Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells.

IL-18, a potent IFN-gamma-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-gamma-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-gamma-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-gamma-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.

Russian experience in screening, analysis, and clinical application of novel interferon inducers.

This review describes a long-standing experience of screening for interferon (IFN) inducers in Russia. IFN inducers represent a special group of potential antiviral compounds. The main requirements for them are (1) high IFN-inducing activity, (2) absence of side effects, (3) wide spectrum of antimicrobial activity, (4) broad therapeutic security and, (5) good solubility in water and biologic liquids. IFN inducers stimulate IFN production in different cells and organs, and that determines the strategy for their application. Amixin (OOO "Lancepharm," Moscow, Russia) induces IFN-alpha/beta production mostly in T cells. Cycloferon (NTFF "Polysan," St. Petersburg, Russia) stimulates B cells and macrophages to produce almost pure IFN-alpha. Double-stranded RNA (dsRNA) and polyphenols of natural origin stimulate IFN production in different populations of immunocytes. Only polymers, such as Larifan (Riga, Latvia), Kagocel ("NIARnedicplus," Moscow, Russia), and Ragosin (N.F. Gamaleya Institute, Moscow, Russia), induce IFN synthesis in muscles, so they may be effective against rabies. Cycloferon, Larifan, and Kagocel, which induce IFN formation in lungs, may be effective against influenza and rhinoviral infections. Cycloferon and Larifan stimulate IFN production in liver and spleen and may be effective against hepatitis B. Oral compounds (Amixin, Kagocel) that stimulate IFN production in intestines may be effective against hepatitis A and enteroviral infections. Low molecular weight inducers (Amixin, Cycloferon, Kagocel) that penetrate the blood-brain barrier may be active against viral encephalitis. At present, clinical trials of IFN inducers are limited, but in the near future, IFN inducers may be used against very different infections and conditions.

Efectos biológicos en células HEp-2, en la multiplicación del adenovirus tipo 6 y en la concentración de interferón en plasma humano en presencia de un RNA de transferencia fúngico / Biological effects in HEp-2 cells, adenovirus type 6 and interferon concentration in human plasma in the presence of a fungal transfer RNA

Introducción: Trabajos previos mostraron la capacidad antiviral in vitro, así como la utilidad como inductor de interferón natural (IFN-n) in vivo de un RNA de transferencia (tRNA) de origen fúngico, por este motivo se sigue estudiando esta molécula como una alternativa para el tratamiento antiviral. Objetivos: Estudiar el efecto del tRNA fúngico en la viabilidad, síntesis de DNA y en la multiplicación del adenovirus tipo 6 (AV-6) en células HEp-2, comparado su efecto con el producido por el polyl:polyC o por IFN-Ó. Valorar su capacidad como unductor a largo plazo de la síntesis de IFN-n in vivo. Material y métodos: Células HEp-2 se incubaron con diferentes concentraciones de las moléculas mencionadas durante 24 horas, y se determinó la viabilidad y la síntesis de DNA celular; adicionalmente cultivos tratados en las mismas condiciones se infectaron con 200 unidades formadoras de placas (ufp) del AV-6, se incubaron cinco días adicionales, y se valoró el grado de protección. In vivo a siete voluntarios clínicamente sanos se les administró intramuscularmente una dosis única de 100 mg del tRNA, y se determinó mediante la técnica de inhibición del efecto citopático (ECP) el nivel sérico de IFN-n, cinco días después de la inoculación. Resultados. El tRNA protegió a las células HEp-2 contra la infección por AV-6, mejor que el polyl:polyC y de forma similar al IFN-Ó. Ninguna de las tres sustancias afectó significativamente la viabilidad celular. En cambio, la síntesis de DNA sí disminuyó de manera directa en relación con la concentración de los inductores, no así con el IFN-Ó. En los plasmas de los sujetos tratados con el tRNA fúngico se encontró un aumento en la concentración de IFN-n (de 84.2 ñ 107.6 a 171.42 ñ 129.5 UI/mL) a los cinco días, aunque la diferencia no fue estadísticamente significativa. Conclusión. El tRNA fúngico mostró actividad antiviral contra el AV-6, no afectó la viabilidad pero sí la síntesis celular de DNA, y con una capacidad de mantener elevada hasta por cinco días la concentración plasmática de IFN-n en humanos

Immunohistochemical and immuno-electron-microscopic detection of interferon-gamma-inducing factor ("interleukin-18") in mouse intestinal epithelial cells.

