Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcyReceptors

Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcyRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcyRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcyR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcyR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcyR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcyRs

Resveratrol increases catecholamine synthesis in macrophages: implications on obesity.

Sympathetic activation of white adipocytes results in the transdifferentiation of white adipocytes into brown like thermogenic adipocytes called beige adipocytes. White adipose tissue (WAT) is composed of several cell types including macrophages; activation of anti-inflammatory M2 macrophages was recently shown to induce beiging of WAT in rodent models. M2 macrophages secrete catecholamines which play a predominant role in the induction of beiging in WAT. In the current study, we demonstrate the novel anti-obesity effects of resveratrol (RES), a phytoalexin, mediated through catecholamine synthesis in RAW 264.7 macrophage cell line. RES significantly increased the mRNA expression of tyrosine hydroxylase, phenylalanine N-methyl transferase, dopamine beta hydroxylase and phenylalanine hydroxylase expression in these cells. In addition, RES increased arginase, a marker for M2 phenotype and decreased LPS-induced iNOS (inducible nitric oxide synthase), a marker for M1 phenotype. Furthermore RES decreased nitric oxde production from LPS-treated RAW264.7 cells. To demonstrate the effect of RES on catecholamine secretion, we examined the cell culture supernatant from RES treated RAW264.7 cells for catecholamine levels. The results indicate that RES significantly increased catecholamine production in RAW264.7 cells. Finally, upregulation of thermogenic markers was seen in mature 3T3-L1 adipocytes incubated with conditioned media from RAW264.7 cells treated with RES. These studies suggest a novel anti-obesity mechanism for RES by increasing catecholamine production in macrophages

Resveratrol Increases Catecholamine Synthesis in Macrophages: Implications on Obesity

Sympathetic activation of white adipocytes results in the transdifferentiation of white adipocytes into brown like thermogenic adipocytes called beige adipocytes. White adipose tissue (WAT) is composed of several cell types including macrophages and activation of anti-inflammatory M2 macrophages was recently shown to induce beiging of WAT in rodent models. M2 macrophages secrete catecholamines which play a predominant role in the induction of beiging in WAT. In the current study, we demonstrate novel anti-obesity effects of resveratrol (RES), a phytoalexin, mediated through catecholamine synthesis in RAW 264.7 macrophage cell line. RES significantly increased the mRNA expression of tyrosine hydroxylase, phenylalanine N-methyl transferase, dopamine beta hydroxylase and phenyl alanine hydroxylase expression in these cells. In addition, RES increased arginase, a marker for M2 phenotype and decreased LPS-induced iNOS (inducible nitric oxide synthase), a marker for M1 phenotype. Furthermore RES decreased nitric oxide production from LPS-treated RAW264.7 cells. To demonstrate the effect of RES on catecholamine secretion, we examined the cell culture supernatant from RES treated RAW264.7 cells for catecholamine levels. The results indicate that RES significantly increased catecholamine production in LPS-treated RAW264.7 cells. Finally, upregulation of thermogenic markers was seen in mature 3T3-L1 adipocytes incubated with conditioned media from RAW264.7 cells treated with RES. These studies suggest a novel anti-obesity mechanism for RES by increasing catecholamine production in macrophages.Support or Funding InformationThis work was supported by the Biomedical sciences graduate research program at Philadelphia College of Osteopathic Medicine – GA Campus

Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcγReceptors

<div><p>Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcγRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcγRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcγR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an <i>in vitro</i> vasculitis model. Dimeric FcγR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcγR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcγRs.</p></div

Inhibition of iNOS in RAW264.7 cell prevents NO release and HUVEC death.

<p>The RAW264.7 cells were pre-treated with 1400W (100ng/ml) 1hr at 37°C. The HUVECs and pre-treated RAW264.7 cells were co-cultured for 4 <b>(A)</b> 8 <b>(B)</b> and 12hrs <b>(C)</b>. The viability of the HUVECs was determined using CellTitre Blue kit method. RAW264.7 cells pre-treated with 2.4G2 were over layered on antibody-coated HUVECs served as specificity control. Uncoated HUVECs, antibody-coated HUVECs and uncoated HUVECs over layered with 2.4G2 pre-treated RAW264.7 cells were served as additional controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P<0.05 considered as significant (*) and P<0.005 as highly significant (**), NS: non-significant.</p

Exogenous nitric oxide initiates intrinsic apoptotic pathway in HUVECs, which was unaltered by decoy FcγR-Igs.

