Oxidative stress, resulting from a disruption of the natural balance between pro- and anti-oxidant systems in favor of the former1,2 has long been reported in patients on maintenance hemodialysis3. In these patients recurrent blood interaction with bioincompatible dialysis membranes triggers polymorphonuclear neutrophil (PMN) and monocyte activation and their subsequent generation of highly reactive oxygen species (ROS) including 
and its derivatives (H2O2, OH° and 1O2) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and OCl- via myeloperoxidase (MPO)-dependent reaction between chloride and H2O24. Moreover, the profound impairment in antioxidant systems as for instance glutathione peroxidase activity5,6,7,8, together with regular perdialytic loss of antioxidant molecules (such as glutathione), favor oxidant-mediated damage9.
Evidence for oxidative stress in hemodialysis patients has long relied solely on elevated circulating levels of lipid oxidation markers10,11,12,13. More recently, increased levels of carbonyl compounds in relation to the formation of advanced glycation end products (AGE) have also been reported in these patients14,15.
In the search for specific markers of protein oxidation16 in the plasma of hemodialysis patients, we recently described a novel family of oxidized protein compounds, which we designated advanced oxidation protein products (AOPP)17 in keeping with their close relationship with AGE compounds such as pentosidine. The generation of AOPP could also be obtained in vitro by exposing proteins such as human serum albumin (HSA) to various oxidants, among which HOCl was the most powerful compound. A close correlation was found between AOPP and carbonyls or dityrosine levels. This latter observation was in keeping with the well-documented capacity of HOCl to induce the formation of tyrosine products18. In a following study19 we showed that (1) in vivo AOPP levels are in fact elevated early in the course of chronic renal failure (CRF), increase with the progression of CRF and are closely related to monocyte activation state, and (2) in vitro, HOCl-modified HSA (referred herein as HSA-AOPP) also retain the capacity to trigger monocyte respiratory burst, and this was not induced by dityrosine alone. Taken together, these findings led us to propose AOPP as both an ultrasensitive gauge of oxidative stress and a novel class of mediators of inflammation in CRF20. More recently, Himmelfarb and McMonagle21 also concluded that albumin was the major plasma protein target of oxidative stress in uremia and that its oxidation contributes to the development of cardiovascular risk.
The role of oxidative stress in the major dialysis-associated complications such as amyloidosis and accelerated atherosclerosis has been well documented22. However, antioxidant therapeutic strategies have not yet been extensively used in hemodialysis patients, with the exception of the recent development of vitamin E–coated dialysis membranes. Among potential antioxidant drugs, N-acetylcysteine (NAC), an aminothiol and synthetic precursor of cysteine and glutathione, might be a good candidate worth testing23. Indeed, NAC has already been used therapeutically in several disorders related to oxidative stress such as chronic bronchitis and acetaminophen poisoning, and it has also recently been shown to protect renal function in conditions of acute24 and CRF25,26. Specifically, NAC is capable of directly scavenging ROS and HOCl27, and after deacetylation, it releases cysteine, which in turn increases the formation of glutathione, within the intracellular pool of antioxidant molecules28.
The present study conducted in vitro was aimed at (1) characterizing the effects of AOPP on monocyte and PMN oxygenation activities, and (2) searching whether these effects could be counteracted by NAC both in normal and uremic (hemodialysis patients) phagocytes.
METHODS
If not otherwise indicated, chemicals were purchased from Sigma-Aldrich Chemical Co. (Saint-Quentin Fallavier, France).
Preparation of AOPP
HSA (Calbiochem, San Diego, CA, USA) was exposed to HOCl (Fluka, Buchs, Switzerland) to produce AOPP17. Briefly, HOCl stock solution was freshly prepared in phosphate-buffered saline (PBS) and concentration was measured by spectrophotometry using a molar extinction coefficient of 350 mol-1/cm-1 at 290 nm at pH 12. HSA (100 mg/mL) was exposed to HOCl (100 mmol/L) for 30 minutes at room temperature and then dialyzed overnight against PBS.
