Induction of C-reactive protein by cytokines in human hepatoma cell lines is potentiated by caffeine.
ABSTRACT: Induction of C-reactive protein (CRP) by conditioned medium from lipopolysaccharide-stimulated human monocytes in two human hepatoma-cell lines, Hep 3B and NPLC/PRF/5, was potentiated 3-6-fold by the methylxanthine caffeine. The induction observed in the presence of conditioned medium plus caffeine was as much as 180-fold, comparable with that seen after many stimuli in vivo. This potentiation was accompanied by an increase in the levels of CRP mRNA. By contrast, no potentiating effect on CRP induction by conditioned medium was found when we tested theophylline, forskolin, 8-bromo cyclic AMP or two Ca2+ ionophores, namely ionomycin and A23187. None of the above compounds, including caffeine, when tested alone, had any detectable effect on the synthesis and secretion of CRP. Our previous study [Ganapathi, May, Schultz, Brabenec, Weinstein, Sehgal & Kushner (1988) Biochem. Biophys. Res. Commun. 157, 271-277], employing defined cytokines, had shown that induction of CRP in Hep 3B cells requires IL(interleukin)-6 plus IL-1, whereas, in the NPLC/PRF/5 cell line, IL-6 alone is effective. Caffeine similarly potentiated induction of CRP by these defined cytokine signals in these two cell lines. Changes in synthesis of other acute-phase proteins, including serum amyloid A (SAA), alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin and albumin, induced by conditioned medium or, in some cases, by IL-6 and/or IL-1 alpha, were only minimally affected by caffeine. Thus these results indicate that the mechanism by which caffeine potentiates CRP induction by cytokines appears to be independent of increases in intracellular concentrations of the two second messengers, cyclic AMP and Ca2+; the precise nature of this mechanism is unclear at the present time. Our results also indicate that the intracellular mechanisms by which cytokines regulate synthesis of CRP may differ from those regulating synthesis of some other acute-phase proteins. The differential response of CRP and SAA to caffeine is of particular interest, since induction of both of these two major acute-phase proteins can be accomplished by identical extracellular signals.
Project description:Okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, inhibited in a dose-dependent manner (5-20 nM) the induction of C-reactive protein (CRP), serum amyloid A (SAA) and fibrinogen by interleukin-6 (IL-6) plus interleukin-1 (IL-1), and of fibrinogen by IL-6 alone, in Hep 3B cells. Induction of alpha 1-proteinase inhibitor (alpha 1-PI) by IL-6 plus IL-1 or IL-6 alone was not significantly affected by OA up to concentrations of 20 nM, above which concentration OA was toxic in Hep 3B cells. OA also inhibited the induction of CRP, fibrinogen and alpha 1-PI by IL-6 in the NPLC/PRF/5 cell line, albeit at a higher concentration (80 nM). These results suggest that the signal transduction mechanisms regulating induction of acute-phase proteins by IL-6, either alone or in combination with IL-1, are mediated by activation of protein phosphatases 1 and/or 2A.
Project description:Human 'acute-phase' serum amyloid A protein (A-SAA) is a major acute-phase reactant (APR) and an apolipoprotein of high density lipoprotein 3 (HDL3). We have examined several parameters of A-SAA biosynthesis in PLC/PRF/5 hepatoma cells in response to monocyte conditioned medium (MoCM) and dual treatment with interleukin-1 beta and interleukin-6 (IL-1 beta + IL-6). Treatment of PLC/PRF/5 cells with MoCM or IL-1 beta + IL-6 caused a dramatic and rapid increase in A-SAA mRNA and protein synthesis; A-SAA mRNA was first detectable at 3 h, with peak levels reached by 24 h. A-SAA mRNA accumulation is accompanied by a gradual and homogeneous decrease in the length of the A-SAA poly(A) tail; the poly(A) tail shortening does not apparently affect the intrinsic stability of A-SAA mRNA. Analysis of RNA isolated from the ribonucleoprotein, monosome and polysome fractions of cytokine-treated PLC/PRF/5 cells showed that most A-SAA mRNA was associated with small polyribosomes, regardless of time post-stimulus, suggesting that the translational efficiency of A-SAA mRNA is constant throughout cytokine-driven induction. Moreover, the transit time of A-SAA protein out of the cell is also constant throughout the time course of induction. These data provide evidence of a paradox with regard to the transcriptional upregulation of A-SAA by IL-1 beta + IL-6 and the relative synthesis of A-SAA protein and suggest a role for post-transcriptional control of A-SAA biosynthesis during the acute phase.
