Role of complement and platelet-activating factor in the stimulation of phagocytosis and reactive oxygen species production during haemodialysis

Karine Gastaldello1,, Cecile Husson1, Robert Wens2, Jean-Louis Vanherweghem1 and Christian Tielemans1

1 Department of Nephrology, Dialysis and Transplantation, Hôpital Erasme, Université Libre de Bruxelles and 2 Department of Nephrology, Hôpital Universitaire Brugmann, Université Libre de Bruxelles, Belgium



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Neutrophil phagocytic functions have been studied extensively in haemodialysis (HD) patients; however, results are contradictory and the mechanisms that modulate phagocytosis and oxidative burst during dialysis are not completely understood.

Methods. The present study investigated neutrophil functions in a selected population of patients before and during clinical dialysis with cuprophane, and polyacrylonitrile (AN69) membranes. We measured phagocytosis of Escherichia coli and intracellular hydrogen peroxide (H2O2) production by flow cytometry in whole blood.

Results. Before dialysis, neutrophils from HD patients showed normal phagocytic capability and H2O2 formation. Phagocytosis of FITC-E. coli was significantly stimulated in cuprophane but not AN69-treated patients. Spontaneous and stimulated H2O2 production was enhanced with both cuprophane and AN69 membranes. We then investigated in vitro the role of complement and platelet-activating factor (PAF) in the activation of neutrophils. Incubation of whole blood with C5a increased phagocytosis but not H2O2 production. On the contrary, the addition of synthetic PAF showed a markedly stimulated H2O2 production without increase in phagocytosis. Moreover, during dialysis with formaldehyde-reused cuprophane, complement activation was abolished and phagocytosis was no longer enhanced, while the stimulation of H2O2 production persisted. In addition, we also excluded a particular role of the membrane itself in the activation of neutrophils.

Conclusion. We demonstrated that in a selected population of HD patients, neutrophils exhibit normal phagocytic capability and normal intracellular H2O2 production. During dialysis, the stimulation of phagocytosis observed with cuprophane is complement dependent, whereas the enhanced H2O2 production observed with both cuprophane and AN69 membranes might be related to PAF production.

Keywords: complement; dialysis membrane; HD; oxidative burst; PAF; phagocytosis



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Haemodialysis (HD) patients are at an increased risk of infection, which remains a leading cause of death in this population. These infections are not due to opportunistic organisms but to common bacteria. Due to the role of neutrophils in the phagocytosis and killing of bacteria, phagocytic functions have been extensively investigated in HD patients. Most of these studies have pointed to a defect in phagocytosis, which is present before the start of dialysis and is enhanced during dialysis with complement-activating membranes (reviewed in [1]). Studies investigating reactive oxygen species (ROS) production in HD patients have yielded conflicting results. Before dialysis, spontaneous and stimulated ROS production by neutrophils is reported to be normal, decreased, or even increased [2,3]. The effect of the dialysis membrane on ROS production is also controversial, spontaneous ROS production is usually reported to be increased after 15–30 min of dialysis with complement-activating membranes, while stimulated ROS production is decreased [2]. Interestingly, these changes are not observed with non-complement-activating membranes. Many factors may contribute to these contradictory observations. First, flaws related to the methodology (use of artificial stimuli instead of bacteria, measurement of luminol-enhanced chemiluminescence that reflects myeloperoxidase-catalysed oxidation rather than the respiratory burst); secondly, activation of the cells by the isolation procedure or selection of functionally effete cells. In addition, factors concomitant to uraemia, which are heterogeneously distributed in the populations under study, might affect phagocytic function, such as iron overload [4], aluminium intoxication, malnutrition [5], elevated intracellular calcium, hyperparathyroidism [6], and erythropoietin deficiency [7]. The aim of this study was to investigate phagocytosis and ROS production in a highly selected population of HD patients in whom we carefully excluded the potentially confounding factors listed above. We studied neutrophil function ex vivo using a direct evaluation technique in whole blood, thus avoiding possibly harmful isolation steps. Since during dialysis, the neutrophil count shows enormous fluctuations, we used a flow cytometric assay in accordance with the most recent recommendations [8], that enabled us to evaluate a fixed number of cells. We compared phagocytosis and subsequent intracellular hydrogen peroxide (H2O2) production by polymorphonuclear cells (PMN) in HD patients and healthy subjects. We also studied the acute changes in phagocytosis and H2O2 production occurring during HD with cuprophane and polyacrylonitrile (AN69), as representatives of complement- and non-complement-activating membranes respectively. To analyse the mechanisms involved in the observed changes, we tested the roles of complement, the membrane itself, and platelet-activating factor (PAF) on phagocytic functions in vitro.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patient characteristics
A total of 32 patients on maintenance HD, and 18 healthy controls participated in the various parts of the study. Our institutional Review Board approved the protocol and informed consent was obtained from all the subjects. All patients had been on HD for at least 1 year. Exclusion criteria were diabetes, immune disorders, ongoing malignancy, haematological (except renal anaemia) or liver diseases, infection during the previous month, and immunosuppressive medication. Moreover, patients were selected for the absence of iron overload (ferritin >500 µg/l), aluminium overload (serum Al >15 µg/ml), hyperparathyroidism (serum intact parathyroid hormone (PTH) >200 pg/ml), and protein malnutrition (dietary protein intake <1 g/kg body weight/day and serum albumin <4 g/dl). All the selected patients had to have a weekly urea Kt/V >=3.6 and were treated with erythropoietin to obtain haemoglobin levels over 10 g/dl.