The novel cytokine interferon-gamma-inducing factor ("interleukin-18") is produced by macrophage-like cells in mice with endotoxin shock and induces the production of interferon-gamma by T cells in vitro. To determine the physiological role for mouse interferon-gamma-inducing factor, we studied its tissue distribution in several organs (intestine, spleen, thymus, kidney, and liver) in healthy mice of different ages, including fetal stages. Activity of the cytokine in the organ extracts of adult mice was measured by enzyme-linked immunosorbent assay, and the cellular distribution of interferon-gamma-inducing factor in organs from fetal and adult mice was determined by immunohistochemistry. Intestinal extracts of adult mice showed the highest concentrations among the organs studied. Other organ extracts of adult mice showed lower concentrations of the cytokine. Immunohistochemical analysis revealed that interferon-gamma-inducing factor was localized in the cytoplasm of intestinal epithelial cells from fetal and adult mice. These results show for the first time that intestinal epithelial cells may be the main producers of interferon-gamma-inducing factor under normal physiological conditions and suggest that its constitutive expression in intestinal epithelial cells may have an important role in the induction of mucosal immunity.

A simple and sensitive bioassay for the detection of human interleukin-18/interferon-gamma-inducing factor using human myelomonocytic KG-1 cells.

Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel cytokine which plays an important role in Th1 responses. Here we describe a simple, sensitive bioassay for human IL-18 using the human myelomonocytic cell line, KG-1, which produces IFN-gamma in response to human IL-18. IFN-gamma production induced by human IL-18 was completely blocked by an antibody against human IL-18. Human IL-18 could be measured in a concentration range from approximately 100 to 10,000 pg/ml, and intra- and inter-assay coefficient variations were both below 15%. It was possible to measure human IL-18 in human serum, cell lysate or culture supernatant by this bioassay. Thus, the human IL-18 bioassay can be expected to be useful in the investigation of the relationship between human IL-18 and various diseases or in analyzing the mechanisms of human IL-18 secretion from IL-18 producing cells.

Phenotypic and genotypic (pulsed-field gel electrophoresis) characteristics of enterotoxin-A-producing Staphylococcus aureus strains.

The phenotypic (antibiotype, serotype, phagetype) and genotypic (SmaI restriction patterns using pulsed-field gel electrophoresis) characters of 162 Staphylococcus aureus epidemiologically unrelated strains were studied. Eighty-two of the isolates produced enterotoxin-A (SEA+), while 80 produced none (SEA-). None of the phenotypic characters observed were characteristic of SEA+ strains. On the other hand, the electrophoretic profiles revealed a non-random distribution of the SEA+ strains (p < 0.01 in groups PI and PIII, and p < 0.03 in group PII). It can therefore reasonably be assumed that the enterotoxin-A-producing strains did not constitute a single clone, but rather, seemed to belong to strains derived from at least three clones with distinct genetic organization.

Subcellular distribution of jacalin in Artocarpus integrifolia seed.

Seeds of Artocarpus integrifolia (jack fruit) contain large amounts of the anti-T lectin, jacalin. The mature seeds of jack fruit were homogenized in 0.25 M sucrose and separated by differential centrifugation into four fractions, viz wall, intermediate, and microsomal pellets and soluble supernatant. The lectin activity was associated with the wall pellet collected at low speed centrifugation. The other three fractions obtained by centrifugation at gradually higher speeds contained a similar lectin but of very low specific activity. The distribution pattern of jacalin remained unchanged in the presence of EDTA and/or Triton X-100 indicating that the lectin was not membrane bound. Immunofluorescent staining of jack fruit seeds showed that jacalin was localized in the cell wall in the intracellular space, which corroborated the results of fractionation studies. The possible relevance of these results to the function of lectin in the plant cell is discussed.

A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock.

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.

Endotoxin-induced serum factor that stimulates gamma interferon production.

