Extracellular 37-kDa antigenic protein from Actinobacillus actinomycetemcomitans induces TNF-alpha, IL-1 beta, and IL-6 in murine macrophages

The extracellular antigens of Actinobacillus actinomycetemcomitans Y4 (serotype b) contain a 37-kDa protein which is a major target for IgGs from patients suffering from severe alveolar bone loss. Since the 37-kDa protein has not been studied sufficiently, our investigation focused on its characteristics, e.g., its localization, specificity, and whether it directly stimulates macrophages to produce cytokines. The 37-kDa protein was purified from the culture supernatant of the Y4 strain by means of chromatofocusing and gel filtration. The 37-kDa protein is a unique glycoprotein which forms immune complexes with monoclonal antibodies against rhamnose-fucose polysaccharide. Patients with A. actinomycetemcomitans-associated periodontitis had higher antibody titers to the purified 37-kDa protein than healthy subjects (p < 0.001). Anti-37-kDa protein antibodies recognized a 37-kDa band in the cytosolic, ribosomal, and total membrane fractions from Y4 cells. Extracellular substances from other strains of A. actinomycetemcomitans (serotypes a and c) also reacted in the Western blots, but Haemophilus spp. or several periodontopathic bacteria did not. These results suggested that the 37-kDa protein is a cytosolic protein that is passed through the cell membrane, and its protein portion is specific for A. actinomycetemcomitans but common to serotypes. This protein induced Il-1 beta, Il-6, and TNF-alpha release from murine macrophages. The Il-6-inducing activity of the 37-kDa protein was higher than that of LPS. These findings suggested that the 37-kDa protein which is released from live cells plays a role in A. actinomycetemcomitans-associated periodontitis, as antigen inducing the release of inflammatory cytokines which are associated with alveolar bone loss.     

IL-1beta and TNF-alpha, but not IFN-alpha, IFN-gamma, IL-6 or IL-8, are secretory mediators in human distal colon

The immunostimulatory effect of intragastrically or parenterally administered beta-(1-->3; 1-->4) glucan, extracted from oats (ObetaG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of ObetaG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non-treated control group. The administration of ObetaG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)-specific immunoglobulins as compared with the non-treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with ObetaG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post-infection the oocyst discharge was significantly diminished (P<0.05-0.01) in the ObetaG-treated groups, except in those treated 6 days after infection, as compared to the non-treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P<0.05) when ObetaG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, ObetaG appeared to up-regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.     

Anti-lipid A monoclonal antibody centoxin (HA-1A) binds to a wide variety of hydrophobic ligands

This note describes the binding specificities of four lipid A monoclonal antibodies (MAbs) including Centoxin (HA-1A); these MAbs display similar binding properties. MAbs reacted with lipid A and heat-killed smooth bacteria, whereas no reactivity was observed with smooth lipopolysaccharide (LPS). Immunoblotting of bacterial extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the MAbs bound to many polypeptide bands including the molecular weight markers. Denaturation of bovine serum albumin (BSA) by boiling or dithiothreitol treatment unmasked antibody epitopes. In addition, binding both to a hydrophobic aliphatic C12 chain covalently coupled to BSA and to single-stranded DNA was observed. The polyreactivity of these clones is most likely mediated by a preferential reactivity with hydrophobic molecular patches.     

Nutritional assessment of children on haemodialysis: value of IGF-I, TNF-alpha and IL-1beta