Comparison of phagocytic functions in HD patients and healthy controls
We compared PMN phagocytosis and H2O2 production in 19 HD patients and 13 healthy subjects. In the HD patients, heparinized (5 IU/ml) whole blood was collected before a routine dialysis session.

Haemodialysis studies
Acute changes in phagocytosis and H2O2 production were studied during a HD session on cuprophane in 10 patients, and on AN69 in nine patients. The patients had been dialysed for at least one month using the membrane under study. Six patients were studied on both cuprophane and AN69 membranes. The dialysers were new GF18E from Gambro, Sweden (Cuprophane, 1.8 m2), and Filtral 20 from Hospal, France (AN69, 2 m2). A bicarbonate-based dialysate containing 1.25 mmol/l of calcium was used in all cases. Anticoagulation was maintained with heparin. Heparinized (5 IU/ml) whole blood was drawn before starting dialysis (T0) and from the efferent line after 5,15, and 180 min (T5, T15, T180). For the evaluation of phagocytic function, blood samples were obtained from seven patients, from both the afferent and the efferent blood lines of the dialyser at 5 and 15 min after initiation of dialysis. Since ROS are quickly degraded, for the measurement of spontaneous H2O2 production during dialysis, blood samples were processed immediately after blood collection. For the measurement of phagocytosis and stimulated ROS production, blood samples were analysed within 1 h of sampling. Blood samples remained at room temperature prior to processing.

Flow cytometric analysis
Analysis was performed using a FACScan flow cytometer and LYSYS-II computer software (Becton–Dickinson, Erembodegem, Belgium) using the blue-green excitation light (488 nm with an argon laser). Three parameters were recorded: forward light scatter (FSC, a measure of cell size), side scatter (SSC, a measure of cell granularity), and fluorescence intensity for phycoerythrin (PE) or fluorescein isothiocyanate (FITC), as appropriate. PMN and mononuclear cells (MNC) were identified using their morphological characteristics displayed on a dot-plot of FSC vs SSC and the analysis was performed on 10 000 cells. Fluorescence was standardized daily with calibrated microbeads (QuantumTM 25 and 27, FCSC, San Juan, PR) which were put into the same histogram channel and gain settings were recorded before the analysis of each set of samples. Results were expressed as mean equivalent of soluble fluorescence (MESF) or change in MESF as compared to prestimulation (in vitro study) or predialytic values.

Assessment of phagocytosis by neutrophils
Quantitative determination of phagocytosis was performed in whole blood by measuring the fluorescence of neutrophils after phagocytosis of FITC-labelled opsonized E. coli (Phagotest®, Orpegen, Heidelberg, Germany). The phagocytosis by neutrophils was evaluated at various bacteria-to-cell ratios in order to determine a saturating concentration, which was 33:1. One hundred microlitres of heparinized whole blood was incubated in an ice bath for 10 min in order to cool the cells down to 0°C before adding the bacteria. Phagocytosis was then induced by adding 20 µl of pre-cooled FITC-E. coli at a concentration of 109/ml. After vortexing, the samples were incubated for 10 min at 37°C in a closed water bath (Shaking water bath SW-20 D, Julabo, Labortechnik GMBH, Germany) and agitated constantly at 120 r.p.m. to allow phagocytosis. At the end of the incubation period, all samples were simultaneously removed from the water bath and placed on ice again in order to stop phagocytosis. One hundred microlitres of ice-cold quenching solution (blue dye) was then added to block the fluorescent signal of the bacteria bound to the neutrophil membrane but not internalized. After washing the samples twice, 2 ml of a lysing solution were added for 20 min at room temperature to lyse red blood cells and to fix white blood cells. After a further wash, 100 µl propidium iodide was added for 10 min at 0°C to stain DNA, and PMN fluorescence was measured within 30 min. The ‘live’ gate was set in the red fluorescence histogram on cells with a DNA content compatible with human diploid cells and the analysis was performed on 10 000 viable neutrophils identified using their morphological characteristics (FSC vs SSC resolution).

