Lipopolysaccharide signaling induces serum amyloid A (SAA) synthesis in human hepatocytes in vitro

To investigate the role of lipopolysaccharide (LPS) in hepatocyte activation, we examined the expression of Toll-like receptor 4 (TLR4), the putative receptor for LPS in human hepatocytes. TLR4 mRNA and protein expression was confirmed in human hepatocytes. Stimulation of human hepatocytes with LPS results in rapid degradation of IkappaB-alpha and mitogen activated protein kinase activation. Human hepatocytes stimulated by LPS produced serum amyloid A protein. Our data suggest that human hepatocytes utilize components of TLR4 signal transduction pathways in response to LPS and these direct LPS-mediated effects on hepatocytes may contribute to liver inflammation and injury.     

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.     

Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6.

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.     

A novel zinc finger protein interacts with receptor-interacting protein (RIP) and inhibits tumor necrosis factor (TNF)- and IL1-induced NF-kappa B activation

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

The effects of pentoxifylline on lipopolysaccharide (LPS) fever, plasma interleukin 6 (IL 6), and tumor necrosis factor (TNF) in the rat

The purpose of these studies was to test whether pentoxifylline, a drug that can inhibit the production and action of cytokines hypothesized to be endogenous pyrogens (for example, interleukin 1 and tumor necrosis factor [TNF]), is antipyretic. We also tested the effects of pentoxifylline on plasma activities of interleukin 6 (IL 6) and TNF in response to an injection of a fever-inducing dose of lipopolysaccharide (LPS). Our results showed that a high dose of pentoxifylline (200 mg/kg) caused hypothermia in control rats and blocked LPS fever, while a low dose (50 mg/kg) did not have these effects. Injection of the high dose of pentoxifylline in control rats caused a rise in plasma IL 6 but not in plasma TNF. However, the peak levels of plasma IL 6 and TNF activities following an injection of LPS were significantly reduced by pretreatment with pentoxifylline. Overall, the data are consistent with the hypothesis that pentoxifylline is an antipyretic drug, which may act at least in part by inhibiting the secretion of pyrogenic cytokines.     

Interleukin -1beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor (TNF) in plasma and pleural fluid of pneumonia, lung cancer and tuberculous pleuritis

Interleukins IL-1beta, IL-6 and TNF are increased in plasma of patients with severe infections and septic shock. Our objective was the evaluation of IL-1beta, IL-6 and TNF in plasma and exudates of pleural fluid and their contribution to the diagnosis. We studied 44 patients, 27 men and 17 women with mean age 66.81 +/- 11.75 years; 16 with pneumonia and parapneumonic effusion, 14 with primary lung cancer and pleural effusion and 14 with tuberculous pleuritis. We measured IL-1beta, IL-6 and TNF in serum and pleural fluid with ELISA. In patients with pneumonia and parapneumonic effusion the mean value of IL-1beta IL-6 and TNF in plasma was 9.05, 19.24 and 21.34 pg/ml and in pleural fluid 10.34, 32.19 and 25.30 pg/ml. In patients with lung cancer the mean values of IL-1beta, IL-6 and TNF were 5.33, 11.74 and 11.51 pg/ml and 6.70, 13.13, 20.89 pg/ml, respectively. In those with tuberculous pleuritis the respective mean values were 10.33, 49.94, 21.27 pg/ml and 14, 56.59, 23.58 pg/ml. In conclusion, IL-1beta and IL-6 were found increased in plasma and tuberculous pleural fluid, indicating an inflammatory status.     

Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-1-beta, tumor necrosis factor, or IFN-gamma

The capacity of IL-1-beta, TNF, and IFN-gamma to stimulate platelet-activating factor (PAF) synthesis by human monocytes is examined in our report. All three cytokines induced PAF synthesis in a novel biphasic pattern with peaks of PAF synthesis 1 to 2 and 6 to 8 h after stimulation of the monocytes. In contrast, calcium ionophore A23187 elicited a single peak of early PAF synthesis. PAF in the early peak was largely retained intracellularly whereas PAF in the late peak was largely released into culture fluids. Combinations of cytokines were subadditive or antagonistic in inducing PAF synthesis. Cycloheximide inhibited the late peak of PAF synthesis indicating that protein synthesis is required for synthesis of the phospholipid PAF. Specific antibodies to TNF or IL-1-beta inhibited the late peak of PAF synthesis induced by IFN-gamma indicating that late PAF synthesis is dependent on cytokine synthesis. The quantities of PAF produced by cytokine-activated monocytes are sufficient to activate human monocytes. Thus, these studies suggest that PAF may mediate in part monocyte activation by cytokines.     

IL-6 plays a critical role in the synergistic induction of human serum amyloid A (SAA) gene when stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time quantitative RT-PCR assay system

Serum amyloid A (SAA) is known to be a precursor of amyloid A (AA) protein in AA (secondary) amyloidosis and SAA1 to be mainly involved in AA amyloidosis. We established an SAA isoform real-time quantitative RT-PCR assay and found that beta-2 microglobulin is more stable as an internal control than GAPDH and beta-actin for our system. Either IL-6 and IL-1beta or IL-6 and TNFalpha, but not IL-1beta and TNFalpha, induced the synergistic induction of SAA1 and SAA2 genes. Anti-IL-6 receptor monoclonal antibody completely inhibited the synergistic induction of SAA1 and SAA2 during triple stimulation with IL-6, IL-1beta, and TNFalpha, but, IL-1 receptor antagonist or anti-TNFalpha monoclonal antibody was only partially inhibited in HepG2, Hep3B, and PLC/PRF/5 cells. Although the SAA1 promoter has no STAT3 consensus sequence, the JAK2 inhibitor-AG490 reduced SAA1 gene expression to 30%, suggesting the involvement of STAT3. We were able to demonstrate that IL-6 plays a critical role in the synergistic induction of human SAA gene when stimulated with proinflammatory cytokines.     

Role of interleukin-6 in regulating synthesis of C-reactive protein and serum amyloid A in human hepatoma cell lines

Bovine erythrocyte acetylcholinesterase, a glycosylinositol phospholipid anchored membrane enzyme, was digested with phosphatidylinositol-specific phospholipase C and the released glycerol-containing moieties were identified and quantitated. About 96% of the total was alkylacylglycerol, of which sn-1-stearyl-2-stearoylglycerol, sn-1-stearyl-2-oleoylglycerol and sn-1-oleyl-2-stearoylglycerol accounted for 69%, 13% and 10%, respectively. These alkylacylglycerols are in marked contrast to the exclusively diacylglycerol species present in phosphatidylinositol from bovine erythrocyte membranes. This difference suggests that assembly of the membrane anchor of Ebo AChE involves a selected cellular pool of diradylglycerols.     

In vitro secretion profiles of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and TNF alpha after selective infection with Escherichia coli in human fetal membranes

Background  

Chorioamniotic membranes infection is a pathologic condition in which an abnormal secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the present study was to evaluate the functional response of human chorioamniotic membranes, as well as the individual contribution of the amnion and choriodecidua after stimulation with Escherichia coli, a pathogen associated with preterm labor.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Kinetics of c-reactive protein (CRP) and serum amyloid A protein (SAA) in patients with community-acquired pneumonia (CAP), as presented with biologic half-life times

Context: In management of community-acquired pneumonia (CAP), excellent biomarkers for inflammation would be helpful in our practice.

Objectives: Kinetics of c-reactive protein (CRP) and serum amyloid A (SAA) was characterized, using their biologic half-life times.

Materials and methods: Time course of CRP and SAA levels in the successfully treated 36 CAP patients were investigated and their half-life times were determined and compared.