BACKGROUND: Protein-energy malnutrition (PEM) is associated with increased morbidity and mortality in haemodialysis (HD) patients. Insulin-like growth factor I (IGF-I) has proved to be a sensitive marker of malnutrition, while interleukin-1 (IL-1beta) and tumour necrosis factor (TNF) have been found to be raised in catabolic states. METHODS: We have investigated the nutritional status of 17 chronic renal failure (CRF) paediatric patients (8 boys, 9 girls) on maintenance HD. Eight predialysis CRF children (5 boys and 3 girls; mean creatinine 5.1+/-3.2 mg/dl) and 10 healthy children served as control groups. PEM was defined according to anthropometric measurements (triceps skinfold thickness (TST), mid-arm circumference (MAC), and mid-arm muscle circumference (MAMC)). These were correlated with serum IGF-I, IL-1, TNF-alpha, transferrin, and albumin (all sampled before the HD session). RESULTS: In the HD group, TST was reduced in 41.2% of the patients, whereas MAC and MAMC were reduced in 82.4 and 76.5% respectively. TST was depleted in only one of the predialysis CRF children. The degree of reduction in MAC and MAMC were 62.5 and 62.5% respectively. Median serum IGF-I level was decreased in both HD and predialysis CRF patients (205.1 interquartile range (IQR) 194.4 microg/l and 258.8 IQR 155.0 microg/l respectively) compared to the healthy children (418.0 IQR 310.5 microg/l) (P=0.0009 and P=0.01 respectively). Within the HD group, IGF-I levels were lower in patients with malnutrition defined according to TST (145.0 IQR 125.5 microg/l) compared to children with normal TST (301.2 IQR 218.8 microg/l) (P=0.05). IGF-I levels of the HD patients with malnutrition according to TST was also lower than the predialysis CRF patients and healthy children (P=0.04 and P=0.002 respectively). Serum IL-1beta was undetectable in all groups. Median serum TNF-alpha levels were higher in HD and predialysis CRF patients compared to healthy children, albeit statistically insignificant. There was no correlation between TNF-alpha, transferrin or albumin and anthropometric parameters. CONCLUSIONS: Our results support the high prevalence of malnutrition in CRF children, which becomes more pronounced when treatment by HD is initiated. We suggest that determination of IGF-I levels in childhood HD patients in conjunction with anthropometric measurements is useful for identification of malnutrition. We have not been able to demonstrate the catabolic effects of cytokines on this state of protein energy malnutrition.     

Protection against lethal endotoxemia by anti-lipid A murine monoclonal antibodies: comparison of efficacy with that of human anti-lipid A monoclonal antibody HA-1A

The protective capacities of murine anti-lipid A monoclonal antibodies (MAbs) 8-2 and 26-20 were examined and compared with those of the human MAb HA-1A with respect to inhibition of lipopolysaccharide (LPS) priming of human polymorphonuclear leukocytes (PMNL) in vitro and protection against lethal endotoxemia in mice. HA-1A did not prevent the priming effect of either rough or smooth LPS, while MAb 26-20 effectively inhibited LPS priming of human PMNL. Also, both murine MAbs protected mice against an otherwise lethal challenge with rough Re LPS of S. minnesota R595 as well as with smooth LPS of E. coli O111:B4. HA-1A exerted no protection against the lethal effects of Re LPS in this in vivo model. The enhanced survival in mice by treatment with MAbs 8-2 and 26-20 was associated with decreased levels of LPS-induced tumor necrosis factor. Neutralization of lipid A as a mechanism of protection was strongly suggested by efficacious inhibition of LPS priming of human PMNL by MAbs 8-2 and 26-20 in vitro.     

Inhibition of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion but not IL-6 from activated human peripheral blood monocytes by a new synthetic demethylpodophyllotoxin derivative

A newly synthesized demethylpodophyllotoxin derivative, 4-O-butanoyl-4'-demethylpodophyllotoxin (BDPT) or BN58705, has recently been shown to exert a potent cytotoxic activity in vitro against a variety of drug-resistant human tumor cell lines. The effect of this agent on effector cells of the immune system, however, has not been examined. The present study investigated the effect of BDPT on the response of activated human peripheral blood derived monocytes (PBM) to secrete cytokines. Activation of PBM overnight with LPS, IFN-gamma, or PMA resulted in secretion into the supernatant of TNF-alpha, IL-1 beta, IL-6, and IL-8 as assessed by ELISA. The addition of BDPT to the stimulated cultures resulted in significant inhibition of TNF-alpha and IL-1 beta secretion, whereas the secretion of IL-6 and IL-8 was not affected. The selective inhibition of TNF-alpha and IL-1 beta secretion by BDPT-treated PBM was observed with all three stimuli tested. The inhibitory effect mediated by BDPT was concentration dependent and was optimal at 6-20 microM. Time kinetic analysis indicated that the inhibition of secretion was rapid and detected as soon as 2 hr following stimulation of the PBM and lasted for as long as 24 hr. A comparison was made between BDPT and pentoxyfilline, a xanthine-derived phosphodisterase inhibitor that was reported to inhibit TNF-alpha and IL-1 beta secretion by PBM. Both BDPT and PTX showed similar time kinetics and patterns of inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-6, IFN-gamma and TNF-alpha production by liver-associated T cells and acute liver injury in rats administered concanavalin A

