IL-1ß, TNF-
and IL-6 release from monocytes in haemodialysis patients in relation to dialytic age
Grazia Malaponte,
Valentina Bevelacqua,
Pasquale Fatuzzo1,
Francesco Rapisarda1,
Giovanni Emmanuele2,
Salvatore Travali2 and
Maria Clorinda Mazzarino
Department of Biomedical Sciences, Section of General Pathology and
2 Section of Clinical Pathology and Molecular Oncology, and
1 Department of Surgery, Section of Surgical Nephrology, University of Catania, Italy
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Abstract
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Background. It has been suggested that changes in immune response to infectious agents in patients on haemodialysis might be due to impaired monocyte function; uraemic and haemodialysed patients overproduce proinflammatory cytokines, such as interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-
) and interleukin-6 (IL-6).
Methods. We quantitated the cytokines released into the plasma and into the supernatants of 24-h cultured purified monocytes, under basal conditions and after stimulation by lipopolysaccharide from Escherichia coli, in 15 healthy subjects (CON), 20 uraemic patients who had not yet started dialysis (CRF) and 60 haemodialysed patients (HD), who were divided into three groups of 20 patients corresponding to short-, medium- and long-term dialysis.
Results. Monocytes from HD patients spontaneously secreted significantly higher levels of cytokines than those from controls and uraemic patients who had not yet started dialysis. After stimulation with lipopolysaccharide (LPS), cytokine levels in culture supernatants of cells from HD patients were significantly lower than those from controls and uraemic patients. Moreover, levels of cytokines in monocyte supernatants and plasma from short-, medium- and long-term haemodialysed patients decreased progressively with dialytic age. Monocytes from haemodialysed patients tended to be constitutively active, but their ability to secrete proinflammatory cytokines was inversely correlated with dialytic age.
Conclusions. These results indicate that prolonged treatment with dialysis can be considered a form of chronic stress that causes the progressive activation of monocytes, which ultimately leads to monocyte exhaustion and dysfunction.
Keywords: chronic renal failure; cytokines; dialytic age; haemodialysis; uraemia
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Introduction
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Bacterial and viral infections represent the most frequent complications in patients with chronic renal failure (CRF) due to the changes in the immunological status of such patients [1]. It has been demonstrated that activation of immunocompetent cells, together with the loss of equilibrium between inflammation-inducing cytokines and their specific inhibitors, is responsible for the dysfunction of the immune system in these patients [2,3]. Moreover, paradoxically, this immunodeficient state is associated with the release of cytokines, such as interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-
) and interleukin-6 (IL-6), which play key roles in inflammatory processes [4]. In addition, it seems that the imbalance is accentuated, rather than corrected, by the dialytic therapy. However, it remains unclear whether a relationship exists between the release of cytokines and the duration of treatment with dialysis, namely dialytic age.
The aims of this study were to investigate: (i) the in vitro release of IL-1ß, TNF-
and IL-6 from purified monocytes of patients with CRF treated with conservative therapy, and of patients in dialytic therapy for different lengths of time; (ii) the in vitro cytokine secretion following the dialytic session; and (iii) the plasma levels of cytokines before and after a single session of dialysis.
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Subjects and methods
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Subjects
This study involved a total of 95 individuals in three groups, whose mean age, sex and clinical data are listed in Table 1
. There were 20 patients with severe CRF (creatinine clearance 14.2±3.1 ml/min) who were being treated with conservative therapy, 60 haemodialysed patients (HD), and 15 healthy subjects as controls (CON). On the basis of the dialytic age, the HD patients were divided further into three subgroups: 20 short-term HD patients, 20 medium-term HD patients and 20 long-term HD patients (mean time from the start of dialysis treatment 11.50±1.53, 33.93±1.89 and 90.80±6.85 months, respectively). Dialysis treatment comprised three 4-h sessions a week using cuprophane membranes (Disscap 160E; Hospal, Mirandola, Italy). The dialysate routinely used contained bicarbonate (34 mmol/l) and acetate (3 mmol/l).
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Table 1. Clinical data for healthy controls (CON) and for patients with chronic renal failure on conservative treatment (CRF), and haemodialysis (HD) patients
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Before and during the study, none of the patients presented with any clinical evidence of acute infection, autoimmune disease or malignancy, and none received drugs that might interfere with the immune response. The nutritional state of all patients was normal.
Before entering this study, all the enrolled patients gave their written informed consent.