AOPP concentrations were measured by a spectrophotometric assay, as described previously17. Briefly, 200 
L of AOPP-HSA or HSA preparation in PBS were placed in a 96-well plate (Nunc-immuno plate, Roskilde, Denmark) and mixed with 20 L acetic acid. AOPP concentrations were measured in a microplate reader MR5000 (Dynatech, Paris, France) at 340 nm and calibrated versus standard reference wells containing 200 L of chloramine-T solution (0 to 100 mol/L), 10 L of 1.16 mol/L potassium iodide, and 20 L of acetic acid. The content of AOPP in the stock HSA-AOPP preparation used for all experiments in this study was 
300 mol/L versus 23 mol/L in native HSA. The concentrations of dityrosine were 9.6 mol/L versus 0.8 mol/L, respectively, and those of carbonyls of 20,940 mol/L versus 100 mol/L, respectively.
In an attempt to concentrate high-molecular-weight AOPP fraction formed in vivo17, 1 mL of hemodialysis plasma (or control plasma) was first diluted 1:4 in PBS and further submitted to differential centrifugation (1000g
20 minutes at 4°C) using a 100 kD cutoff centricon (Amicon, Beverly, MA, USA).
Isolation of PMN and monocyte
PMN and monocytes were isolated from ethylenediaminetetraacetic acid (EDTA) anticoagulated venous blood of healthy controls (N = 11) recruited among volunteer blood donors of the Etablissement du Sang (Paris) and hemodialysis patients (N = 16) treated three times a week (4- to 5-hour sessions) with dialyzers equiped with cellulosic (N = 5), or synthetic (N = 11) membranes. The dialysate was of standard ionic composition with bicarbonate buffer.
Mononuclear cells were obtained by density gradient centrifugation over Ficoll-Hypaque 400 (Pharmacia Fine Chemicals, Uppsala, Sweden) gradient as described previously29. Isolation of monocytes from the mononuclear cells, harvested at the interface of the gradient, was performed by depletion of nonmonocyte using the magnetic cell sorting (MACS) cell isolation system (Miltenyi Biotec, Gladbach, Germany). Briefly, T cells, natural killer (NK) cells, B cells, dendritic cells, and basophils were labeled using a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56 and anti-immunoglobulin E (IgE) antibodies. After washing, labeled cells were mixed with MACS microbeads coupled to an antihapten monoclonal antibody. Magnetically labeled cells were depleted by retaining them on a MACS column placed in the magnetic field of the VarioMACS, yielding to a highly purified monocyte suspension (90% to 95%). Monocytes were adjusted at a concentration of 2.5 106cells/mL in phenol-red free Hank's balanced salt solution (HBSS) (Eurobio, Paris, France).
PMN and erythrocytes sedimented through the Ficoll gradient were resuspended in HBSS to the original volume of blood layered on the gradient, mixed in a 2:3 ratio with a 5% solution of Dextran 500 (Pharmacia) in saline and maintained 45 minutes at 4°C. The PMN containing supernatant was then gently aspirated and centrifuged (200g at 4°C for 10 minutes). After lysis of residual erythrocytes by 1.6% NaCl, PMN (>95%) were resuspended (5 106 /mL) in HBSS.
NADPH oxidase and myeloperoxidase-dependent oxygenation activities
Monocyte and PMN NADPH oxidase and myeloperoxidase (MPO)-dependent oxygenation activities were measured by chemiluminescence (CL) using a single-photon luminometer (AutoLumat LB953, Berthold Co., Wildbad, Germany), as described previously30. Briefly, two distinct activity-specific lipopolysaccharide (LPS) free chemiluminogenic substrates obtained from Luminostix (San Antonio TX, USA) were used: lucigenin (10,10-dimethyl-9,9-biacridium dinitrate), which selectively measures NADPH oxidase–dependent extracellular superoxide anion formation, and the cyclic hydrazide luminol (5-amino-2,3-dihydro–1,4-phthalazinedione), which measures intracellular MPO-dependent formation of H2O2 and HOCl31. One hundred microliters of PMN suspension (5 105 cells) were distributed into polystyrene tubes containing 100 L of a luminigenic substrate and 50 L of either HBSS (resting CL) or agents to be tested, including HSA or HSA-AOPP [final concentration (fc) of 2 mg/mL)]; 4-phorbol,12-miristate,13-acetate (PMA) (fc, 16 mol/L), serum AB opsonized zymosan (2 109 particles/mL, fc, 2 mg/mL). Luminescence activities were measured in duplicate over 40 minutes and expressed as integrated total counts.
