Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease.

Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.     

Production of an interleukin-1 inhibitor by cell line P388D1 murine macrophages stimulated with Haemophilus actinomycetemcomitans lipopolysaccharide.

Murine macrophages of the P388D1 cell line stimulated with lipopolysaccharide (LPS) from Haemophilus actinomycetemcomitans Y4 released an interleukin-1 (IL-1) inhibitor, as well as IL-1. Maximal IL-1 activity in culture supernatants was detected after 24 h of culture. On the other hand, IL-1 inhibitor activity reached a maximum level after 72 h of culture. An IL-1 inhibitor was partially purified from the culture supernatant of P388D1 cells stimulated with Y4 LPS for 72 h by ammonium sulfate precipitation, followed by Sephacryl S-200 gel chromatography. A 160-kilodalton peak inhibitory to IL-1 and a 14-kilodalton peak showing IL-1 activity were separated by Sephacryl S-200 column chromatography. The partially purified IL-1 inhibitor significantly suppressed the proliferation of C3H/HeJ murine thymocytes that had been induced with murine and human IL-1 in the presence of a submitogenic dose of concanavalin A. The IL-1 inhibitor more strongly suppressed human recombinant IL-1 beta than human recombinant IL-1 alpha. This inhibitory activity of the partially purified preparation was unaffected by the presence of trypsin inhibitor and the protease inhibitor aprotinin. The IL-1 inhibitor did not exhibit either IL-2 or IL-2 inhibitor activity. The inhibitor suppressed C3H/HeJ thymocyte proliferation induced by IL-1 in the presence of a saturated concentration of IL-2 instead of a suboptimal concentration of concanavalin A. These results indicate that prolonged culture of Y4 LPS-stimulated murine macrophages releases a specific inhibitor of IL-1.     

Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes.     

Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.     

Detection of antigens with affinity for host cell membrane polypeptides in culture supernatants of Trypanosoma cruzi.

Parasite antigens which bind to host cell molecules of approximately 32 and 34 kilodaltons (kDa) were identified in supernatant fluids obtained from axenic cultures of Trypanosoma cruzi. These parasite components were first detected in culture supernatants obtained after 2 weeks in culture. Immunoblot analysis of culture supernatants exhibiting binding activity revealed the presence of several parasite antigens ranging in molecular mass from approximately 26 to 290 kDa. Gel filtration (Sephacryl S-300) analysis of culture supernatants revealed four major peaks, but only the highest-molecular-mass peak (containing several parasite antigens ranging from 27 to 250 kDa) possessed binding activity for the host cell molecules.     

Cloning of the Gene Encoding the Actinobacillus actinomycetemcomitans Serotype b OmpA-Like Outer Membrane Protein

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.     

Restoration of the lipopolysaccharide-responsive phenotype in C3H/HeJ peritoneal macrophage-P388D1 cell hybrids

The fusion of thioglycollate-elicited peritoneal macrophages from the lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mouse strain to a hypoxanthine phosphoribosyltransferase (HPRT)-negative variant of the murine macrophage cell line P388D1 has resulted in the derivation of eight hybrid clones following HAT selection. Propidium-Iodide staining followed by flow cytometry has demonstrated that the DNA content of the hybrids represents the sum of the parents. Codominant expression of class I antigens from both parental haplotypes is observed in the hybrids. While class II antigens are inducible following a 72-hr induction with gamma interferon-containing supernatants, the amount of each haplotype varies between clones. These hybrids demonstrate Fc-mediated erythrophagocytosis in contrast to P388D1. In distinction to the C3H/HeJ primary peritoneal-macrophage parent, LPS treatment of the hybrids results in the increased release of both interleukin-1 (IL-1) and cachectin/tumor necrosis factor (TNF) into culture supernatants. Therefore, cell fusion has resulted in the stable restoration of the LPS-responsive phenotype in C3H/HeJ macrophage hybrids. These macrophage hybrids should serve as useful models in understanding the regulation of macrophage effector functions in response to environmental stimuli.     

Suppression of murine macrophage interleukin-1 release by the polysaccharide portion of Haemophilus actinomycetemcomitans lipopolysaccharide.