The relationship between the development of acute hepatitis and the production of TNF-alpha IFN-gamma and IL-6 by liver-associated T lymphocytes following intravenous injection of concanavalin A (Con A) was studied in rats. Following a single injection of Con A, there was a dose and time-dependent correlation in the serum levels of serum alanine aminotransferase (ALT), IL-6, IFN-gamma and TNF-alpha. These increases correlated with an increase in the numbers of CD4+, CD8+ and CD25+ T cells in blood and CD4+ and CD25+ T cells in the liver perfusate, but not with CD8+ T cells in liver perfusate. Increased levels of IL-6, IFN-gamma and TNF-alpha were constitutively produced by liver-associated CD4+ T cells when cultured. In Con A-stimulated cultures, liver-associated CD4+ T cells secreted increasing levels of TNF-alpha in a time-dependent manner following Con A injection, but TNF-alpha production by peripheral blood lymphocytes was transient with peak levels detected at 1 h which then declined over 24 h. Histological examination of the liver revealed fatty change, hepatocyte degeneration and necrosis, with an associated cell infiltrate of neutrophils and CD4+ T cells both in the portal areas and around the central veins. These results support the hypothesis that Con A-induced liver damage is mediated by CD4+ T cells acting within the liver, at least in part through the secretion of TNF-alpha, IFN-gamma and IL-6.     

In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus

Cytokines are known to play a role in T-cell lymphopoiesis as potent growth or differentiation factors, but many experiments focusing on their role in the thymus have been conducted only in vitro. We have thus used frozen sections obtained from fetal thymuses of normal C57BL 6 mice to investigate by immunohistochemistry the presence of interleukin-1 beta (I4-1 beta), IL-2. IL-4. IL-6. interferon-7 (IFN-7) and tumour necrosis facor-alpha (TNF-alpha). The results reveal that apart from IL-2, which was not detected, all these cytokines display a time-dependent expression pattern in the normal fetal thymus. First, production of IL-4, IL-6 and TNF-alpha is detected around days 13 14; this is followed by a second wave on days 16 17, with a production of IL-1 beta, IL-4 and IL-6, and finally, just before birth (day 19), by a third wave of IL-1 beta, IL-4, IL-6, IFN-7 and TNF-alpha production. This supports the hypothesis that cytokines play a rote in T-cell lymphopoiesis.     

TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages

Macrophages are present in inflammatory tissue sites where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of macrophages to participate in such matrix destruction, we studied the effects of three cytokines present in inflammatory tissue sites, TNF-alpha, IL-1beta, and IFN-gamma, on the production of three matrix-degrading metalloproteinases, interstitial collagenase, stromelysin, and 92-kDa gelatinase, as well as their natural inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases number 1), by human monocyte-derived macrophages differentiated in vitro. Spontaneous production of interstitial collagenase and stromelysin by these cells was minimal, and was not influenced by the cytokines. In contrast, the cells secreted substantial basal amounts of 92-kDa gelatinase, the secretion of which was stimulated (2- to 15-fold; on average 5-fold) by both TNF-alpha and IL-1beta, while the production of TIMP-1 was unaffected. IFN-gamma suppressed the production of the 92-kDa gelatinase induced by TNF-alpha- and IL-1beta. TNF-alpha and IL-1beta regulated the expression of 92-kDa gelatinase by monocyte-derived macrophages at the pretranslational level. The results show that expression of 92-kDa gelatinase, but not its natural inhibitor TIMP-1, by human tissue-type macrophages is selectively up-regulated by proinflammatory cytokines; which suggests that these cells, when actually present in an inflammatory environment, will actively participate in the destruction of the extracellular matrix.     