Study of the effect of PMN priming by platelet-activating factor (PAF) on AOPP-induced activation of NADPH oxidase and MPO oxygenation activities was performed by using PAF (1 g)-coated tubes as described32 and following the same steps as above.
Effect of NAC and thiol compounds on PMN and monocyte oxygenation activities
The effect of NAC on PMN and monocyte oxidative responses was tested according to the technical conditions described in33. Briefly, 5 105 cells were set to incubate (30 minutes at 37°C under constant agitation) with NAC (fc, 0.01 mg/mL up to 1mg/mL) or HBSS as controls. It was verified in preliminary experiments that (1) 30 minutes was an optimal time of incubation; (2) cell viability was not affected up to a 2 mg/mL of NAC; and (3) washing after cell treatment had no influence on the effect of NAC. To quantify the action of NAC, results were expressed as % control CL response = 100 CL (cells + NAC) - CL (cells + HBSS)/CL (cells + HBSS).
The effect of glutathione and dithiothreitol (fc, 5 mmol/L for both, previously reported as optimal in34) was tested in the same conditions as those used for NAC.
Statistical analysis
Results were expressed as mean 
SEM and compared using analysis or variance (ANOVA) or Mann-Whitney or Wilcoxon tests for unpaired and paired data, respectively. Differences were considered significant when the P value was 0.05 or less.
RESULTS
AOPP triggers oxidative responses of PMN in a dose-dependent manner
Both NADPH oxidase, measured by lucigenin-amplified CL, and MPO-dependent activity, measured by luminol-amplified CL, of normal PMN were significantly increased in a dose-dependent manner with increasing AOPP concentrations Figure 1. Most important, there was a linear relationship between HSA-AOPP concentrations and lucigenin or luminol CL intensity (r correlation coefficient of 0.98 and 0.95, respectively).
Figure 1.
Dose-dependent effect of human serum albumin-advanced oxidation protein products (HSA-AOPP) on neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent oxygenation activities. The AOPP stock solution was diluted in order to achieve a final protein concentration ranging from 2000 to 62.5 g/mL. Each AOPP dilution was used to trigger polymorphonuclear nertrophils (PMN) respiratory burst measured by lucigenin-chemiluminescence (CL) (A) or luminol-CL (B). Data from a typical experiment (reproduced five times) are expressed as integrated CL responses over 40 minutes (mean SD of duplicate measurements).
The possibility that LPS contamination contributes to the observed effect of HSA-AOPP on PMN oxygenation activities could be ruled out as follows: (1) LPS concentration as measured by Limulus amoebocyte lysate (LAL) test was similar in HSA-AOPP (0.53 EU/mL) and in HSA (0.58 EU/mL), which itself did not trigger any CL response as opposed to HSA-AOPP and as compared to HBSS Table 1; (2) LPS (from 0.5 EU/mL up to 50 EU/mL) had no enhancing effect on lucigenin or luminol CL production by PMN as compared to HBSS alone; and (3) polymyxin B did not significantly inhibit HSA-AOPP–induced PMN lucigenin CL [572,850 8582 counts/40 minutes versus 458,100 6380 counts/40 minutes, respectively (N = 4, NS)] or PMN luminol CL response [608,500 counts/40 minutes versus 569,700 8138 counts/40 minutes (N = 4, NS].
Table 1 - Comparative study of monocyte and neutrophil oxygenation activities in healthy subjects.
Full table Priming by PAF enhances PMN response capacity to HSA-AOPP
As previously described32, PAF-elicited priming of PMN selectively increased luminol-amplified, but not lucigenin-amplified, CL responses to opsonized zymosan (CR1/CR3 receptor-mediated). In contrast, PAF enhanced HSA-AOPP–induced PMN responses via both NADPH oxidase and MPO-dependent pathways, although the increase was less pronounced for the former than for the latter Figure 2.