Assessment of intracellular H2O2 production by neutrophils
Quantitative determination of intracellular H2O2 production by neutrophils was based on a flow cytometric method using the fluorogenic substrate dihydrorhodamine 123 (DHR) and adapted for whole blood measurement [8]. DHR is taken up into the cell at 37°C and is then oxidized by H2O2 in the presence of peroxidases into green fluorescent rhodamine 123. Dihydrorhodamine 123 solution was freshly prepared 30 min before each assay by reconstitution of a substrate disk with 1 ml of washing solution (Bursttest®, Orpegen, Heidelberg, Germany). In preliminary experiments (Table 1Go), we determined the optimal time point for addition of DHR to measure the production of ROS. In whole blood, we observed that addition of DHR after E. coli or phorbol myristate acetate (PMA) induced H2O2 production was optimal; while in experiments using isolated PMN, we demonstrated that a 5 min period of PMN loading with DHR was optimal for maximal enhancement of PAF induced H2O2 production. In whole blood, H2O2 production was evaluated with stimuli (E. coli- and PMA-stimulated H2O2 production) and without stimuli (spontaneous H2O2 production). For the measurement of stimulated-H2O2 production, 100 µl of heparinized blood was incubated in an ice bath for 10 min in order to cool the cells to 0°C. These blood samples were then incubated with pre-cooled E. coli (20 µl at a concentration of 109/ml) or PMA (1.6x10-6 mol/l) for 10 min at 37°C in the shaking water bath. Afterwards, cells were loaded with 20 µl of the DHR solution (25 µg/ml of washing solution with 1% DMSO) for another 10 min at 37°C in the shaking water bath. For the measurement of spontaneous H2O2 production, pre-cooled blood samples were incubated only with 20 µl of DHR solution for 10 min at 37°C as described above. At the end of the incubation time with DHR, all samples were simultaneously taken out of the water bath and the reaction was stopped by adding 2 ml of lysing solution for 20 min at room temperature. After washing the samples twice, 100 µl of propidium iodide was added for 10 min at 0°C to stain DNA, and neutrophil R123 fluorescence was analysed within 30 min. The ‘live’ gate was determined as described above.


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Table 1. Optimal time point for addition of DHR 123 to measure ROS production in whole blood

 

Measurements of endotoxin levels
Endotoxin levels were measured using a chromogenic limulus amoebocyte specific assay LAL-Coatest® endotoxin (Chromogenix, Mölndal, Sweden). Results are expressed as EU/ml. The sensitivity of the assay is 0.015 EU/ml in the dialysate.

Effect of the dialysis membrane on neutrophil functions
To test the direct influence of the dialysis membrane on PMN function, we incubated whole blood with flat membranes (2 cm2) of ethylene oxide sterilized cuprophane (Akso-Nobel, Obemburg, Germany) and AN69 (Hospal, France). All in vitro experiments were carried out in non-adherent polypropylene tubes at 37°C in a shaking water bath. Pieces of cuprophane and AN69 membranes were rinsed with physiological saline and incubated with 2 ml of normal heparinized whole blood for 10, 30, and 60 min at 37°C in a 5% CO2 atmosphere. C3a desArg levels were measured after 10, 30, and 60 min of incubation. To avoid any further complement activation after sampling, blood was collected in tubes with EDTA, kept on ice and centrifuged immediately at 4°C (1500 g, 15 min). Plasma was removed and stored at -80°C. Concentrations of C3a desArg were measured (method described in detail in [9]) with a commercial radioimmunoassay (Amersham, Buckinghamshire, UK). As a negative control, blood samples were incubated for 10, 30, and 60 min without membrane. At the end of the incubation period, phagocytosis of FITC-E. coli and H2O2 production in response to E. coli was evaluated as described above. Strict aseptic measures were observed in handling the specimens, and pyrogen-free media were used in all instances to avoid artefactual activation of leukocytes.