Project description:Maximal induction of the acute-phase proteins C-reactive protein (CRP) and serum amyloid A (SAA) in the human hepatoma cell line Hep3B requires the combination of interleukin (IL)-6 and IL-1. In contrast, IL-1 inhibits fibrinogen induction by IL-6. To explore the possible participation of the sphingomyelin-ceramide pathway in the transduction of cytokine effects, the role of this pathway in expression of CRP, SAA and alpha-fibrinogen was investigated. The cell-permeable ceramide analogues C2 and C6 each greatly potentiated induction of both CRP and SAA mRNA by IL-6+IL-1beta but did not affect the responses of alpha-fibrinogen to IL-6 or to IL-6+IL-1beta. The combination of IL-6+IL-1beta led to increased turnover of sphingomyelin in Hep3B cells. D609, an inhibitor of ceramide production by acidic but not neutral sphingomyelinases, substantially inhibited induction of CRP and SAA by IL-6+IL-1beta. The ability of C2 and C6 to potentiate the effects of cytokines suggests that the sphingomyelin-ceramide pathway participates in induction of CRP and SAA by IL-6+IL-1beta under these experimental conditions, most likely by transducing the effects of IL-1beta. C2 and C6 were unable to substitute for IL-1beta in enhancing IL-6 effects on CRP and SAA, consistent with other reports indicating that the sphingomyelin-ceramide pathway is only a single component of multiple necessary converging pathways for induction of many genes. In contrast, this pathway does not appear to participate in mediating the inhibitory effects of IL-1beta on fibrinogen induction by IL-6.
Project description:BACKGROUND:To explore the correlations between SAA, CRP, and clinical indices of patients with acutely exacerbated chronic obstructive pulmonary disease (AECOPD). METHODS:A total of 120 patients with AECOPD and another 120 with remitted COPD were enrolled in an AECOPD group and a COPD remission group, respectively. Meanwhile, 120 healthy subjects were included as a control group. SAA, CRP, PCT, Fbg, IL-8, IL-6, TNF-?, and IP-10 levels were detected. FEV1 and FEV1 /FVC were measured. RESULTS:Compared with control group, the serum levels of SAA, CRP, PCT, Fbg, IL-8, IL-6, TNF-?, and IP-10 significantly increased in COPD remission group (P < 0.05). The levels of AECOPD group significantly exceeded those of COPD remission group (P < 0.05). The levels of AECOPD patients with different GOLD grades were significantly different (P < 0.05). AECOPD group had significantly lower FEV1 and FEV1 /FVC than those of COPD remission group (P < 0.05). The CAT score of AECOPD patients was (18.41 ± 2.55) points. The levels of SAA, CRP, PCT, Fbg, IL-8, IL-6, TNF-?, and IP-10 were negatively correlated with FEV1 and FEV1 /FVC, and positively correlated with CAT score. The area under receiver operating characteristic curve of SAA was largest (0.931). The cutoff values for SAA, CRP, PCT and Fbg were 18.68 mg/L, 14.70 mg/L, 0.39 ?g/L, 3.91 g/L, 0.46 ?g/L, 24.17 ?g/L, 7.18 mg/L, and 83.19 ng/L, respectively. CONCLUSIONS:Serum levels of SAA, CRP, PCT, Fbg, IL-8, IL-6, TNF-?, and IP-10 in AECOPD patients were elevated, which may undermine pulmonary functions. SAA can be used as an effective index for AECOPD diagnosis and treatment.
Project description:BACKGROUND:Platelet-rich fibrin (PRF) serves as a reservoir of bioactive molecules to support wound healing and bone regeneration. The beneficial action of PRF might involve macrophage polarization from proinflammatory M1 toward pro-resolving M2 phenotypes. This study aims to evaluate the effect of PRF on macrophage polarization. METHODS:Murine primary macrophages and RAW 264.7 cells were exposed to saliva and lipopolysaccharides (LPS) with and without PRF lysates obtained by repeated freeze-thawing or the secretome of PRF membranes, termed PRF conditioned medium. The expression of the M1 marker genes interleukin 1? (IL1?) and interleukin 6 (IL6) along with the M2 markers arginase-1 and chitinase-like 3 (Chil3 or YM1) were evaluated by real time polymerase chain reaction. Immunoassay and immunofluorescence staining were performed for IL6 and p65 translocation, a subunit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), respectively. RESULTS:We report here that PRF lysates and PRF conditioned medium, the latter containing the secretome, greatly decreased the proinflammatory response of primary macrophages and RAW 264.7 cells as indicated by the expression of IL1? and IL6. The anti-inflammatory activity of PRF lysates was further confirmed by IL6 immunoassay. Moreover, PRF lysates suppressed the translocation of p65 from the cytoplasm into the nucleus after incubation with saliva. In support of M2 polarization, PRF lysates and PRF conditioned medium enhanced the expression of arginase-1 and YM1 in primary macrophages. CONCLUSION:Our results indicate that PRF holds an anti-inflammatory activity and shifts the macrophage polarization from an M1 toward an M2 phenotype.