<p>Concentration-dependent apoptosis was induced by nitric oxide in cultured HUVECs (1x 10⁶ cells) stimulated with different concentrations of <i>S</i>-nitroso-N-acetyl-penicillamine (SNAP) (0–0.5mM) for 24 hr with or without CD16A-Ig was analyzed at mRNA level. Total RNA extracted from the SNAP treated cells was used to make cDNA. QRTPCR analysis of apoptosis related genes <b>(A)</b> Bak <b>(B)</b> Bax <b>(C)</b> Cytochrome-C and <b>(D)</b> Caspase-3 were carried out. Data shown are the average of six individual experiments; each experiment was carried in triplicate. P<0.01 were considered as significant (*) and P<0.005 were as highly significant (**), NS: non-significant.</p

Exogenous nitric oxide induces caspase-3 activation and upregulation in HUVECs.

<p><b>(A)</b> Cells treated with different concentrations of <i>S</i>-nitroso-N-acetyl-penicillamine (SNAP) (0- 2mM) for 24hr. Untreated HUVECs served as specificity control. Cells were lysed and total proteins extracted. Western blot was carried out using an antibodies specific for caspase-3 and β-actin (internal control). <b>(B)</b> Caspase-3 enzymatic activity was performed using total protein extracts. Western blot pictures are representative of three individual experiments. Caspase-3 enzyme activity graph s is average of three individual experiments. P<0.05 considered as significant (*) and P<0.005 as highly significant (**), NS: non-significant.</p

Immune-complex induced nitric oxide secretion is prevented by dimeric FcγR-Ig molecules.

<p><b>(A)</b> RAW 264.7 cells were cultured with different concentrations (0–300μg/ml) of soluble ICs in serum free RPMI1640 medium for 12 and 24 hrs. Untreated RAW264.7 cells and cells treated with antigen, antibody and 2.4G2 mAb served as specificity controls. The culture medium was collected at specified time points, and the nitrite concentration was determined using the Griess reagent. <b>(B)</b> RAW 267.4 cells were cultured with 100 and 200μg/ml of soluble ICs for different time points (0–24 hr). The culture medium was collected at specified time points, and the nitrite concentration was determined using the Griess reagent. <b>(C)</b> CD16A-Ig and CD32A-Ig blocked the binding of ICs to RAW 264.7 cells thereby inhibited the NO production. In all the experiments RAW 264.7 cells pretreated with 2.4G2 mAb, antigen alone, antibody alone, cells treated with medium alone served as specificity controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P < 0.05 considered as significant (*) and P< 0.005 as highly significant (**), NS: non-significant.</p

Hypothetical model of inhibition of IC-mediated, NO-induced apoptotic pathway in HUVECs by decoy FcγR-Igs.

<p>Circulating ICs deposits in the blood vessels during IC-mediated inflammatory disorders. Effector cells upon binding to the IC get activated. These cells result in the secretion of pro-inflammatory factors like cytokines and toxic superoxide radicals such as nitric oxide in copious amounts. Nitric oxide ability to interact with cellular components in the endothelium results in triggering apoptotic signaling pathway and subsequently cell death and tissue damage. Decoy FcγR-Igs competes with cell surface FcγRs expressed on effector cells and block the access to the ICs deposits in various organs including blood vessels. This will eventually inhibit effector cell mediated endothelial inflammation and damage observed in autoimmune vasculitis.</p

FcγR-Ig blocks the macrophage mediated antibody-coated endothelial cell death.

<p><b>(A)</b> RAW 264.7 cells were co-cultured with HUVECs coated with anti-human fibronectin Ab or HUVECs alone in the presence or absence of CD16A-Ig and CD32A-Ig at 37°C. <b>(A)</b> The cells were analyzed after 2hr for binding of RAW264.7 cells with HUVECs using plate inversion method. <b>(B)</b> After 8hr, the HUVECs viability was analyzed using CellTiter-Blue Assay method. <b>(C)</b> After 12hr caspase-3 assay was carried out by calorimetric method. Blocking mAbs (2.4G2), CD16A-Ig, and CD32A-Ig were pre-incubated for 1 hr at 4°C and then continuously present during their incubation at 37°C. In all the experiments RAW 264.7 cells pretreated with 2.4G2 mAb, antigen alone, antibody alone, HUVEC treated with medium alone served as controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P value < 0.05 considered as significant (*) and P< .005 as highly significant (**), NS: non-significant.</p