Lipopolysaccharide (LPS) was extracted from whole cells of Haemophilus actinomycetemcomitans Y4 by the hot phenol-water procedure. LPS was cleaved into its lipid A and polysaccharide moieties by hydrolysis in 1% acetic acid. The major component sugars of the polysaccharide were glucose, heptose, rhamnose, galactose, and fucose. LPS and lipid A from H. actinomycetemcomitans induced the release of interleukin-1 (IL-1) by LPS-responsive C3H/HeN murine peritoneal macrophages and cell line macrophages (P388D1 and J744.1), but not by LPS-nonresponsive C3H/HeJ peritoneal macrophages. The polysaccharide was unable to induce the release of IL-1. It suppressed the IL-1 release from LPS- and lipid A-stimulated macrophages, but not the production of cell-associated and intracellular IL-1. The addition of rhamnose, a sugar component of the polysaccharide, abrogated the inhibitory effect of the polysaccharide on IL-1 release. These results suggest the participation of a lectinlike molecule in IL-1 release.     

Enhancement of in vitro lipopolysaccharide-stimulated interleukin-1 production by levamisole

The production of interleukin-1 (IL-1) by the P388D1 mouse macrophage cell line and by adherent peritoneal exudate cells (PMs) was examined. In vitro IL-1 production by P388D1 cells treated with lipopolysaccharide (LPS) was enhanced by coculture with levamisole (0.1 to 10 microM). Oral administration of levamisole (3 mg/kg) to mice also resulted in potentiation of in vitro IL-1 production by thioglycollate-elicited peritoneal macrophages in response to in vitro LPS stimulation. Potentiation was approximately twofold. IL-1 production in the absence of LPS by either the P388D1 cells or the PMs was nil, and levamisole did not directly stimulate IL-1 production in these cases. IL-1 activity in the culture supernatants was measured by thymocyte comitogenic assays. The immunochemical identify of the thymocyte comitogenic activity as IL-1 alpha was confirmed by neutralization with a specific goat anti-mouse IL-1 alpha antiserum. These results suggest that one mechanism by which levamisole acts to normalize and restore immune responses may be enhancing the signals which enable activated macrophages to secrete IL-1.     

Xid mouse lymphocytes respond to TI-2 antigens when co-stimulated by TI-1 antigens or lymphokines

Spleen cells from male (CBA/N x DBA/2) F1 hybrid mice do not significantly respond to in vitro stimulation by trinitrophenyl-conjugated polyacrylamide beads (TNP-PAA), whereas the same antigen elicits high PFC responses in female F1 hybrid cells. Therefore, this antigen could be classified as a T-independent type 2 (TI-2) antigen. When male spleen cells were co-stimulated by TNP-PAA and TI type 1 antigen, either LPS or Brucella abortus, they produced vigorous anti-TNP responses. A similar increase of the in vitro responsiveness of male F1 hybrid spleen cells to TNP-PAA antigen was provoked by the addition of supernatants from P 388-D1 cells stimulated by muramyl-dipeptide (MDP) mainly containing interleukin-1 (IL-1) or supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated EL-4 cells that contained T-cell factors. The PFC response to another TI-2 antigen, TNP-Ficoll, was also significantly enhanced after co-stimulation by P 388-D1 supernatants. The response to TI-2 antigens being macrophage dependent, the influence of supernatants of peritoneal macrophages from male and female F1 hybrids incubated with TNP-Ficoll on the PFC response of normal DBA/2 mouse spleen cells to sheep erythrocytes was assessed. It was found that macrophage supernatants from female hybrids regularly increased by more than two times this anti-SRBC PFC response, whereas macrophage supernatants from male F1 hybrids did not. Moreover, in a specific proliferation test measuring IL-1 activity, when macrophage supernatants from female F1 produced a 13-fold increase of thymidine incorporation, supernatants from male F1 only produced a three-fold increase. It is concluded that, in addition to the known defects of B cells from Xid mice, their macrophages are also defective.     