Figure 2.
Effect of platelet-activating factor (PAF) priming on human serum albumin-advanced oxidation protein products (HSA-AOPP)–induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent oxidative responses of normal neutrophils. Lucigenin (A, left panel) and luminol (B, left panel) amplified chemiluminescence (CL) production in normal polymorphonuclear neutrophils (PMN) previously treated (30 minutes at 22°C) with PAF or Hank's balanced salt solution (HBSS) and tested in presence of the indicated agents. On the left, CL response kinetics (in a representative case). On the right, results of integrated CL production over 40 minutes (mean SEM of six experiments).
PMN respond to plasma-derived AOPP
Plasma of hemodialysis patients (HD1 and HD2) containing 142 mol/L and 209 mol/L of AOPP, respectively, and a control plasma were enriched in high-molecular-weight fractions by differential centrifugation and tested for their capacity to trigger normal PMN respiratory burst (lucigenin CL production). As shown on Figure 3, both HD1 and HD2 triggered CL responses as compared to a pool of control plasma high-molecular-weight fraction, which itself did not significantly enhanced basal CL production. However HD1- or HD2-induced CL responses remained lower than that induced by HSA-AOPP.
Figure 3.
Plasma-derived advanced oxidation protein products (AOPP) trigger neutrophil respiratory burst. Plasma from two hemodialysis patients (HD1 and HD2) and control plasma were centrifuged in order to obtain an AOPP-enriched fraction which was used to trigger polymorphonuclear neutrophils (PMN) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as measured by lucigenin-chemiluminescence (CL). Human serum albumin (HSA)-AOPP was used as positive control. Results of a representative experiment.
Full figure and legend (16K)Comparative study of PMN and monocyte responses to HSA-AOPP and to receptor-dependent or receptor-independent stimuli
NADPH oxidase oxygenation activity
Regardless of the compound tested, lucigenin-amplified CL production was higher in PMN than in monocytes. However, the difference was statistically significant only for stimulated CL Table 1. For monocytes, HSA-AOPP–induced responses did not significantly differ from those induced by opsonized zymosan or PMA. For PMN, HSA-AOPP–induced responses were also of the same order of magnitude as those induced by PMA but were significantly higher than those induced by opsonized zymosan (P < 0.01).
MPO-dependent oxygenation activity
Likewise, HSA-AOPP triggered significantly higher luminol-amplified CL responses than native HSA in both monocyte and PMN or medium alone Table 1. However, HSA-AOPP–induced responses of both monocyte and PMN remained significantly lower than those induced by opsonized zymosan or PMA (P < 0.001, for both).
NAC inhibits HSA-AOPP–induced oxidative responses of both PMN and monocytes
Preincubation of monocytes or PMN with NAC (1 mg/mL) significantly reduced their capacity to elicit HSA-AOPP–induced responses via either NADPH oxidase or MPO-dependent pathways Figure 4. At a lower concentration of 0.1 mg/mL, NAC still inhibited both types of oxygenation activities of monocyte but not those of PMN. At a NAC concentration of 0.01 mg/mL, a 30% increase of both CL responses was observed in PMN but not in monoctyes. Thus, the dose of 1 mg/mL of NAC was used in the following experiments.
Figure 4.
Dose-dependent effect ofN-acetylcysteine (NAC) on human serum albumin-advanced oxidation protein products (HSA-AOPP)–induced monocyte and neutrophil NADPH-oxidase and MPO-dependent oxidative responses. HSA-AOPP–induced monocyte (A) and neutrophil (B) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [lucigenin-amplified chemiluminescence (CL) responses, top panels] and myeloperoxidase (MPO)-dependent oxygenation activities (luminol-amplified CL responses, bottom panels) were measured after cell incubation (30 minutes at 37°C) with Hank's balanced salt solution (HBSS) or NAC at the indicated concentrations. Results (mean SEM of six experiments) were expressed as % control CL response = 100 CL (cells + NAC) - CL (cells + HBSS)/CL (cells + HBSS).
