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.     

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.     

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.     

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.     

Antilipid a monoclonal antibody HA-1A: immune complex clearance of endotoxin reduces TNF-alpha, IL-1 beta and IL-6 production

When the projecting point of saphenous nerve in second somatosensory cortex (S II) of cat was stimulated, the evoked potentials elicited by C-fiber inputs of saphenous nerve recorded in the primary somatosensory cortex (C-CEP) might be either inhibited or facilited according to whether the superficial and/or the deeper layer of the cortex was stimulated. The inhibition was expressed as a decrease of amplitude and prolongation of latency of C-CEP; while the facilitation, as an increase of amplitude and duration of C-CEP. When the superfaicial layer of S II was stimulated by weaker current, both inhibitory and facilitatory effects could be observed, but only inhibitory effect was observed, when the deep layer was stimulated. With the same intensity of stimulation, inhibitory effect was more pronounced when the deep layer rather than the superficial layer was stimulated. It is suggested that S II may play a role in the modulation of C-CEP of S I.     

In vitro IL-1 beta and TNF-alpha release from THP-1 monocytes in response to metal ions

OBJECTIVES: Previous studies have shown that biomaterials can activate macrophages to produce cytokines and promote an inflammatory response. Although the toxicity of many metal ions has been extensively investigated, little is known about the ability of these ions to alter cytokine release from macrophages. Yet the release of these ions from biomaterials has been well documented. Previous studies indicated that alterations in cytokine release might be expected because metal ions alter protein production in macrophages at sub-toxic concentrations. Thus, the hypothesis of this study was that metal ions can alter the secretion of cytokines from macrophages at sub-toxic concentrations. METHODS: The release of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from macrophages was investigated when the macrophages were exposed to metal ions, with or without lipopolysaccharide (LPS), a component of dental plaque. Human THP-1 macrophages were exposed to ions of Ag, Au, Cu, Hg, and Ni for 24 h. In half of the cultures, LPS was added for the last 4 h. The release of IL-1 beta and TNF-alpha into the medium was measured using enzyme-linked immunosorbent assays. ANOVA and Tukey multiple comparison intervals were used to compare the various experimental conditions. RESULTS: None of the metal ions elevated the IL-1 beta or TNF-alpha levels after 24 h, but Ni ions significantly elevated the IL-1 beta and TNF-alpha levels after 72 h. With LPS added, Ag, Cu, and Ni significantly amplified the LPS-induced production of IL-1 beta but only Ni amplified the TNF-alpha response. These alterations in cytokine response occurred with metal ion concentrations which have been previously shown to be released from dental alloys in vitro and in vivo. SIGNIFICANCE: It appeared plausible that macrophage-cytokine mediated inflammatory responses may be altered by the presence of some metal ions in tissues, particularly Ni.     

Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae

OBJECTIVE: To demonstrate the use of aggregated, locally collected birth notification data to examine trends in birth-weight specific survival for singleton and multiple births. DESIGN: Retrospective analysis of 171,527 notified births and subsequent infant survival data derived from computerised community child health records. Validation of data completeness and quality was undertaken by comparison with birth and death registration records for the same period. SETTING: Notifications of births in 1989-1991 to residents of the North Thames (East) Region (formerly North East Thames Regional Health Authority). OUTCOME MEASURES: Birthweight specific stillbirth, neonatal, and postneonatal death rates. RESULTS: There was close correspondence between the notification and registration data. For 96% of the registered deaths a birth notification record was identified and for the majority of these the death was already known to the Community Child Health Computer. Completeness of birth-weight data, particularly at the lower end of the range, was substantially better in birth notification data. Comparison with the most recent published national data relating to birthweight specific survival of very low birthweight singleton and multiple births suggests that the downward trend of mortality is continuing, at least in this Region. CONCLUSIONS: The use of routinely collected aggregated birth notification data provides a valuable adjunct to existing sources of information about perinatal and infant survival, as well as other information regarding process and outcome of maternity services. Such data are required for comparative audit and may be more complete than that obtained from registration or hospital generated data.     

Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from Actinobacillus actinomycetemcomitans were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degreesC and heat shocked at 43 degreesC were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 microgram/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.     

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)     

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.     

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.     

Nitric oxide prevents IL-1beta and IFN-gamma-inducing factor (IL-18) release from macrophages by inhibiting caspase-1 (IL-1beta-converting enzyme)

Procytokine processing by caspase-1 is required for the maturation and release of IL-1beta and IFN-gamma-inducing factor (IGIF) (or IL-18) from activated macrophages (Mphi). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1beta-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1beta, but not TNF-alpha, in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine. Stimulated peritoneal Mphi from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1beta if exposed to N(G)-monomethyl-L-arginine, whereas Mphi from iNOS knockout mice (iNOS-/-) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1beta and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1beta and IFN-gamma than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1beta and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1beta and IGIF release.     

Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis

We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue.     

Effect of liposomal and free bisphosphonates on the IL-1 beta, IL-6 and TNF alpha secretion from RAW 264 cells in vitro

PURPOSE: In order to evaluate the possible antiinflammatory action of bisphosphonates, the effect of the drugs on the secretion of proinflammatory cytokines (IL-1 beta, IL-6 and TNF alpha) from macrophages was studied. Liposomes or high concentration of extracellular calcium was used to enhance the intracellular delivery of bisphosphonates. METHODS: RAW 264 cells were used as macrophage model, and they were induced with lipopolysaccharide to produce the cytokines. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. RESULTS: As a free drug, clodronate and pamidronate, but not etidronate, inhibited LPS-stimulated secretion of the cytokines from macrophage-like RAW 264 cells. Low concentrations of pamidronate, however, induced the IL-6 secretion, and the cytokine inhibitory action at the higher concentrations of pamidronate was attributed to cytotoxicity of the compound. The cytokine induction or toxic effects were not observed with clodronate or etidronate. When the drugs were encapsulated in negatively charged unilamellar liposomes, the inhibitory potency of both clodronate and etidronate enhanced by a factor of 10-20, while that of pamidronate was not increased. The complex formation of bisphosphonates with extracellular calcium, although enhancing the uptake of the compounds by macrophages, did not considerably increase their cytokine inhibitory potency. CONCLUSIONS: Bisphosphonates have inhibitory action on cytokine secretion by macrophages. The non-cytotoxic cytokine inhibition by liposome encapsulated clodronate could be beneficial in local inflammatory diseases, where the inflammation is sustained by the excessive amounts of inflammatory cytokines produced by activated macrophages.     

IL-2, but not IL-4 and other cytokines, induces phosphorylation of a 98-kDa protein associated with SHP-2, phosphatidylinositol 3'-kinase, and Grb2

Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.