Hypersensitivity pneumonitis: whole Micropolyspora faeni or antigens thereof stimulate the release of proinflammatory cytokines from macrophages

Hypersensitivity pneumonitis (HP) is an allergic granulomatous interstitial lung disease resulting from a reaction of selected individuals to repeated inhalations of certain antigens. HP is characterized by chronic inflammation, and the development of the disease seems to be immunologically mediated. In farmer's lung, the source of provoking antigen has been found to be actinomycetes such as Micropolyspora faeni. In this study, we show that M. faeni, or antigens thereof, stimulate strong release of proinflammatory cytokines from blood monocytes and alveolar macrophages obtained from nonfarmer volunteers and naive mouse peritoneal macrophages. Interleukin-1 (IL-1) was produced by human alveolar macrophages and murine peritoneal macrophages in response to whole M. faeni and antigens thereof. IL-1 activity was detected in the supernatants at 12 h of incubation and was maximal by 24 to 36 h (200 to 400 U/ml of IL-1). A rabbit antiserum to IL-1 alpha and IL-1 beta neutralized the thymocyte-stimulating activity of the supernatants. Moreover, M. faeni (1 to 100 micrograms of antigen) elicited a strong secretion of tumor necrosis factor-alpha (TNF-alpha) from human alveolar macrophages and monocytes as well as mouse peritoneal macrophages, where 1 micrograms of M. faeni elicited the secretion of approximately 100 U of TNF-alpha from 2 x 10(5) macrophages, and 100 micrograms stimulated the release of approximately 1,000 U of bioactive TNF-alpha. One particle of whole M. faeni per cell was sufficient to induce copious release of TNF-alpha from macrophages or monocytes (100 U of bioactive TNF-alpha; 1,000 pg/ml of antigenic TNF-alpha as seen by radioimmunoassay). Both IL-1 and TNF-alpha productions stimulated by M. faeni were not abrogated by inclusion of polymyxin B. We propose that the direct stimulation of cytokines by M. faeni or antigens thereof may play an important role in HP.     

Factors in AMLR culture supernatant which mediate the cytotoxic T-cell response to hapten-altered self: interaction with macrophages

Supernatants from autologous mixed lymphocyte reaction (AMLR) cultures mediated the cytotoxic T-cell response in a system containing T cells and mitomycin C-treated, TNP-modified syngeneic thymocytes. No cytotoxic activity developed in the absence of the AMLR supernatants. The removal of thymic adherent cells abrogated the effect of the AMLR supernatants in the generation of cytotoxic cells. AMLR helper activity was restored following the addition of small numbers of splenic adherent cells. These results led to speculation that the AMLR supernatant interacted with accessory cells. Examination of the supernatant revealed significant levels of colony stimulating factor (CSF), and interferon-gamma (IFN-gamma). Functionally, incubation of the AMLR supernatant with P388D1, a macrophage tumor line, resulted in the production of interleukin 1 (IL-1). CSF is a major inducer of IL-1 synthesis. In addition, the AMLR supernatant induced the expression of Ia antigens on the P388D1 cells. IFN-gamma has been reported to mediate this activity. Although both lymphokines were present and appeared to have an effect on macrophages, it was unknown if both were required for cytotoxic T-cell development in this system. Samples of AMLR supernatant were incubated for 10 min at various temperatures. The ability of the AMLR supernatants to mediate cytotoxic T-cell activation was sensitive to incubation at 80 or 90 degrees C. Interestingly, at these temperatures CSF activity in the bone marrow colony-forming cell assay was enhanced. In separate experiments, AMLR culture supernatant was dialyzed against a pH 2 buffer. The induction of IA antigens by these supernatants was sensitive to dialysis at this pH. The similarity with IFN-gamma provides further evidence that this activity was mediated by IFN-gamma in the AMLR supernatants. The ability of the AMLR supernatants to mediate the cytotoxic T-cell response to altered self was also pH 2 sensitive. In contrast, IL-1 inducing capability and bone marrow colony growth, both CSF activities, were not reduced by pH 2 dialysis. Taken together, these data demonstrate a primary role for an IFN-like molecule, present in the AMLR supernatant, on cytotoxic T-cell activation. CSF involvement in this response is suggested by its IL-1 inducing activity, but cannot be definitively proven in the present study.     

