The Effect of C-Reactive Protein Isoforms on Nitric Oxide Production by U937 Monocytes/Macrophages

Inflammation is regulated by many endogenous factors including estrogen, a steroid hormone that declines with increasing age, leading to excessive inflammation in the elderly. C-reactive protein (CRP) is an acute phase inflammatory protein that exists in two forms, native CRP (nCRP) and monomeric CRP (mCRP), which mediate distinct biological activities. It is unclear how each CRP isoform mediates nitric oxide (NO), a signalling molecule generated by NO synthase (NOS). This study investigated whether CRP isoforms have distinct effects on NO production by unstimulated and lipopolysaccharide (LPS)-activated monocytes/macrophages and whether estrogen mediates CRP-induced NO production in an in vitro model of ageing. NO and inducible NOS (iNOS) were measured (n=12) by the Griess assay and an enzyme-linked immunosorbent assay (ELISA) respectively following incubation (24 hours) of human-derived U937 monocytes/macrophages with CRP isoforms ([nCRP] = 500 and 1000µg/ml; [mCRP] = 100 and 250µg/ml) in the absence or presence of 17 beta-estradiol (1x10-7, 1x10-8 and 1x10-9M). The response to each CRP isoform and estrogen was dependent on the differentiation and activation status of cells. Monocytes with or without prior LPS-activation significantly increased (P0.05) on NO/iNOS production by unactivated or LPS-activated macrophages whereas nCRP significantly (P<0.05) reduced NO/iNOS production by macrophages, with or without prior LPS-activation. The nCRP isoform had opposing actions on monocytes, significantly (p<0.01) increasing and reducing NO/iNOS by unactivated and LPS-activated monocytes respectively. Estrogen significantly (P<0.01) reversed nCRP-mediated NO inhibition by unactivated macrophages but decreased CRP-induced NO by unactivated monocytes treated with nCRP or mCRP and LPS-activated monocytes treated with mCRP. NO was differentially mediated by CRP isoforms in a cell-type/state specific manner, with production corresponding to concomitant changes in iNOS levels. Collectively, the findings indicate nCRP and estrogen predominantly reduce NO production whereas mCRP increases NO production. This supports growing evidence that mCRP exacerbates inflammation whilst nCRP and estrogen dampen the overall inflammatory response. Therapeutic strategies that restore estrogen levels to those found in youth and promote the stability of nCRP or/and prevent the formation of mCRP may reduce NO production in age-related inflammatory conditions

TISSUE-SPECIFIC EXPRESSION AND CELLULAR LOCALIZATION OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED NAD:ARGININE ADP-RIBOSYLTRANSFERASE.

Mono ADP-ribosyltransferases (ADPRT) transfer the ADP-ribose moiety O fNAD to acceptor proteins. A glycosylphosphatidylinositol(GPI)- anchored ADPRT, originalyisolated from rabbit skeletal muscle, is conserved across species as evidenced on immunoblots where antibodies against rabbit skeletal muscle transferase reacted with partially purified ADPRT from canine,bovine and humanskeletal muscle. On Northern analysis, the skeletalmuscle ADPRTcDNA hybridized with a 1.6-kb band, strongly with mouse cardiac and skeletal muscle poly(A)+RNA and weakly with a 1.6-kb band from lung and lymphocyte mRNA. ADPRT activity was detected in human cells obtained by bronchoalveolarlavage (BAL).On immunoblots, a 40-kDa protein, reactive with anti-ADPRT antibodies, bound [32P]-NAD in an overlay assay. On immunohistochemical staining and confocal microscopic study of lung tisue, the epithelial layer was strongly reactive, with the immunoreactivity localized on the surface of intermediate bronchial epithelial cells and on the apical surface of ciliated cells, consistent with previous observations that GPI-linked proteins are targeted to the apical surface of polarized cells. Immunoreactivity was reduced by incubation of cells with phosphatidylinositol (PI)-specific phospholipase C, which releases the protein from the PI anchor. These data are consistent with the hypothesis that a GPI-anchored ADPRT, in addition to be found in muscle and lymphocytes, is a constituent of pulmonary epithelial cell membranes

Augmentation of human neutrophil and alveolar macrophage LTB(4) production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H(2)O(2)) and leukotriene B(4) (LTB(4)) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated. 2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H(2)O(2) in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml(−1))- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB(4) in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC). 3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H(2)O(2) when incubated with exogenous H(2)O(2) concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB(4) when incubated with exogenous LTB(4). 4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H(2)O(2) had no significant effect on LTB(4) production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H(2)O(2) to H(2)O and O(2), caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB(4) measured in the supernatants of OZ- and fMLP-stimulated neutrophils. 5. H(2)O(2) (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB(4) (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml(−1)). 6. In conclusion, the anti-oxidant drug, NAC, increases LTB(4) production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H(2)O(2). LTB(4) undergoes a H(2)O(2)-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB(4), suggesting that NAC may increase LTB(4) production by blocking the H(2)O(2)-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8