Evaluation of opsonizing capacity of the uraemic serum
To evaluate the opsonizing ability of the uraemic patients' serum we compared results obtained with both a Phagotest using non-opsonized FITC-E. coli and with the Phagotest described in the previous section. The latter uses FITC-E. coli that is pre-opsonized with both immunoglobulins and complement of pooled normal sera.

Effect of complement activation on phagocytic function
To assess the possible role of complement activation on the intradialytic changes in phagocytic function, we performed additional in vitro and ex vivo experiments.

In vitro. Heparinized whole blood from 13 patients was collected before starting HD. One hundred microlitres of blood was incubated with recombinant C5a (rHuC5a) (Sigma Chemical Co, St Louis, MO) at a concentration of 250 ng/ml for 15 min at 37°C. We chose to use the dose of C5a (250 ng/ml) corresponding to the blood level reached at 15 min during in vivo dialysis with the cuprophane membrane [9]. Afterwards, phagocytosis and H2O2 production were determined as described above.

Ex vivo. Phagocytic function was evaluated in three patients during utilization of a new cuprophane membrane and after the fifth reuse of the same dialyser with formaldehyde (a procedure known to blunt complement activation). Reuse was performed using an automated device with quality control (Renatron II, Renal Systems Inc.). To confirm that complement activation was abolished after the fifth reprocessing, we measured C3a desArg levels in samples collected both before and after 15 min of dialysis at the efferent line. The concentrations of C3a desArg were measured as described above.

PMN isolation
Human PMN were isolated from the heparinized whole blood of six HD patients by single-step gradient centrifugation (PolymorphprepTM, Nycomed, Oslo, Norway) at room temperature. The neutrophils were recovered, washed twice and resuspended in HEPES-buffered Hanks' Balanced Salt Solution (HBSS, Gibco BRL, Life Technologies, Paisley, UK) containing 1.4 mmol/l Ca2+, and supplemented with 0.5% human serum albumin (HAS; Sigma Chemical Co). The cell count was adjusted to 107 cells/ml HBSS. All isolation procedures and subsequent PMN functional assays were conducted in polypropylene test tubes. Preparations contained more than 95% PMN as determined by light microscopy.

Role of PAF in the activation of neutrophils
To assess whether PAF might be implicated in the changes in phagocytic function occurring during HD, we conducted in vitro experiments to test the effect of PAF on isolated PMN. We used isolated neutrophils since PAF is promptly inactivated in the plasma by a specific PAF acetyl hydrolase to form the biologically inactive lyso-PAF [10]. We investigated the effects of a synthetic PAF L-{alpha}-phosphatidylcholine, ß-acetyl-{gamma}-O-alkyl (Sigma Chemical Co) on PMN phagocytosis and H2O2 production. Various concentrations of PAF (10-6, 10-7, 10-8, 10-9 mol/l) were prepared from the stock solution (10-3 mol/l DMSO) by dilution with HAS-saline. The neutrophils (106 PMN in 100 µl HBSS) were pre-warmed for 10 min at 37°C before stimulation. For the measurement of phagocytosis, pre-warmed PMN were stimulated with PAF (10-6, 10-7, 10-8, 10-9 mol/l) or with medium alone for 5 min at 37°C in the shaking water bath. Thereafter, PMN supplemented with 10% autologous plasma were incubated with 20 µl of FITC-E. coli (109/ml) for 10 min as described above. For the determination of intracellular H2O2 production by neutrophils, we used primed PMN since PAF alone did not significantly increase H2O2 production. Preliminary studies demonstrated that a 2-min period of PMN priming by Cytochalasin B (5 µg/ml) at 37°C was optimal for maximal enhancement of PAF induced H2O2 production. Following the loading period with DHR, primed neutrophils were stimulated with PAF (10-6, 10-7, 10-8, 10-9 mol/l) for 5 min at 37°C. H2O2 production was then determined as described above. Unstimulated PMN were run in parallel as a control.

Statistical analyses
Data are expressed as mean±SEM. Baseline levels of phagocytosis and H2O2 production, as well as changes occurring during HD were analysed by paired or unpaired two-tailed tests as required using the non-parametric Wilcoxon signed-rank test and Mann–Whitney U test respectively. Arteriovenous changes across the dialyser were compared using the Wilcoxon signed-rank test for paired samples. Multiple groups comparison was done with two-way variance analysis. Statistical significance was accepted for P<0.05.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Baseline levels of phagocytosis and H2O2 production
To determine whether neutrophils from selected HD patients have impaired phagocytic functions, we compared blood samples of healthy subjects with blood samples collected before a routine dialysis session of HD patients. There were no significant differences in the baseline level of phagocytosis or H2O2 production between neutrophils from normal subjects and HD patients (Table 2Go).