Results & Discussions: SAA and CRP declined in an exponential mean and the biologic half-life times of SAA levels was 34.9?±?28.7?h, significantly shorter than that of CRP, 46.4?±?21.7?h (p?=?0.0014). Conclusion: The kinetic evidence, presented as biologic half-life times of CRP and SAA, helps us make a clinical assessment of CAP patients.     

The human C-reactive protein gene (CRP) and serum amyloid P component gene (APCS) are located on the proximal long arm of chromosome 1

The genes encoding two pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are located on the proximal long arm of human chromosome 1. Mapping of the CRP and SAP genes between the centromere and band q32 was achieved by Southern blot analysis of DNA from a panel of human × Chinese hamster somatic cell hybrids carrying defined fragments of human chromosome 1. Both genes were localized more precisely between bands q12 and q23 by in situ hybridization to human metaphase chromosomes.     

Herbal melanin modulates tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) production.

Recent studies have indicated that cytokines can enhance immunogenicity and promote tumor regression. However, the means for modulating cytokine production are not yet fully investigated. In this study we report the effects of a herbal melanin, extracted from Nigella sativa L., on the production of three cytokines [tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF)], by human monocytes, total peripheral blood mononuclear cells (PBMC) and THP-1 cell line. Cells were treated with variable concentrations of melanin and the expression of TNF-alpha, IL-6 and VEGF mRNA in cell lysates and secretion of proteins in the supernatants were detected by RT-PCR and ELISA. Melanin induced TNF-alpha, IL-6 and VEGF mRNA expression by the monocytes, PBMC and THP-1 cell line. On the protein level, melanin significantly induced TNF-alpha and IL-6 protein production and inhibited VEGF production by monocytes and PBMC. In the THP-1 cell line melanin induced production of all three cytokine proteins. These observations raise the prospects of using N. sativa L. melanin for treatment of diseases associated with imbalanced cytokine production and for enhancing cancer and other immunotherapies.     

Genome-wide associated loci influencing interleukin (IL)-10, IL-1Ra, and IL-6 levels in African Americans

Interleukins (ILs) are key mediators of the immune response and inflammatory process. Plasma levels of IL-10, IL-1Ra, and IL-6 are associated with metabolic conditions, show large inter-individual variations, and are under strong genetic control. Therefore, elucidation of the genetic variants that influence levels of these ILs provides useful insights into mechanisms of immune response and pathogenesis of diseases. We conducted a genome-wide association study (GWAS) of IL-10, IL-1Ra, and IL-6 levels in 707 non-diabetic African Americans using 5,396,780 imputed and directly genotyped single nucleotide polymorphisms (SNPs) with adjustment for gender, age, and body mass index. IL-10 levels showed genome-wide significant associations (p < 5 × 10(-8)) with eight SNPs, the most significant of which was rs5743185 in the PMS1 gene (p = 2.30 × 10(-10)). We tested replication of SNPs that showed genome-wide significance in 425 non-diabetic individuals from West Africa, and successfully replicated rs17365948 in the YWHAZ gene (p = 0.02). IL-1Ra levels showed suggestive associations with two SNPs in the ASB3 gene (p = 2.55 × 10(-7)), ten SNPs in the IL-1 gene family (IL1F5, IL1F8, IL1F10, and IL1Ra, p = 1.04 × 10(-6) to 1.75 × 10(-6)), and 23 SNPs near the IL1A gene (p = 1.22 × 10(-6) to 1.63 × 10(-6)). We also successfully replicated rs4251961 (p = 0.009); this SNP was reported to be associated with IL-1Ra levels in a candidate gene study of Europeans. IL-6 levels showed genome-wide significant association with one SNP (RP11-314E23.1; chr6:133397598; p = 8.63 × 10(-9)). To our knowledge, this is the first GWAS on IL-10, IL-1Ra, and IL-6 levels. Follow-up of these findings may provide valuable insight into the pathobiology of IL actions and dysregulations in inflammation and human diseases.