotoxicity when incubated with fresh peripheral blood lymphocytes in a standard 4 h assay, or on LAK cytotoxicity when incubated only during the final 4 h effector phase. Lymphocyte protein synthesis was generally unaffected by the addition of this extract. Thus, a water-soluble snuff extract appears to suppress LAK activity by inhibiting DNA synthesis. Altered LAK function in the oral mucosa might permit the development of snuff-associated carcinogenesis.

Immunohistochemical mapping of epidermal growth-factor receptors in normal human oral soft tissue.

Epidermal growth-factor receptor (EGF-r) has been identified on basilar cells of stratified squamous epithelia and skin adnexa in man. Recent studies have mapped EGF-r to various oral cells in animals; however, complete mapping of EGF-r in normal human oral mucosa has not been done. Normal tissues from eight sites in human oral mucosa were examined for their expression of EGF-r using avidin-biotin peroxidase complex with mouse anti-EGF-r monoclonal antibody. Immunoreactivity was detected in palatal gingiva, buccal gingiva, soft palate, lateral tongue, dorsal tongue and floor of the mouth. The connective tissues of the periodontal ligament and dental pulp were non-reactive. EGF is known to exist in most body fluids, particularly saliva. In normal human mucosa, EGF is localized to connective tissue subjacent to epithelium. With the receptor in the overlying epithelium, a possible epithelial-mesenchymal interaction may exist between the receptor and ligand. A paracrine mode of action may be postulated, functioning to regulate the complex biological functions of the human oral tissues.

Assessment of dental student studying strategies.

This study evaluated the studying strategies of second-year and third-year dental students as related to end-quarter exam preparation. Focus groups were convened to elicit current student studying strategies that were incorporated into a three-question survey and administered to second- and third-year students. Strategic use of study resources was rank ordered in the first question; course characteristics influencing study prioritization were rank ordered in the second question; and a third question addressed strategic time management. Overall, both second- and third-year students ranked the studying resource "notepool" first, although students of high academic standing ranked "texts and syllabi" first. Regarding course characteristics, both classes gave high ranking to "instructor expectations," "performance on midterm," and "course structure." Second-year students rated "performance on midterm" as significantly more important to prioritization than did third-year students (p = 0.01, t-test). As to time management, a statistically significant number of second-year students (p < 0.01, chi-square) ranked "studying for exams mid-quarter" first while third-year students (p = 0.05) ranked "studying the week before finals" first. Second- and third-year students of high academic standing indicated that they began studying at the beginning of the quarter. The data suggest that studying strategies change as students progress in school and that students of high and low academic ranking differ in the strategies they employ.

Bacterial activation of human natural killer cells: role of cell surface lipopolysaccharide.

Culture of human peripheral blood lymphocytes with gram-negative bacteria associated with periodontal disease caused a rapid increase in the cytotoxic potential of natural killer (NK) cells. The NK cells were activated to kill NK-resistant targets, the peak cytotoxicity occurring on day 1 of culture. The addition of anti-Tac, anti-CD3, or anti-OKT-11 antibodies to block activation via the interleukin-2 (IL-2), T-cell, or E rosette receptors had a minimal effect on this inductive process. Anti-IL-2 antiserum was effective in blocking a significant amount, but not all, of the cytotoxicity in bacterium-activated cultures. Modest IL-2 production (5 to 6 National Institutes of Health units) was measured in lymphocyte cultures activated by bacteria, but proliferation was not induced during a 1-week period. When polymixin B sulfate was added to bind and block lipopolysaccharides, bacterium-induced cytotoxicity was completely abrogated for all activating bacteria. In addition, when culture supernatants from Actinobacillus actinomycetemcomitans were tested, activation still occurred. However, again, this activation was totally inhibited by polymixin B sulfate. Monocytes were also activated by bacteria to produce tumor necrosis factor (TNF). To exclude the possibility that TNF was responsible for cytotoxicity, an antiserum to TNF was added to cocultures of bacteria and lymphocytes with adherent cells removed. The antiserum had no effect on the inductive process. In addition, exogenous TNF did not kill M14 targets. These results suggest that bacterial cell surface lipopolysaccharides provide a major activation signal for NK cells to enhance cytotoxicity.

Roles of interferon and cellular adhesion molecules in bacterial activation of human natural killer cells.

Interaction of lipopolysaccharide (LPS) from enteric and oral bacteria with natural killer (NK) cells enhanced cytotoxicity against NK-sensitive and NK-resistant targets. This activation occurred without expansion of the NK cell population or without changes in the leukocyte function-associated antigen family of cellular adhesion molecule (CAM) expression on NK cells. Significant interferon (IFN) titers were measured in LPS-lymphocyte supernatants, and antibody to IFN-alpha blocked LPS activation. LPS-induced NK cytotoxicity was inhibited by antibodies to individual alpha chains of CAM and, more profoundly, by antibody to the beta chain of CAM. However, LPS, when preincubated with NK cells, did not compete with subsequent anti-CAM antibody binding as detected by flow cytometry. Anti-CAM antibodies had no effect on NK activation by IFN, but antibodies to either CD11a or CD11c abrogated IFN production induced by LPS. These findings suggest that LPS binds NK cells at non-CAM sites, resulting in the release of IFN. IFN then acts in an autocrine manner independent of CAM to enhance NK cytotoxicity. Interaction of anti-CAM antibodies with CAM may provide a negative signal in regulating LPS-induced IFN production.

The regulatory effects of monocytes on human natural killer cells activated by lipopolysaccharides.

Lipopolysaccharides (LPS) rapidly enhance cytotoxicity of human natural killer (NK) cells against tumor targets. The regulatory effects of peripheral blood monocytes (MO) on this activation were measured. When lymphocytes were kept at a constant number in culture containing LPS from oral and enteric bacteria, increasing the percentage of MO caused a dose-dependent suppression of NK cytotoxicity. This suppression was reversed by adding the prostaglandin (PG) inhibitor indomethacin which indicates that PGE was released by MO stimulated by LPS. PGE is known to suppress NK activity by its effects on cAMP. MO separated from lymphocytes by transwell membranes also suppressed NK cells in the presence of LPS but this action was again reversed by indomethacin. This suggests that cell-to-cell contact is not necessary for MO to suppress NK cytotoxicity when stimulated by LPS. The role of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in NK suppression was studied. Antibodies to IL-1 and TNF did not alter the suppression mediated by MO on NK activity. Adding IL-1 or TNF to cell cultures without MO or LPS had no effect on NK activity after 24 h. TNF, but not IL-1, enhanced NK activity in the presence of LPS in cultures without MO. When PGE was preincubated with only lymphocytes for 2 h, the activating effects of a secondary stimulation, interleukin-2 (IL-2), were inhibited. IL-1 had no effect on IL-2 activation when pre-incubated with PBL but TNF slightly enhanced IL-2-induced NK cytotoxicity.