Reconstituted high-density lipoprotein modulates activation of human leukocytes

An anti-inflammatory effect of reconstituted High Density Lipoprotein (rHDL) has been demonstrated in atherosclerosis and in sepsis models. An increase of adhesion molecules as well as tissue factor expression on endothelial cells in response to inflammatory or danger signals are attenuated by the treatment with rHDL. Here we show the inhibitory effect of rHDL on the activation of human leukocytes in a whole blood assay as well as on monocyte-derived human dendritic cells (DC). Multiplex analysis of human whole blood showed that phytohaemagglutinin (PHA)-induced secretion of the cytokines IL-1β, IL-1RA, IL-2R, IL-6, IL-7, IL-12(p40), IL-15 and IFN-α was inhibited. Furthermore, an inhibitory effect on the production of the chemokines CCL-2, CCL-4, CCL-5, CXCL-9 and CXCL-10 was observed. Activation of granulocytes and CD14+ monocytes by PHA is inhibited dose-dependently by rHDL shown as decreased up-regulation of ICAM-1 surface expression. In addition, we found a strong inhibitory effect of rHDL on toll-like receptor 2 (TLR2)- and TLR4-mediated maturation of DC. Treatment of DC with rHDL prevented the up-regulation of cell surface molecules CD80, CD83 and CD86 and it inhibited the TLR-driven activation of inflammatory transcription factor NF-κB. These findings suggest that rHDL prevents activation of crucial cellular players of cellular immunity and could therefore be a useful reagent to impede inflammation as well as the link between innate and adaptive immunity

Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data

LTA induced activation of NF-κB is inhibited by rHDL.

<p>NF-κB activation was measured in cell extracts at 1 hour after LTA induced activation by a transcription factor ELISA. Cells were preincubated 30 min before LTA stimulation with rHDL (40<b> </b>μg/ml). A representative experiment performed in duplicates from one donor out of three is shown. The bars represent mean ± SD.</p

rHDL inhibits the PHA induced production of proinflammatory cytokines in human whole blood.

<p>Cytokines were measured by multiplex analysis in supernatants of overnight cultures for IL-1β (<i>A</i>), IL-6 (<i>B</i>), IL-7 (<i>C</i>), IL-12(p40) (<i>D</i>), IL-15 (<i>E</i>), IFN-α (<i>F</i>) and TNF-α (<i>G</i>). n.d.: not detectable. Mean values ± SD are shown as column graphs (n = 4). *p<0.05; **p<0.01 vs. PHA or medium (paired Student's <i>t</i>-test).</p

rHDL inhibits the production of proinflammatory chemokines in human whole blood.

<p>Chemokines were measured by multiplex analysis of overnight culture for CCL-2 (<i>A</i>), CCL-3 (<i>B</i>), CCL-4 (<i>C</i>), CCL-5 (<i>D</i>), CXCL-8 (<i>E</i>), CXCL-9 (<i>F</i>) and CXCL-10 (<i>G</i>). Mean values ± SD are shown as column graphs (n = 4). *p<0.05; **p<0.01 vs. PHA or medium (paired Student′s <i>t</i>-test).</p

Effect of rHDL on MoDC maturation given prior to or after TLR stimulation.

<p>rHDL (40 μg/ml) was given to the cells at the indicated time point prior to or after stimulation with LTA (5 μg/ml) for 24 hours. The cells were then evaluated for the expression of CD86 by flow cytometry. The results shown are from one donor and representative of two independent experiments with cells from different donors.</p

rHDL prevents phenotypic maturation of human MoDC in response to LTA in a dose dependent manner.

<p><i>A</i>, rHDL dose-dependently prevents LTA-induced (5 μg/ml) MoDC maturation. Histograms show the typical expression profiles of CD80, CD86 and CD83. Grey histograms show the typical expression profiles of the indicated surface molecules for rHDL plus LTA treated MoDC. White: LTA only treated cells; Dotted line: no stimulus. <i>B</i> and <i>C</i>, To compare the levels of up-regulation of the indicated surface molecules, the median fluorescence intensity (MFI) ratios were calculated by dividing the median fluorescence of LTA- and/or rHDL-treated MoDC by the median fluorescence of immature MoDC and indicated as fold increase in the MFI. Mean values ± SD are shown as column graphs (n = 2 for <i>B</i>, n = 3 for <i>C)</i>. *p<0.05; **p<0.01 vs. mature MoDC (unpaired Student′s <i>t</i>-test).</p

<i>In vivo</i> pharmacokinetics of different IgG preparations, proline and glycine in rats.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, repectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and analyzed for total IgG by Nephelometry and Proline and Glycine concentration by HPLC. The elimination profile for IgG is shown in (A) and for proline and glycine in (B).</p

<i>In vivo</i> comparison of the binding to Aβ, Actin, tetanus toxoid and Varicella Zoster Virus by ELISA.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, respectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and the binding capacity to oligomeric Aβ (A and B), actin (C and D), Tetanus Toxoid (E and F), and VZV (G and H) was analyzed by ELISA. (*p<0.05, **p<0.01). Each ELISA was performed 3 times n = 3 (with all animals n = 30) except VZV t = 2 min n = 2 (with all animals n = 30) due to limited sample volume.</p