Cell isolation, culture and stimulation of monocytes
Heparinized samples (2530 ml) of blood were taken from both controls and patients with CRF on conservative therapy, and also from all HD patients at the start (Ts) and end (Te) of a dialysis session. Blood was immediately centrifuged at 300 g for 15 min at room temperature. Aliquots of plasma were frozen at -70°C prior to analysis. Peripheral blood mononuclear cells were isolated as described by Böyum [5]. Monocytes were prepared by centrifugation at 500 g for 30 min at room temperature on an isosmotic solution of 46% Percoll (Pharmacia, Uppsala, Sweden). The purity of monocytes was analysed on a FACScan flow cytometer (Becton Dickinson, Milan, Italy) with monoclonal antibodies CD14 and CD11c/CD18 (Becton Dickinson). The estimated purity was
95%, and >90% of monocytes were positive for non-specific acid esterase activity. Cell viability was estimated, by the trypan blue exclusion test, to be close to 95%. Monocytes (2x106) were incubated for 20 h at 37°C in 5% CO2/95% air atmosphere, in hydrophobic Petriperm dishes (Heraeus, Hanau, Germany). They were cultured without stimulus for evaluation of the spontaneous release of cytokines, and also with 1 µg/ml lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5; Sigma Chemical Co., St Louis, MO, USA) for the evaluation of the release of cytokines after stimulation [6].
The supernatant was collected after the 20 h incubation and filtered through a filter with 0.2 nm pores (Sigma Chemical Co.). It was stored at -70°C prior to analysis of cytokines. All reagents used were LPS-free, as determined by the Limulus Amoebocyte Lysate (LAL) assay (Kabi vitrum, Munich, Germany).
IL-1ß, TNF-
and IL-6 assay
Levels of IL-1ß, TNF-
and IL-6 were determined in duplicate samples with a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA), in accordance with the manufacturer's instructions. The lower limit of detection was 4.5 pg/ml for IL-1ß, 8 pg/ml for TNF-
and 3.5 pg/ml for IL-6.
Extraction of RNA and the reverse transcriptionpolymerase chain reaction
Total RNA was extracted from monocytes that had been cultured for 4 h by the guanidium isothiocyanate/phenol/chloroform method, as described by Chomczynski and Sacchi [7]. For examination of the expression of IL-1ß, TNF-
and IL-6 mRNA, 1 µg of total RNA was reverse-transcribed in 20 µl of reaction mixture (50 mM TrisHCl pH 8.3, 75 mM potassium chloride, 3 mM magnesium chloride, 10 mM dithiothreitol) that contained 200 U of Moloney murine leukemia virus reverse transcriptase (RT; Amersham, Cleveland, OH, USA) and random hexamers (1 µg/reaction) as first-strand cDNA primers (Pharmacia), in the presence of 10 U of placental ribonuclease inhibitor (Pharmacia). Reactions were allowed to proceed at 25°C for 15 min and then at 37°C for 45 min. Thereafter, the DNA in an aliquot of each reaction mixture was amplified by the polymerase chain reaction (PCR) in 100 µl of a reaction mixture that contained 2.5 U of Taq DNA polymerase (AmpliTaq; Perkin Elmer Cetus, Emeryville, CA, USA) and 12.5 pmol of a primer pair specific for an individual cytokine. In all amplification reactions, primer pairs specific for ß-actin or phosphoglycerate kinase were included for co-amplification of a housekeeping gene to provide an internal control for quantitation of levels of mRNA (Table 3
). Samples were incubated under the conditions indicated in Table 3
and then held at 72°C for 10 min for final extension. They were stored at 4°C prior to analysis. Control reactions without RT were also performed to exclude the possibility of contamination by genomic DNA. Amplified products after RTPCR were separated by electrophoresis on a gel composed of 2% Nusieve and agarose (3:1 w/w; FMC BioProducts, Rockland, ME, USA) in 0.045 M Trisborate/1 mM EDTA buffer. They were visualized after staining with ethidium bromide, and a 100-bp ladder (Promega, Madison, WI, USA) was used as a source of size markers.
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Table 3. Linear regression analysis of the correlation between plasma levels of IL-1ß, TNF- and IL-6 in short-, medium- and long-term HD patients, and the secretion in vitro of the same cytokines
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Statistical analysis
Statistical analysis was performed with the SPSSv 6.1 software package (SPSS Inc., Chicago, IL, USA). Each result was calculated as the mean±standard error of the mean (SEM). Evaluation of the statistical significance of differences for paired and unpaired values was performed using the Student's t-test. Correlations were examined by linear regression analysis. Values of P<0.05 were considered to represent statistical significance.