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Table 2. Comparison of phagocytic function between healthy subjects and HD patients

 

Acute changes in phagocytosis during HD
Due to leukopenia associated with cuprophane dialysis, the bacteria–neutrophil ratio varies during the session. The kinetics of phagocytosis evaluated at various bacteria to neutrophil ratios showed that concentrations of E. coli used at the start of dialysis were already saturating (data not shown). Haemodialysis associated changes in phagocytic function are shown in Figure 1Go. Phagocytosis of FITC-E. coli (Figure 1Go, left panel) was markedly enhanced 15 min after initiation of dialysis with cuprophane membranes and returned to predialytic values by 180 min. In contrast, phagocytosis of FITC-E. coli did not change during the course of AN69 dialysis (Figure 1Go, left panel). These results show that PMN phagocytosis is stimulated during dialysis with the cuprophane membrane, whereas this is not the case for neutrophils from AN69-treated patients.



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Fig. 1. Left panel: Intradialytic changes in PMN phagocytosis of FITC-E. coli. Right panel, intradialytic changes in PMN E. coli-stimulated H2O2 production. Whole blood was obtained before, and after 5, 15, and 180 min of dialysis with • cuprophane (n=10) or with {blacksquare} AN69 (n=9) membranes. Data are presented as mean±SEM. *P<0.05, **P<0.02 compared to predialytic values.

 

Acute changes in spontaneous intracellular H2O2 production during HD
Neutrophils from patients undergoing HD with the cuprophane membrane showed a significant increase in spontaneous H2O2 production after 15 min of dialysis, when compared with predialytic values (MESF: T0, 4750±673; T15, 5740±701; n=8, P=0.004). Interestingly, neutrophils from AN69-treated patients also showed a significant increase in spontaneous H2O2 production after 15 min of dialysis, compared to predialytic values (MESF: T0, 3201±531; T15, 4390±431, n=8, P=0.008). Comparison between cuprophane and AN69 showed no significant difference at any time point (Cuprophane vs AN69: T0, P=0.9; T15, P=0.89; n=8). These results indicate that spontaneous intracellular H2O2 production is stimulated during dialysis with both cuprophane and AN69 membranes.

Acute changes in stimulated H2O2 production during HD
Cuprophane dialysis induced a significant increase in E. coli-stimulated H2O2 production by neutrophils 5 min into dialysis, and this increase was maintained over the course of dialysis (Figure 1Go, right panel). In addition, E. coli-stimulated H2O2 production increased significantly during dialysis with AN69 (Figure 1Go, right panel). Comparison between cuprophane and AN69 showed no significant difference at any time point. Since H2O2 production is likely to be proportional to the level of stimulation, in this case phagocytosis of E. coli that was already upregulated following exposure to cuprophane membrane, we evaluated the H2O2 production in response to another stimulus, PMA. PMA-stimulated H2O2 production was similarly significantly enhanced after 15 min of dialysis with both cuprophane (MESF: T0, 232 356±46 914; T15, 381 646±62 718; n=9, P=0.004 for T15 compared with T0), and AN69 membranes (MESF: T0, 225 229±36 371; T15, 442 852±43 983; n=9, P=0.004 for T15 compared with T0). These results demonstrate that stimulated-H2O2 production is enhanced during dialysis with both cuprophane and AN69 membranes. These results show that neutrophils harvested during HD are still able to further enhance their oxidative metabolism in response to a stimulus, despite their enhanced spontaneous H2O2 production.

Endotoxin levels
Endotoxin was never detectable in the control medium (<0.015 EU/ml). The endotoxin concentrations of the dialysate were assessed throughout the study and were always less than 1 EU/ml.

