C-reactive protein mediates CD11b expression in monocytes through the non-receptor tyrosine kinase, Syk, and calcium mobilization but not through cytosolic peroxides

Objective: C-Reactive protein (CRP) can modulate integrin surface expression on monocytes following Fcγ receptor engagement. We have investigated the signal transduction events causing this phenotypic alteration. Methods: CRP-induced signalling events were examined in THP-1 and primary monocytes, measuring Syk phosphorylation by Western blotting, intracellular Ca2+ ([Ca2+]i) by Indo-1 fluorescence and surface expression of CD11b by flow cytometry. Cytosolic peroxides were determined by DCF fluorescence. Results: CRP induced phosphorylation of Syk and an increase in [Ca2+]i both of which were inhibitable by the Syk specific antagonist, piceatannol. Piceatannol also inhibited the CRP-induced increase in surface CD11b. In addition, pre-treatment of primary monoytes with the Ca2+ mobiliser, thapsigargin, increased CD11b expression; this effect was accentuated in the presence of CRP but was abolished in the presence of the [Ca2+]i chelator, BAPTA. CRP also increased cytosolic peroxide levels; this effect was attenuated by antioxidants (ascorbate, α-tocopherol), expression of surface CD11b not being inhibited by antioxidants alone. Conclusion: CRP induces CD11b expression in monocytes through a peroxide independent pathway involving both Syk phosphorylation and [Ca2+]i release. © Birkhäuser Verlag, 2005.

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 μg/ml for 0-60 min at 37°C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 μg/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.

Pro-inflammatory cytokines IL6, TNF-α, IL1β, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis

Objective: To evaluate the serum levels and diagnostic value of cytokines and acute phase proteins in patients with infective endocarditis (IE). Patients and methods: Serum samples from 63 patients diagnosed with IE and 71 control patients were analysed for the following markers: interleukin-6 (IL6), tumour necrosis factor-α (TNF-α), interleukin 1-β (IL1β), procalcitonin (PCT), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). Results: Serum levels of IL6, IL1β and CRP were significantly elevated in patients with IE as compared to controls. PCT, TNF-α and LBP were not elevated. Conclusion: Serum CRP and IL6 are elevated in IE. IL 6 may aid in establishing the diagnosis. There was no correlation between IL 6 levels and CRP, causative microorganism, echocardiographic features or outcome. © 2007 The British Infection Society.

Multiparameter flow cytometric analysis of C -reactive protein binding to human-derived cell lines and peripheral blood monocytes

C-reactive protein (CRP), a normally occurring human plasma protein may become elevated as much as 1,000 fold during disease states involving acute inflammation or tissue damage. Through its binding to phosphorylcholine in the presence of calcium, CRP has been shown to potentiate the activation of complement, stimulate phagocytosis and opsonize certain microorganisms. Utilizing a flow cytometric functional ligand binding assay I have demonstrated that a monocyte population in human peripheral blood and specific human-derived myelomonocytic cell lines reproducibly bind an evolutionarily conserved conformational pentraxin epitope on human CRP through a mechanism that does not involve its ligand, phosphorylcholine. ^ A variety of cell lines at different stages of differentiation were examined. The monocytic cell line, THP-1, bound the most CRP followed by U937 and KG-1a cells. The HL-60 cell line was induced towards either the granulocyte or monocyte pathway with DMSO or PMA, respectively. Untreated HL-60 cells or DMSO-treated cells did not bind CRP while cells treated with PMA showed increased binding of CRP, similar to U-937 cells. T cell and B-cell derived lines were negative. ^ Inhibition studies with Limulin and human SAP demonstrated that the binding site is a conserved pentraxin epitope. The calcium requirement necessary for binding to occur indicated that the cells recognize a conformational form of CRP. Phosphorylcholine did not inhibit the reaction therefore the possibility that CRP had bound to damaged membranes with exposed PC sites was discounted. ^ A study of 81 normal donors using flow cytometry demonstrated that a majority of peripheral blood monocytes (67.9 ± 1.3, mean ± sem) bound CRP. The percentage of binding was normally distributed and not affected by gender, age or ethnicity. Whole blood obtained from donors representing a variety of disease states showed a significant reduction in the level of CRP bound by monocytes in those donors classified with infection, inflammation or cancer. This reduction in monocyte populations binding CRP did not correlate with the concentration of plasma CRP. ^ The ability of monocytes to specifically bind CRP combined with the binding reactivity of the protein itself to a variety of phosphorylcholine containing substances may represent an important bridge between innate and adaptive immunity. ^

Relationship of Omega-3 Fatty Acids on C-Reactive Protein and Homocysteine in Haitian and African Americans with and without Type 2 Diabetes