Ia antigen expression and IL-1 activity in murine tumour-associated macrophages.

Tumour-associated macrophages (TAM) isolated from five murine sarcomas had a relatively high frequency of I-A+ cells, with mean values of 27% (mFS6), 52% (MN/MCA1), 68% (N3), 62% (N4) and 98% (J3) for TAM compared to 12% for resident peritoneal macrophages. Expression of I-E in TAM was also high (29%) in the only sarcoma (N4) examined in this respect. Expression of I-A by TAM declined in culture but exposure to lymphokine supernatants maintained and increased the frequency of I-A+ cells in TAM. Transplantation of tumours into nude mice caused a marked decrease in the percentage of I-A+ TAM in the case of the N4 sarcoma (8% compared to 48%), whereas for the MN/MCA1 sarcoma the diminution was only marginal (from 53 to 41%), TAM from murine sarcomas did not constitutively release appreciable levels of interleukin-1 (IL-1) activity. Upon stimulation with bacterial lipopolysaccharides or silica, TAM showed a limited capacity to produce and release IL-1 activity compared to peritoneal macrophages. Thus the expression of I-A antigens and the IL-1-producing capacity are uncoupled in TAM from murine sarcomas. These properties of TAM could play an important role in the generation of anti-tumour immunity and/or of suppressive T-cell circuits.     

Hemolysin-Positive Enteroaggregative and Cell-Detaching Escherichia coli Strains Cause Oncosis of Human Monocyte-Derived Macrophages and Apoptosis of Murine J774 Cells

Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetaching Escherichia coli (EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1β (IL-1β) into culture supernatants. The mode of cell death differed in the two types of macrophages. Damage to macrophage plasma membrane integrity without changes in nuclear morphology resulted in cytolysis of HMDM. This mechanism of cell death has been previously described for virulent Shigella infection of HMDM and is termed oncosis. In contrast, infection of J774 cells by EAggEC and CDEC strains resulted in apoptosis. The presence of α-hemolysin (Hly) in EAggEC and CDEC strains appears to be critical for both oncosis in HMDM and apoptosis in J774 cells. Bacteria lacking Hly, including Hly⁻ EAggEC strains as well as enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli strains, behaved like avirulent Shigella flexneri in that the macrophage monolayers were intact, with no release of lactate dehydrogenase activity or IL-1β into the culture supernatants.     

Interleukin-1 induces mucus secretion from mouse intestinal explants

Secretion of goblet cell (GC) mucus occurs during immune reactions in the gut. As human macrophages produce a substance that induces mucus secretion from lung explants, we tested the effect of macrophage-derived factor(s) on mucus secretion from intestinal explants. Fragments of mouse duodenum were incubated with macrophage culture supernatants and purified interleukin-1 (IL-1) preparations, and the amount of mucus released was estimated by an enzyme-linked lectin assay. Both the culture supernatants and the IL-1 preparations induced dose- and time-dependent mucus release. Lipopolysaccharide-induced culture fluids were shown to contain IL-1. Thus, stimulation of mucus secretion from GC can be added to the list of biological activities attributable to IL-1.     

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.     

Impaired production and lack of secretion of interleukin 1 by human breast milk macrophages.

Interleukin 1 (IL-1) production by human breast milk macrophages (HMMø) in response to bacterial lipopolysaccharides (LPS) was investigated in 49 healthy mothers and compared with that of blood monocytes (HMo). IL-1 activity in 24 h HMMø culture supernatants was in most cases below the assay detection limits and much smaller than that of stimulated HMo (72 +/- 17 u/ml). Intracellular IL-1 activity in response to LPS in HMMø raised from less than 2 +/- 1 u/ml to 19 +/- 12 u/ml and was similar to that found in stimulated HMo (16 +/- 4 u/ml). Neither the low IL-1 production by HMMø nor its release into supernatants could be increased by stimulating with higher LPS concentrations (40-400 micrograms/ml) or when longer culture times were assayed (24-72 h). Inhibiting the production of prostaglandins by adding indomethacin to HMMø cultures, caused no effect either on IL-1 production or on its secretion to supernatants. The presence of inhibitors for the IL-1 thymocyte proliferative assay, in supernatants from HMMø, was excluded by mixing experiments with a known amount of IL-1. Thus, we conclude that HMMø produce four or five times less IL-1 than HMo in response to LPS stimulus. Furthermore, HMMø are completely unable to release the IL-1 produced.     