Seminal cytokine concentrations (IL-1beta, IL-2, IL-6, sR IL-2, sR IL-6), semen parameters and blood hormonal status in male infertility

Several lines of evidence indicate that cyokines are involved in male fertility. They are secreted by different parts of the male genital tract and may exert effects on steroidogenesis, spermatogenesis and sperm functions. We measured the concentrations of interleukins (IL-beta, IL-2, IL-6) and those of interleukin soluble receptors (sR IL-2, SR IL-6) in semen of fertile subjects (n = 21) and of patients with a range of andrological diseases (n = 119). The seminal concentrations of cytokines were analysed according to semen parameters as well as to the blood hormonal profiles of follicle stimulating hormone, luteinizing hormone and testosterone. An increase of IL-1beta was observed in the group of patients with infertility. No difference was found between the different subgroups defined on the basis of progressive motility, percentage of abnormal forms and diagnosis of infection. The seminal cytokine concentrations were independent of the blood hormonal status. Our data suggest that the determination of interleukins (-1beta, -2 and -6) or interleukin soluble receptors (sR IL-2, sR IL-6) in human spermatozoa does not provide convenient information in male routine infertility work-up.     

The role of IL-4, IL-10, and TNF-alpha in the immune suppression induced by ultraviolet radiation

The two main catalytic residues Cys25 and His159 of the monomeric cysteine protease papain are located on different walls of a cleft formed by two domains. This topology suggests a possible relationship between relative domain organization and catalytic mechanism. The effect on enzymatic parameters of structural modifications at various locations of the two-domain interface of papain was examined by individual or double replacements by Ala of pairs of interacting residues. Most modifications had no effect on enzyme activity. However, the enzyme's substrate turnover (kcat) decreased following simultaneous alteration of the two most conserved residues, forming an apolar contact located 15 A away from the active site. The pH activity profile of the double mutant was unchanged, indicating a conserved ionization state of the active site thiolate-imidazolium ion pair. This state is strongly dependent on the distance separating the two residues, thus suggesting that the active site geometry has not been significantly altered. Efficient enzymatic activity in papain requires more than a correct active site geometry and is influenced by domain packing properties in a region remote from the active site.     

IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-alpha, IFN-gamma, and IL-12 production

IL-13 is a potent down-modulator of macrophage proinflammatory activity in vitro, similar in this context to the anti-inflammatory cytokines IL-4 and IL-10. Since IL-10 effectively confers protection to mice from LPS-induced lethal endotoxemia through inhibition of proinflammatory cytokine production, we investigated whether IL-13 may also be capable of providing protection in this experimental model of endotoxic shock. A single injection of recombinant murine IL-13 (rmIL-13; 0.5-10 microg) significantly increased survival in a dose-dependent manner when a lethal i.p. injection of endotoxin was administered to BALB/c mice. This effect appeared to be IL-13 specific, since survival was not affected in mice that received heat-inactivated rmIL-13. rmIL-13 provided significant protection to mice even when given 30 min after LPS injection; however, this protection decreased in a time-dependent manner as the administration of rmIL-13 was delayed by 1, 2, and 5 h following LPS injection. The protective effect of IL-13 was correlated with significant decreases in the production of the inflammatory mediators TNF-alpha, IFN-gamma, and IL-12 as well as a decrease in the anti-inflammatory mediator IL-10. Our data suggest that IL-13 provides protection from LPS-induced lethal endotoxemia in a manner that is similar to but independent from that of IL-10, and therefore can be added to the list of cytokine immunomodulators that might be beneficial in the treatment of septic shock.