Figure 5 compares the effect of NAC (1 mg/mL) on HSA-AOPP–mediated CL responses to that on other stimulating agents. Whereas NAC inhibited PMA-induced MPO-dependent oxygenation activities of both PMN and monocytes, it had no effect on PMA-induced NADPH oxidase activity of monocytes and an enhancing effect on that of PMN. In contrast, regardless of the cell type, NAC exerted an enhancing effect on opsonized zymosan-induced NADPH oxidase and MPO-dependent oxygenation activities. Such an effect was still observed at a higher NAC concentration (up to 2 mg/mL) (data not shown).
Figure 5.
Comparative effect ofN-acetylcysteine (NAC) (1 mg/mL), opsonized zymosan, and phorbol myristate acetate (PMA) on human serum albumin-advanced oxidation protein products (HSA-AOPP)–induced monocyte and neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent oxidative responses. Monocyte (A) and neutrophil (B) NADPH oxidase [lucigenin-amplified chemiluminescence (CL) responses, top panels] and MPO-dependent oxygenation activities (luminol-amplified CL responses, bottom panels) triggered by HSA-AOPP, opsonized zymosan, or PMA were measured after cell incubation (30 minutes at 37°C) with Hank's balanced salt solution (HBSS) or NAC (1 mg/mL). For each stimulus, results (mean SEM of six experiments) were expressed as % control CL response = 100 CL (cells + NAC) - CL (cells + HBSS)/CL (cells + HBSS).
In an attempt to investigate the mechanisms by which NAC may inhibit AOPP-mediated phagocyte activation, we tested the effect of thiol-related compounds (e.g., glutathione and dithiothreitol) (both at 5 mmol/L) on HSA-AOPP–induced responses of PMN. As shown in Figure 6, both compounds induced a significant inhibition of CL response. Interestingly, the inhibition induced by glutathione was of the same order of magnitude as that induced by NAC (5 mmol/L), and this was true for both types of oxygenation activities. The inhibition exerted by dithiothreitol was also similar to that obtained with glutathione and NAC upon MPO activity but appeared much lower than that upon NADPH oxidase activity.
Figure 6.
Comparative effect ofN-acetylcysteine (NAC), glutathione (GSH) and dithiothreitol (DTT) on human serum albumin-advanced oxidation protein products (HSA-AOPP)–induced neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent oxidative responses. Neutrophil NADPH oxidase [lucigenin-amplified chemiluminescence (CL) responses] (A) and MPO-dependent oxygenation activities (luminol-amplified CL responses) (B) triggered by HSA-AOPP were measured after cell incubation (30 minutes at 37°C) with either Hank's balanced salt solution (HBSS) or NAC or GSH or DTT at 5 mmol/L. For each stimulus, (mean SEM of six experiments) were expressed as % control CL response = 100 CL (cells + NAC) - CL (cells + HBSS)/CL (cells + HBSS).
HSA-AOPP trigger MPO-dependent oxygenation activity of uremic PMN
As shown in Table 2, both basal and stimulated CL productions were higher in hemodialysis patients than in healthy subjects (shown in Table 1). Unlike with control PMN (shown in Figure 1), PAF pretreatment of uremic PMN did not significantly influence opsonized zymosan-induced MPO oxygenation activity, in keeping with an already primed state32. In contrast, such a treatment significantly enhanced HSA-AOPP–induced responses of hemodialysis patient PMN (by 117%).
Table 2 - Effect of N-acetyl cysteine (NAC) on myeloperoxidase (MPO)-dependent oxygenation activity of polymorphonuclear neutrophils (PMN) from hemodialysis patients.
Full table Surprisingly, PAF induced a profound decrease in basal CL production and this was not due to loss of cell viability (95% of viable PMN after PAF treatment).
NAC selectively inhibits HSA-AOPP–induced MPO-dependent oxidative response of uremic PMN
NAC (1 mg/mL) significantly reduced HSA-AOPP–induced luminol-amplified CL response of unprimed as well as of PAF-primed PMN. In contrast, NAC had no effect on opsonized zymosan-induced CL response Table 2.
DISCUSSION
Our previous studies (recently reviewed in35) have led us to identify AOPP as novel markers of oxidative damage to proteins and novel mediators of inflammation, notably involved in the monocyte activation state associated with uremia. AOPP formed in vitro also behaved as potent mediators of monocyte activation, triggering both their respiratory burst and tumor necrosis factor-
(TNF-) synthesis.