Background: Omega-3 fatty acids (n-3) may be protective of cardiovascular risk factors for vulnerable populations. The purpose of this study was to assess the association between n-3 with, C-reactive protein (CRP), and homocysteine (HCY) in Black minorities with and without type 2 diabetes. Methods: A cross-sectional study was conducted with 406 participants: Haitian Americans (HA): n=238. African Americans (AA): n=172. Participants were recruited from a randomly generated mailing lists, local diabetes educators, community health practitioners and advertisements from 2008-2010. Sociodemographics and anthropometrics were collected and used to adjust analyses. All dietary variables were collected using the semi-quantitative food frequency questionnaire (FFQ) and used to quantify vitamin components. Blood was collected to measure CVD risk factors (blood lipids, HCY, and CRP). Results: African Americans had higher waist circumferences and C-reactive protein and consumed more calories as compared to Haitian Americans. Omega 3 fatty acid intake per calorie did not differ between these ethnicities, yet African Americans with low n-3 intake were three times more likely to have high C-reactive protein as compared to their counterparts [OR=3. 32 (1. 11, 9. 26) p=0.031]. Although homocysteine did not differ by ethnicity, African Americans with low omega 3 intake (<1 g/day) were four times as likely to have high homocysteine (>12 mg/L) as compared to their counterparts, adjusting for confounders [OR=4.63 (1.59, 12.0) p=0.004]. Consumption of n-3 by diabetes status was not associated with C-reactive protein or homocysteine levels. Conclusions: Consumption of n-3 may be protective of cardiovascular risk factors such as C-r

Obesity and Serum High Sensitivity C-Reactive Protein Levels Among Elderly Turkish Immigrants in the Netherlands with Type 2 Diabetes

The study examined the associations of anthropometric measures of obesity with high sensitivity C-reactive protein (hs-CRP) levels in Turkish immigrants with type 2 diabetes (T2D) living in the Netherlands. A total of 110 participants, physician-diagnosed with T2D, aged 30 years and older were recruited from multiple sources from The Hague, Netherlands. Serum hs-CRP levels were measured with immunoturbidimetric assay. Glycated hemoglobin (A1C) was determined by high-pressure liquid chromatography. Measures of obesity: body weight, body mass index (BMI), waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), and waist-to-height ratio (WHtR) were determined. Statistical analysis included descriptive statistics, Pearson’s correlations and multiple linear regressions (MLR) stratified by gender. Hs-CRP was log transformed to achieve normality. Subjects with hs-CRP levels >10 mg/L (n = 17) were excluded from the analysis. Females had a higher BMI (p = 0.007), HC (p < 0.001), and WHtR (p = 0.011) as compared to males. Conversely, males had a higher weight (p = 0.007), and WHR (p < 0.001) than females. MLR showed that after controlling for covariates, log hs-CRP was positively associated with BMI (B = 0.039, SE = 0.019, β = 0.287, p < 0.05), WC (B = 0.025, SE = 0.011, β = 0.332, p < 0.05) and WHtR (B = 4.015, SE = 1.464, β = 0.376, p < 0.01) in females only. Gender-specific associations between obesity measures and hs-CRP level need to be further investigated in the Turkish immigrant population. Hs-CRP assessment may be added as a standard of care for T2D treatment within this population.

A Staggered Decameric Assembly of Human C-Reactive Protein Stabilized by Zinc Ions Revealed by X-ray Crystallography

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Association between ferritin, high sensitivity c-reactive protein (hsCRP) and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects

Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM); 30 obese subjects without diabetes (OB) and 30 healthy subjects (Cn). We isolated peripheral mononuclear cells (PMCs) and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-αIL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL-6), and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Three-dimensional flow-through protein platform

We have developed a new protein microarray (Immuno-Flow Protein Platform, IFPP) that utilizes a porous nitrocellulose (NC) membrane with printed spots of capture probes. The sample is pumped actively through the NC membrane, to enhance binding efficiency and introduce stringency. Compared to protein microarrays assayed with the conventional incubation-shaking method the rate of binding is enhanced on the IFPP by at least a factor of 10, so that the total assay time can be reduced drastically without compromising sensitivity. Similarly, the sensitivity can be improved. We demonstrate the detection of 1 pM of C-reactive protein (CRP) in 70 mu L of plasma within a total assay time of 7 min. The small sample and reagent volumes, combined with the speed of the assay, make our IFPP also well-suited for a point-of-care/near-patient setting. The potential clinical application of the IFPP is demonstrated by validating CRP detection both in human plasma and serum samples against standard clinical laboratory methods.

Free energy calculations of the interactions of c-Jun-based synthetic peptides with the c-Fos protein

The c-Fos–c-Jun complex forms the activator protein 1 transcription factor, a therapeutic target in the treatment of cancer. Various synthetic peptides have been designed to try to selectively disrupt the interaction between c-Fos and c-Jun at its leucine zipper domain. To evaluate the binding affinity between these synthetic peptides and c-Fos, polarizable and nonpolarizable molecular dynamics (MD) simulations were conducted, and the resulting conformations were analyzed using the molecular mechanics generalized Born surface area (MM/GBSA) method to compute free energies of binding. In contrast to empirical and semiempirical approaches, the estimation of free energies of binding using a combination of MD simulations and the MM/GBSA approach takes into account dynamical properties such as conformational changes, as well as solvation effects and hydrophobic and hydrophilic interactions. The predicted binding affinities of the series of c-Jun-based peptides targeting the c-Fos peptide show good correlation with experimental melting temperatures. This provides the basis for the rational design of peptides based on internal, van der Waals, and electrostatic interactions.

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.