Actinobacillus actinomycetemcomitans Y4 capsular polysaccharide induces IL-1beta mRNA expression through the JNK pathway in differentiated THP-1 cells

Capsular polysaccharide from Actinobacillus actinomycetemcomitans Y4 (Y4 CP) induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures, as reported in previous studies. We also found that Y4 CP inhibits the release of interleukin (IL)-6 and IL-8 from human gingival fibroblast (HGF). Thus Y4 CP induces various responses in localized tissue and leads to the secretion of several cytokines. However, the effects of Y4 CP on human monocytes/macrophages are still unclear. In this study, THP-1 cells, which are a human monocytic cell line, were stimulated with Y4 CP, and we measured gene expression in inflammatory cytokine and signal transduction pathways. IL-1beta and tumour necrosis factor (TNF)-alpha mRNA were induced from Y4 CP-treated THP-1 cells. IL-1beta mRNA expression was increased according to the dose of Y4 CP, and in a time-dependent manner. IL-1beta mRNA expression induced by Y4 CP (100 microg/ml) was approximately 7- to 10-fold greater than that in the control by real-time PCR analysis. Furthermore, neither PD98059, a specific inhibitor of extracellular signal-regulated kinase nor SB203580, a specific inhibitor of p38 kinase prevented the IL-1beta expression induced by Y4 CP. However, JNK Inhibitor II, a specific inhibitor of c-Jun N-terminal kinase (JNK) prevented the IL-1beta mRNA expression induced by Y4 CP in a concentration-dependent manner. These results indicate that Y4 CP-mediated JNK pathways play an important role in the regulation of IL-1beta mRNA. Therefore, Y4 CP-transduced signals for IL-1beta induction in the antibacterial action of macrophages may provide a therapeutic strategy for periodontitis.     

Regulatory Effects of Cytokine Production in Atopic Allergic Reaction by Gammi-Danguieumja

Gammi-danguieumja (GD) is clinically used in South Korea for treating atopic dermatitis. However, its effects in experimental models remain unknown. We investigated a possible effect of GD on cytokines production using human T cell line (MOLT-4) or human mast cell line. As a result, GD (0.01 mg/mL)-containing medium in stimulated culture supernatants increased IL-2 and IFN-γ, and decreased IL-4 secretion in MOLT-4. GD (0.01–1 mg/mL)-containing medium in stimulated culture supernatants dose-dependently and significantly decreased IL-8, IL-13, and tumor necrosis factor-alpha secretion on the phorbol 12-myristate 13-acetate and A23187-stimulated HMC-1. In addition, GD inhibited histamine release from activated mast cells. These results suggest that GD contributes to the regulation of atopic allergic reactions.     

Release of a chemotactic monokine upon treatment with lymphocyte supernatants

Macrophages produced and/or released a chemoattractant(s) for neutrophils after having been treated for 2 hr with soluble products from two lymphoma cell lines. Culture supernatants of the EL-4 and Yac-1 cell lines, but not sarcoma l, Bc100, or the macrophagelike cell line P388d1, triggered the release of the chemoattractant. The macrophage-derived chemoattractant (MDC) was detectable within 2 hr following triggering and culture supernatants had maximal activity by 48 hr. The triggering of the macrophages to release the chemoattractant and the activity of the chemoattractant was not dependent upon any component of fetal bovine serum. Activation of complement was also not involved, since activated serum did not competitively inhibit the chemotactic activity of the macrophage-derived chemoattractant. The chemoattractant was macromolecular, stable to heating at 90 degrees C for 15 min, sensitive to pronase and chymotrypsin, and was affected by treatment with low pH.