In the present study, we show that HSA-AOPP obtained by exposure of HSA to HOCl trigger in a HOCl dose-dependent manner oxidative responses not only of monocytes but also of PMN and via both NADPH oxidase and MPO-dependent pathways. In PMN, NADPH oxidase oxygenation activity triggered by HSA-AOPP was significantly higher than those induced by complement opsonized zymosan (which acts via CR1 and CR3 ligation). Like HSA-AOPP, uremic plasma fractions enriched in AOPP obtained by fast protein liquid chromatography (FPLC) also retained the capacity to activate monocytes. However, this in vitro data should not be misinterpreted in the context of uremia. It is highly probable that plasma-derived AOPP also triggered significant phagocyte activation in vivo, but that inhibitory mechanisms might also counter regulate such proinflammatory reaction.
In contrast to lucigenin, the CL probe luminol used to measure MPO-dependent activity penetrates inside the cells and thus mainly measures intracellular oxidant formation31,36. Surprisingly, intracellular MPO-derived oxygenation activity induced by AOPP was much lower than that obtained with opsonized zymosan or PMA, regardless of the cell type. Taking into account the fact that PMN could be activated by LPS, it was verified that AOPP-induced respiratory burst was not due to LPS contamination, thus definitively ruling out an artifact.
These observations support our working hypothesis that AOPP behave as mediators of inflammation, which, however, still need to be precisely defined. Indeed, like AGE, which under their name comprise a wide variety of heterogeneous compounds37, AOPP might also be considered not as single species but rather as a family of compounds. Among these, both dityrosine and carbonyls, which are produced following exposure of albumin to HOCl15,18,21 were found to be present in our HOCl-modified HSA preparation, and in our in vivo study of a cohort of uremic patients were closely correlated to AOPP levels19. Interestingly, in these previous studies we also observed that these protein oxidation derivatives were not, when tested alone, capable to trigger monocyte respiratory burst, as was the whole AOPP preparation. This observation does not rule out the possibility that other HOCl-mediated albumin oxidation products compounds (e.g., oxidized fatty acids or reactive aldehydes attached to HSA, and protein nitration-derived products) could as single exert such an effect and their proper contribution to AOPP biochemical composition and mediated biologic effects remains to be determined.
Our results showing that AOPP generated in vivo and extracted from uremic plasma are capable to trigger neutrophil respiratory burst in a similar manner as does HOCl-treated control plasma are in support of their physiopathologic relevance for the phagocyte activation associated with uremia. Since AOPP are preferentially formed by chlorinated oxidants17,19 and MPO is the sole enzyme capable of producing such oxidants in vivo, this AOPP-induced activation of neutrophil MPO-dependent activity is also in favor of our working hypothesis that these products as numerous mediators may by themselves amplify their own formation.
We also showed that PMN priming by PAF enhances their potential to mount oxidative responses to AOPP. PAF has been found to trigger up-regulation of CR1, CR3, and Fc
RII, the receptors ligated upon opsonized zymosan stimulation32,38,39. Since the ligation of opsonized zymosan with opsonin receptors induces rapid receptor internalization, up-regulation of opsonin receptors by PAF results in a selective increase in intracellular oxidants, as evidenced by enhanced MPO-derived activities, without increasing extracellular superoxide production. In contrast, PAF did not increase PMA-induced respiratory burst because it is receptor independent. Interestingly, PAF significantly increased AOPP-induced extracellular superoxide anion release and intracellular MPO activities, thus suggesting that AOPP triggers respiratory burst via the ligation of a specific receptor, which is distinct from opsonin receptor. This receptor might be stored within intracellular pools, but would be easily mobilizable toward the plasma membrane We therefore hypothesized that AOPP-induced respiratory burst could be a pertinent parameter to be modulated via appropriate therapeutic strategies aimed at reducing oxidative stress in hemodialysis patients. In this context, NAC was chosen as a candidate for antioxidant therapy. Regardless of cell type, NAC at a dose of 0.5 to 1 mg/mL inhibited AOPP-induced oxygenation activities via both NADPH oxidase and MPO-dependent pathways. With regard to other stimulating agents we also found that, while NAC did not inhibit NADPH oxidase activation induced by PMA, it reduced MPO-dependent activity of both PMN and monocytes. In contrast, NAC had no inhibitory effect and even exerted an enhancing effect, on both opsonized zymosan-induced types of oxygenation activities. As opposed to PMA, which activates phagocyte oxidative metabolism via a receptor-independent pathway, opsonized zymosan triggers phagocyte oxidative responses via CR1 and CR3 receptor-dependent pathways31. Interestingly, an enhancing effect of NAC on the phagocytic activity of neutrophils has been observed40.