Serum from Mycobacterium bovis BCG-infected mice that had been challenged with lipopolysaccharide (LPS) exhibited a marked ability to induce gamma interferon (IFN-gamma) in cultures of spleen cells of normal mice in the presence of interleukin-2 (IL-2). The inducing activity became detectable in the circulatory system about 90 min after LPS challenge, disappeared at around 5 h, and was observable upon 640x dilution of the serum. Addition of monoclonal anti-IL-2 receptor antibody to the culture inhibited the induction by the serum. The activity induced high levels of IFN-gamma even in nylon wool-nonadherent cells, while concanavalin A failed to do so. Serum from uninfected mice challenged with LPS contained no such activity. The molecular weight of the active substance, estimated by gel filtration, was about 70,000. There were pronounced differences among mouse strains in the activities of the sera prepared, which paralleled the amounts of IFN-gamma produced in vivo. However, the levels of IFN-gamma produced in whole spleen cells and in nylon wool-nonadherent cells from mice of various strains were the same when stimulated with competent serum. These results indicate the existence of an unidentified factor that induces IFN-gamma in cooperation with IL-2 in macrophage-depleted splenocytes. They also suggest that IFN-gamma production in vivo is not genetically controlled at the lymphocyte level but, rather, at the level of synthesis of the unknown factor.

Investigation on extracellular slime from Pseudomonas aeruginosa as interferon inducer in mice.

The investigation concerns interferon (IFN) production in the sera and spleens of 129/Ao/Boy mice induced with Pseudomonas aeruginosa slime extract. Interferon was present in the serum as early as 2 h after i.v. and i.p. injection of the immunogenic dose of the slime - 100 micrograms/mouse. Likewise, in the spleen the same dose induced interferon production at the second hour after its administration. In the spleen interferon was synthetized longer, even up to 7 days. On the other hand, it disappeared from the serum after 24 h. In vitro investigations on interferon induction in peritoneal cells and spleen revealed that after slime extract stimulation, only non-adherent cells are capable of IFN production; while adherent cells are not. Interferon synthesis in peritoneal cells in vitro was much enchanced if for the experiments, cells isolated 2 - 4 h after i.v. administration of mice with Ps. aeruginosa slime extract, were used. Besides, the stimulatory effect of the extract on interferon production was well marked in the Newcastle virus-induced peritoneal cells. For comparison, interferon obtained after induction with slime extract in vivo (in the serum) and in vitro (in peritoneal cells) was tested for its properties. The interferons although both acidstable, displayed significant differences. IFN obtained in vitro from peritoneal cells culture appeared thermolabile and susceptible for neutralization with gamma-globulin of rabbit serum against interferon from Newcastle virus-induced L929 cells (anti-MuIFN alpha/beta). IFN from serum was thermostabile, undergoing only slight neutralization with anti-MuIFN alpha/beta globulin.

[Physicochemical and biological properties of double-stranded RNA--an interferon inducer]. / Fiziko-khimicheskie i biologicheskie svoistva dvuspiral'noi RNK--induktora interferona.

Physico-chemical and biological properties of an interferon inducer, a natural double-stranded RNA (dsRNA) obtained from RNA-genome bacteriophage-infected E. coli are described. Three independent methods i.e. sedimentation analysis, gel chromatography, and direct measurement of electronograms, were used to determine an average length of dsRNA molecules which was found to correspond to about 432 nucleotide pairs. dsRNA melting temperature in 0.15 M NaCl is 103 degrees C, and the resistance to RNase A is above 85%. Additional purification of the preparation by the methods eliminating bacterial endotoxin and other possible admixtures does not decrease acute toxicity of dsRNA. The influence of dsRNA on the cytolytic activity of natural killers (NK) in normal subjects and patients with viral diseases of the liver was demonstrated. dsRNA enhances the activity of NK in normal subjects as well as in patients with acute virus hepatitis B in the abatement stage of the disease and in patients with chronic persisting hepatitis. At the peak of acute virus hepatitis B NK stimulation was detected in only 40 +/- 16% of the patients. In patients with chronic active liver diseases dsRNA enhanced NK activity in 50 +/- 22% of cases. A similar effect on NK cell activity is exerted by human leukocyte interferon.