Evaluation of phagocytic function at the inlet and the outlet of the dialyser
Because apparent changes in neutrophil function may indicate a direct effect of the extracorporeal circulation on neutrophils, or changes in the population of circulating cells [8], we studied neutrophils drawn simultaneously from the inlet and the outlet of the dialyser. In the group of patients dialysed with cuprophane membranes, there was a significant arteriovenous increase in FITC-E. coli phagocytosis across the dialyser at 15 min (n=7, 224 309±86 239, P=0.014); while there was no change in the phagocytic capability of neutrophils after passing across the AN69 dialyser (n=7, 90 498±114 399, P=0.34). Similarly, a comparison of changes in E. coli-stimulated H2O2 production from the afferent to efferent samples was performed. This revealed that there was a significant increase in ROS production at the 5- and 15-min time-points during cuprophane dialysis (T5 and T15 respectively: 85180±8409, P=0.008 and 105 637±41 663, P=0.008, n=7). This increase in ROS production was also observed at the 15-min time-point during AN69 dialysis (66934±27 831, n=7, P=0.04). These results show that both phagocytosis of FITC-E. coli and E. coli-stimulated H2O2 production were enhanced in neutrophils passing through the cuprophane membrane, while only E. coli-stimulated H2O2 production was enhanced in neutrophils passing through the AN69 membrane.

We also assessed the intradialytic changes in neutrophil function at the afferent line of the dialyser. E. coli-stimulated H2O2 production was significantly increased after 15 min of dialysis compared to T0 with both cuprophane and AN69 membranes, while no significant change in phagocytic capability was observed. This demonstrates that the process of intradialytic neutrophil activation is a systemic inflammatory reaction and is not limited to blood travelling through the dialyser.

Specific effect of the dialysis membrane on neutrophil function
Since we observed a significant arteriovenous increase in H2O2 production with both membranes and a significant arteriovenous enhancement of phagocytic capability with cuprophane membranes, the next point to be considered was the possibility that these membranes had a direct activating effect on neutrophil function. However, following incubation of blood with cuprophane and AN69 flat membranes for 10, 30, and 60 min, there was no difference in PMN phagocytosis compared with blood incubated without membrane (cuprophane, P=0.57, P=0.4, P=0.99; AN69, P=0.6, P=0.89, P=0.61 for T10, T30, and T60 respectively vs T0). Similarly, following incubation of blood with cuprophane and AN69 flat membranes, there was no difference in H2O2 production compared with blood incubated without membrane (cuprophane, P=0.99, P=0.43, P=0.7; AN69, P=0.8, P=0.6, P=0.4 for T10, T30, and T60 respectively vs T0). C3a desArg levels were increased after 10, 30, and 60 min of incubation of whole blood with cuprophane by 24, 21, and 21% respectively as compared to T0. It should be noted that phagocytosis was not increased in this model, while cuprophane activates the complement and the phagocytosis in vivo. However, this is not an unexpected finding given the weak activation of complement in this model (increase of 24% for T10 compared to T0) compared to levels obtained during in vitro or in vivo dialysis [9] (increase of 500% for T10 compared to T0). These data indicate that activation of neutrophil functions during dialysis is not related to an activating effect of the dialysis membrane itself.

Evaluation of the opsonizing capacity of uraemic serum
There were no significant differences in phagocytosis of opsonized vs non-opsonized FITC-E. coli. There was also no significant change in the opsonizing capacity of the uraemic serum during the course of dialysis (Figure 2Go).



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Fig. 2. Phagocytosis of non-opsonized FITC-E. coli (– – –) vs pre-opsonized FITC-E. coli (—) with immunoglobulins and complement of pooled normal sera. Whole blood was obtained during dialysis with • cuprophane (n=5) or with {blacksquare} AN69 (n=5) membranes. There were no significant differences in the level of phagocytosis between opsonized versus non-opsonized E. coli. Data are presented as changes in MESF±SEM as compared to predialytic values.

 

Effect of complement activation on phagocytic functions
As phagocytosis is enhanced during cuprophane but not during AN69 dialysis, we thought that the trigger might be complement activation. To address the role of complement activation, we incubated whole blood with recombinant C5a at a concentration of 250 ng/ml, which is the level reached at 15 min during in vivo dialysis with the cuprophane membrane [9]. Results are shown in Figure 3Go and indicate that C5a significantly increased PMN phagocytosis of FITC-E. coli (MESF: baseline level of phagocytosis, 436 743±21 398, C5a-stimulated phagocytosis: 529 698±17 405; P=0.004), whereas H2O2 production was not significantly modified (MESF: baseline level of H2O2 production, 164 801±9805, C5a-stimulated H2O2 production: 184 634±12 618; P=0.13). To further confirm the role of complement activation in the enhanced PMN phagocytosis during cuprophane dialysis, we compared the changes in phagocytosis and E. coli-stimulated H2O2 production induced by new and formaldehyde-reused (5th reuse) cuprophane membranes (n=3). As expected, the reuse procedure abolished complement activation (plasma C3a desArg ng/ml: first use, T0: 755±220; T15, 9846±154 and fifth reuse: T0, 721±140; T15, 982±38 ng/ml; P<0.0001, first vs fifth reuse at T15 min). After reuse, PMN phagocytosis was no longer enhanced at T15, whereas enhanced H2O2 production by neutrophils persisted (Figure 4Go). These data demonstrate that complement activation is involved in the stimulation of PMN phagocytosis observed with first-use cuprophane but not in the enhanced H2O2 production. Moreover, the fact that H2O2 production is enhanced with new or reused cuprophane, and with AN69 membranes suggests another underlying mechanism than complement activation.