The inhibitory effect of NAC on AOPP- and PMA-induced but not on opsonized zymosan-induced respiratory burst strongly suggests that NAC acts via the modulation of intracellular signaling, and not via a nonspecific intracellular oxidant scavenging effect. Of note, both glutathione and dithiothreitol, which, like NAC, also act mainly intracellularly and upon signal transduction pathways34, exerted a similar inhibitory effect on AOPP-induced PMN oxygenation activities. Of interest is the consideration that signaling involved in AOPP and in PMA-induced respiratory burst might share some common steps since they are both inhibited by NAC, whereas opsonized zymosan-induced respiratory burst is not. Although NAC did not inhibit opsonized zymosan-induced respiratory burst, we cannot rule out that NAC might interfere, in vivo, with some mechanisms involved in host defense.
The last part of our study, which was conducted with PMN of a homogeneous series of dialysis patients, showed that basal luminol-amplified CL is much higher than those of controls. This suggests that PMN of hemodialysis patients are in a primed state. As to the effect of PAF on uremic PMN, we observed an unexpected phenomenon namely a significant decrease in basal CL, which did not occur with control PMN and remains to be elucidated. We herein provide evidence that uremic PMN retain a normal capacity to mount MPO-dependent oxidative responses to AOPP and that these responses are selectively enhanced by PAF priming. Interestingly, the respiratory burst induced by AOPP in uremic PMN was, as observed with normal PMN, selectively inhibited following pretreatment by NAC. It must be mentioned that the abnormal decrease of plasma glutathione in uremia may further aggravate the deleterious effect of potential AOPP-mediated ROS production.
There is an increasing body of evidence of the involvement of ROS in the pathogenesis of renal disease, and its most harmful complication (i.e., accelerated atherosclerosis). Interestingly, recent reports have stressed on AOPP as a most relevant index of phagocyte-derived oxidative stress and associated cardiovascular complications, not only in uremic patients41 but also in nonuremic patients with coronary disease42. Interestingly, in our own study, AOPP but not another oxidative stress marker such as malondialdehyde (MDA) were found to be an independent risk factor for atherosclerosis and AOPP were highly correlated with ferritin.
Thus, antioxidant administration seems a promising approach43 to prevent oxidative stress-related complications in end-stage renal disease (ESRD) patients9,14,22. Among the numerous antioxidants tested, there is a great deal of recent evidence to suggest that NAC administration may protect renal function24,25,26.
AOPP-induced respiratory burst appears to be a promising target for modulating the inflammatory state in CRF. More important, the fact that the effect of NAC differs according to the stimulating agent and the intracellular signaling is of special relevance in the context of therapeutic intervention. Indeed, our results are in keeping with the hypothesis that, in vivo, NAC will selectively inhibit oxygenation activities triggered by AOPP, a circulating inflammatory mediator, without decreasing intracellular oxidants, which are induced via the ligation of opsonin receptors, and required for host defense. Clinical trials should be developed in order to verify whether this new indication of an old drug is relevant and whether NAC can be useful in the treatment of uremic patients.
Notes
During the publication process of this paper, Tepel et al44 reported that the oral administration of NAC to chronic hemodialysis patients over 14.5 months was associated with a significant reduction of cardiovascular events, in keeping with the probable clinical relevance of our study.
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Acknowledgments
Part of this work (in uremic patients) was supported by a grant (B.D.L.) from the Baxter Extramural program.