Electrochemistry of double-stranded complexes of synthetic polyribonucleotides having interferonogenic and antiviral activity.

Double-stranded (ds) complexes of poly(C) with poly(G) and poly(G,I) were studied using differential pulse polarography (DPP) and differential pulse voltammetry at a pyrolytic graphite electrode (DPV). The complex formed by copolymer was found to be DPP inactive. On the other hand, poly(G).poly(C) yielded a small DPP peak corresponding to single-stranded (ss) poly(C). It was suggested that ss poly(C) present in the solutions of poly(G).poly(C) appeared due to the existence of segments in poly(G) during the complex-forming process in which guanine residues were unable to be hydrogen-bonded with bases in poly(C). Polynucleotide complexes investigated in this report yielded a DPV peak corresponding to electrooxidation of guanine residues, which was markedly lower than that yielded by ss polymers. Moreover, this DPV peak yielded by the complex prepared from an equimolar mixture of poly(G) and poly(C) was still markedly higher than that yielded by poly(G,I).poly(C), or by poly(G).poly(C) prepared in the excess of poly(C). The lowering of the DPV peak was explained as being particularly due to the presence of the polynucleotide segments with an intact and regular secondary structure. The results of our electrochemical analysis of the complexes investigated were compared with their biological activity reported earlier. This comparison calls attention to the fact that biological effectiveness of these biopolymers may be dependent on details of their secondary structure which may be monitored using the methods of electrochemical analysis.

Ia antigen: a murine B lymphocyte receptor for transformed cell induction of interferon-alpha/beta.

A receptor on the surface of nonsensitized mouse spleen cells that recognizes a glycoprotein from transformed mouse L-929 cells is described. The interaction of the receptor and glycoprotein inducer results in the production of MoIFN alpha/beta. An assay was developed to assess certain biologic and physicochemical characteristics of the receptor. The receptor and glycoprotein inducer bound in a concentration-dependent manner, which tends to indicate a direct interaction between the two. The receptor was not ubiquitous; spleen cells but not normal mouse embryo cells appeared to be the source. It was specific for MoIFN alpha/beta inducers from transformed cells, but not from other MoIFN alpha/beta or gamma inducers such as NDV, LPS, PWM, or SEA. The receptor appeared to be a cell surface protein in that its activity was abolished by trypsinization of whole spleen cells. Previous studies indicated that the receptor was probably located on B cells. Gel filtration indicated that the receptor had a m.w. of 30,000 to 60,000. Because the receptor appeared to be: 1) B lymphocyte associated, 2) a surface protein, and 3) 30,000 to 60,000 daltons, a similarity to Ia antigen was suggested. This possibility was confirmed by showing binding of the receptor to an anti-IaK antibody-Sepharose affinity column. PAGE analysis of the affinity-purified receptor revealed a single protein band with a m.w. of approximately 60,000. ELISA of the above gel slices with anti-Ia antibody further confirmed the specificity of the column. A physical association of the receptor and inducer was demonstrated by showing binding of the glycoprotein inducer to a receptor (Ia antigen)-Sepharose affinity column. Furthermore, the receptor (Ia antigen) was highly purified by a glycoprotein inducer-Sepharose affinity column.

Effect of coal rank on the interferon system.

Studies on the induction of interferon by influenza virus in monkey kidney (LLC-MK2) cell monolayers pretreated with coal dust revealed that the inhibitory activity of the mineral on this process was related to coal rank. Maximal inhibition of viral interferon induction was noted with high rank coal and the degression of this activity was related to coal's position in the carboniferous series; anthracite, bituminous, subbituminous, lignite, and peat. The cytocidal activity of each rank of coal, however, was comparable in relation to the corresponding quantities of coal dust that were tested indicating that findings related to interferon production are not explicable on the basis of remaining viable cells. Coal dust, irrespective of rank, also did not affect the ability of exogenous interferon to confer antiviral cellular protection. An inverse relationship mediated by coal of different rank occurred between influenza virus growth and interferon levels which suggested that increased virus growth reflected the ability of higher rank coal to affect adversely interferon production.