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Fig. 3. Effect of rHuC5a on PMN phagocytosis of FITC-E. coli and on E. coli-stimulated H2O2 production in whole blood. Blood was preincubated in vitro with (hatched bars) or without (open bars) rHuC5a 250 ng/ml for 15 min at 37°C. Data are presented as mean±SEM (n=13). *P=0.004 compared with baseline levels of phagocytosis.

 


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Fig. 4. Evaluation of PMN phagocytosis of FITC-E. coli and of E. coli-stimulated H2O2 production in whole blood during the first-use of a cuprophane membrane and after 5th reuse of the dialyser with formaldehyde. Blood was obtained before dialysis (open bars) and at 15 min after the initiation of dialysis (hatched bars) at the efferent line of the dialyser. Data are presented as mean±SEM (n=3). *P=0.05, **P=0.015, ***P=0.0007 compared with predialytic values.

 

In vitro effect of PAF on phagocytic functions
To further investigate the basic mechanisms involved in the activation of neutrophil function, we evaluated the role of PAF, since synthesis and release of PAF occurs within the first minute of interaction between blood and cuprophane or AN69 membranes [11,12], as well as enhancement of oxidative metabolism. We hypothesized that PAF could be the factor triggering neutrophils to generate H2O2 during dialysis. Neutrophils isolated from HD patients were incubated in the presence of PAF. The PAF doses of 10-6, 10-7, 10-8, 10-9 mol/l were chosen because they are consistent with in vivo physiological PAF concentrations [13,14]. PAF at concentrations from 10-9 to 10-6 mol/l did not increase FITC-E. coli phagocytosis by PMN compared to unstimulated cells. In cytochalasin B pre-treated PMN, PAF increased spontaneously and PMA-stimulated H2O2 production in a dose-dependent manner (Table 3Go). These results demonstrate that in vitro PAF is capable of enhancing H2O2 production but not phagocytic capacity.


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Table 3. Effects of PAF on phagocytic functions

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this selected population of HD patients, neutrophils obtained before a routine dialysis session, exhibit normal phagocytic capacity and H2O2 production compared to healthy controls. During dialysis, we observed important changes in phagocytosis and H2O2 production, which are dependent on the HD membrane. PMN phagocytosis was enhanced in cuprophane-treated but not AN69-treated patients. In contrast, we observed a rapid, sustained increase in H2O2 production with both cuprophane and AN69 membranes. This dissociation between phagocytosis and H2O2 production is not surprising. It should be recalled that stimulation of ROS production by neutrophils does not necessarily imply that phagocytosis is also stimulated. Indeed, phagocytosis activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to produce ROS at the inner surface of the phagosome [2]. But, in addition to the stimulus of phagocytosis itself, the NADPH oxidase is both primed and activated by many other factors [15] such as N-formylated methionyl peptides, C5a, leukotriene B4 (LTB4), and most interestingly PAF.

In this study we tried to dissect the potential mechanisms involved in the intradialytic changes we observed. First, we excluded a direct activating effect of the dialysis membrane itself. Then we assessed the role of complement in the stimulation of phagocytosis during cuprophane dialysis. Two lines of evidence indicate that complement activation is involved in the stimulation of phagocytosis observed with cuprophane. First, rHuC5a at concentrations reached during in vivo cuprophane dialysis enhanced PMN phagocytosis. Second, the reuse procedure that abolished complement activation also abolished the enhancement of PMN phagocytosis induced by first-use cuprophane membrane.