Project description:Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects.
Project description:BACKGROUND: Although elevated levels of C-reactive protein (CRP), interleukin (IL)-6, serum amyloid A protein (SAA) and total homocysteine (tHcy) have been associated with the increased likelihood of cardiovascular events, the relative or combined utility of these biomarkers in predicting atherosclerosis and death in an angiography cohort is unknown. METHODS: A cohort of 1117 consecutive patients (797 men and 320 women), referred to 2 Vancouver teaching hospitals for selective coronary angiography, was recruited between 1993 and 1995. Angiography results were obtained for 1019 patients. In 2004 we determined that of 1050 patients who could be traced, 231 had died, 95 of CAD-related causes. We compared the relative utility of baseline measurements of CRP, IL-6, SAA and tHcy as well as of lipids for predicting angiographic CAD and all-cause and CAD-related death. RESULTS: The risk of death increased across quartiles for CRP, IL-6, SAA and tHcy. When comparing the highest and lowest quartiles, the greatest hazard ratios were associated with IL-6 (2.57, 95% confidence interval [CI] 1.62-4.09) and tHcy (2.36, 95% CI 1.53-3.65). A Cox regression model containing all plasma biomarkers and traditional risk factors indicated that age, angiographic CAD and baseline plasma levels of IL-6 and tHcy remained independent predictors of CAD-related death, whereas age, sex, smoking, diabetes and apolipoprotein B levels were independent predictors of angiographic CAD. Kaplan-Meier survival curves indicated a utility in combining measures of CRP, SAA, IL-6 and tHcy for predicting risk of all-cause and CAD-related death. INTERPRETATION: A comparison of elevated levels of CRP, IL-6, SAA and tHcy with traditional CAD risk factors indicated that IL-6 and tHcy were the strongest independent biomarkers for CAD-related death. Elevated levels of multiple biomarkers were associated with an increasing rate of all-cause and CAD-related death.
Project description:BACKGROUND: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. METHODS: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. RESULTS: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. CONCLUSIONS: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.
Project description:The aim of this study was to investigate associations of adiponectin, leptin, C-reactive protein (CRP), interleukin (IL)-6, and serum amyloid A (SAA), individually or in combinations, with risk of incident type 2 diabetes in a Aboriginal Canadian [corrected] population.Of the 606 Sandy Lake Health and Diabetes Project cohort subjects who were free of diabetes at baseline, 540 (89.1%) participated in 10-year follow-up assessments. Concentrations of fasting adiponectin, leptin, CRP, IL-6, SAA, and covariates were measured at baseline. Fasting glucose and a 75-g oral glucose tolerance test were obtained at baseline and follow-up to determine incident type 2 diabetes, defined as clinically diagnosed type 2 diabetes or as fasting plasma glucose > or =7.0 mmol/l or 2-h postload plasma glucose > or =11.1 mmol/l at follow-up.Low adiponectin, high leptin, and low adiponectin-to-leptin ratio at baseline were associated with increased risk of incident type 2 diabetes after adjustment for age, sex, triglycerides, HDL cholesterol, hypertension, and impaired glucose tolerance (odds ratio 0.63 [95% CI 0.48-0.83], 1.50 [1.02-2.21], and 0.54 [0.37-0.77], respectively). When the models were additionally adjusted for waist circumference or BMI, however, only low adiponectin remained significantly associated with increased incident diabetes (0.68 [0.51-0.90]). Combinations of leptin, CRP, IL-6, and/or SAA with adiponectin, assessed using either the ratio or joint effects, did not improve diabetes prediction.Low baseline adiponectin is associated with increased risk of incident type 2 diabetes independent of leptin, CRP, IL-6, SAA, and metabolic syndrome variables including obesity.
Project description:The acute-phase response to tissue injury and inflammation is accompanied by a dramatic increase in the hepatic synthesis of plasma proteins known as acute-phase reactants (APRs). This response is mediated by cytokines produced in part by activated macrophages at the site of inflammation; glucocorticoids have also been implicated as playing a regulatory role. The effects of the cytokines interleukin (IL)-1 beta and -6, alone or in combination, and in the absence or presence of the synthetic glucocorticoid dexamethasone, on the levels of APR mRNAs in the human hepatoma cell line PLC/PRF/5 were analysed. The accumulation of APR mRNAs [the complement components C3, factor B and Cl inhibitor; the major APRs C-reactive protein (CRP) and serum amyloid A protein and the CRP analogue serum amyloid P protein] was determined in dose-response and time-course studies. The APRs differed from each other in their responses to IL-1 beta alone, IL-6 alone, and IL-1 beta plus IL-6. Dexamethasone enhanced the cytokine-driven induction of a subset of APR mRNAs. These studies detail the heterogeneity of the 'in vitro' acute-phase response to defined mediators.