However, several lines of evidence indicate that complement activation is not involved in the stimulation of H2O2 production observed during dialysis. First, incubation of whole blood with rHuC5a (at a concentration similar to the one observed during in vivo cuprophane dialysis) did not significantly enhance H2O2 production by neutrophils in vitro. Second, H2O2 production is enhanced during dialysis with the non-complement-activating AN69 membrane. However, it should be noted that while complement activation during AN69 dialysis is minimal in the blood phase, it is not zero. Actually, AN69 membrane activates complement, but the vast majority of the C3a and C5a molecules generated in the presence of AN69 remains bound to the membrane surface [16]. We can therefore not exclude the possibility that complement activation products, which are adsorbed on the membrane surface, play a role in the activation of H2O2 production observed during dialysis with AN69 membrane. Third, formaldehyde-reused cuprophane, which does not activate complement, is able to stimulate H2O2 production by neutrophils. Taken together, these observations suggest that another triggering mechanism might be involved in the activation of H2O2 production observed during dialysis.

Eventually we hypothesised that the enhanced H2O2 production observed dialysis with both complement- and non-complement-activating membranes could be related to PAF. Indeed, synthesis of PAF occurs within the first minute of interaction between blood and cuprophane [13] or AN69 [17] membranes, as well as enhancement of oxidative metabolism. Moreover, it has been previously shown that PAF has an effect on neutrophil function in the setting of HD [1113]. PAF is a potent inflammatory phospholipid synthesized by many types of tissues and cells including monocytes, macrophages, platelets, endothelial cells, and PMN. PAF has been implicated as an endogenous mediator of platelet aggregation and degranulation, cellular chemotaxis, and PMN priming, ultimately leading to organ injury in inflammatory disorders. We used PMN isolated from HD patients, since we have no information on PAF receptor expression in uraemic neutrophils, and because there could be a desensitization or a decrease in specific binding following repeated exposure to PAF. In vitro, phagocytosis was not significantly modified by adding synthetic PAF at the concentrations used. In agreement with a previous report [18], we observed in resting neutrophils that PAF did not stimulate H2O2 production, whereas, in neutrophils pre-treated with cytochalasin B, PAF increased spontaneous and PMA-stimulated H2O2 production in a dose-dependent manner. In this in vitro model, H2O2 production in response to the phagocytosis of E. coli cannot be interpreted since cytochalasin B blocks the phagocytosis. Although these observations strongly suggest that PAF could be the factor triggering oxygen radical production, several weaknesses must be considered. First, since PAF is rapidly inactivated in plasma by PAF acetyl hydrolases [10], the relevance of PAF in neutrophil activation in vivo might be minimized. However, release of PAF in plasma may nevertheless occur following inactivation of plasma antiproteinases, or in areas of close contact between cells and the extracellular matrix or artificial membranes from which antiproteinases are excluded [19]. Moreover, the release of PAF can be documented in plasma from the efferent line of cuprophane [13] and AN69 [17] dialysers during the first minute of interaction between blood and HD membranes. This early synthesis of PAF might be a relevant factor in priming PMN to generate H2O2 production. A second potential weakness of the study is that we did not correlate the changes in H2O2 production to changes in PAF. However, it is likely that following synthesis, PAF is retained rather than being released and acts locally, thereby preventing activation of PMN in the fluid phase. Indeed, it has been shown that PMN adhesion to endothelial cells via CD11b–intercellular adhesion molecule interactions may trigger the synthesis and the expression of membrane-bound PAF, which remains active, thus potentiating local PMN adhesion and function [14,20]. Another important consideration concerning PAF enhancement of PMN responses is the possibility that other cells respond to PAF and produce substances which modulate PMN response [21]. For example, stimulated platelets may produce arachidonic acid metabolites, which are in turn modified by PMN enzymes. In addition, PMN are exposed to a multitude of bioactive substances during dialysis and these various substances acting in concert or sequentially on PMN can profoundly influence their responses.

In summary, we have shown that before dialysis, neutrophils from HD patients exhibit normal phagocytic capability and normal H2O2 production. The intradialytic stimulation of phagocytosis is related to the activation of complement by cuprophane membranes. On the other hand, neutrophil H2O2 production is stimulated by both complement-activating and non-complement-activating membranes, suggesting that another trigger is involved. We suggest that PAF could be responsible for this oxygen radical production.



   Acknowledgments
 
This project was supported by FNRS grant No. 3.4548.98 and by Fondation Erasme.



   Notes
 
Correspondence and offprint requests to: Karine Gastaldello, Department of Nephrology, 808 Route de Lennik, B-1070 Brussels, Belgium.kgastald{at}ulb.ac.be Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 17. 2.00
Revision received 21. 4.00.