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Results
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Release of IL-1ß, TNF-
and IL-6 from purified monocytes, and the effect of dialysis treatment
The mean values for the spontaneous and LPS-induced release of IL-1ß, TNF-
and IL-6 from purified monocytes, obtained from all subjects enrolled, are shown in Figure 1
. Note that the levels of spontaneously released IL-1ß, TNF-
and IL-6 (Figure 1A
, B
and C
, respectively) for controls and for patients with CRF on conservative therapy were very low, which was in contrast to HD patients, whose monocytes released spontaneously high levels of cytokines (P<0.0001). Surprisingly, cytokine release decreased significantly in relation to dialytic age, particularly in the long-term HD patients. The differences between spontaneously released cytokine levels for short-term vs medium- and long-term, and for medium- vs long-term HD patients were significant, with a P value ranging from 0.02 to 0.0001.

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Fig. 1. Levels of IL-1ß (A), TNF- (B) and IL-6 (C) released into the culture supernatant of purified monocytes, with or without stimulation by LPS. Monocytes were prepared from the blood of healthy subjects (CON), patients with CRF on conservative therapy and short-, medium- and long-term HD patients. The results are shown as means±SEM.
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LPS stimulation of monocytes from CON and CRF patients determined a sharp increase in IL-1ß, TNF-
and IL-6 levels, as is well known. Also, in HD patients, LPS led to an increase in cytokine levels, although not to the same extent; moreover the LPS-induced release decreased significantly with the months of treatment (P values ranged from 0.05 to 0.0001).
Figure 1
also show the spontaneous and LPS-induced release of IL-1ß, TNF-
and IL-6 from monocytes collected from HD patients at the end (Te) of the dialysis sessions, comparing them with the basal levels (Ts).
At the end of any dialytic session (Te), both the spontaneous and the LPS-induced release of cytokines showed a moderate increase compared with the Ts values (P values ranged from 0.05 to 0.01), supporting the hypothesis that dialytic treatment by itself promotes pro-inflammatory cytokine release. As was shown for the basal values, cytokine release was inversely correlated to dialytic age, with lower values in long-term HD patients than in medium- and short-term patients (P values ranged from 0.05 to 0.0001).
Analysis of levels of IL-1ß, TNF-
and IL-6 mRNA in monocytes by semiquantitative RTPCR
Cytokine mRNA expression was evaluated in purified monocytes, cultivated either in the presence or absence of LPS; the experiments were carried out several times for each subject, and for HD patients before and after the dialytic treatment. Figure 2
shows the mean expression of IL-1ß, TNF-
and IL-6 mRNAs evaluated in all patients, as calculated by densitometry in comparison to the steady-state levels of endogenous stable reference mRNAs.

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Fig. 2. Levels of IL-1ß, TNF- and IL-6 mRNAs expression in monocytes, stimulated (+) or not () with LPS, from all CRF and HD (short-, medium- and long-term groups) patients. After normalization of the specific genes to the appropriate housekeeping gene (actin or phosphoglycerate kinase) the results are plotted as means of normalized arbitrary units. LPS stimulated the expression of cytokine mRNAs, but its effects were reduced as the dialytic age increased (S, short term; M, medium term; L, long term). Bars indicate standard deviations.
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Figure 3
is a typical example of our RTPCR experiments. No gene expression for IL-1ß, TNF-
and IL-6 was detected under basal conditions in monocytes from controls and from patients with CRF, while transcripts for all these cytokines were easily observed in monocytes from HD patients. Our study also shows that the steady-state levels of IL-1ß, TNF-
and IL-6 mRNAs in unstimulated monocytes from short-term HD patients were slightly higher than those in monocytes from medium- and long-term HD patients. Although cytokine expression levels in LPS-stimulated monocytes were clearly increased in all HD samples compared with the unstimulated cultures, the previously observed, dialytic age-dependent down-regulation was appreciable.

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Fig. 3. Representative cases of cytokine mRNA expression in each group, as evaluated by semi-quantitative RTPCR. Actin (ACT) and phosphoglycerate kinase (PGK) are utilized as housekeeping genes.
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Plasma levels of IL-1ß, TNF-
and IL-6
Plasma levels of IL-1ß, TNF-
and IL-6 from controls, patients with CRF on conservative therapy and from short-, medium- and long-term HD patients are shown in Figure 4
. The levels of IL-1ß, TNF-
and IL-6 in plasma from controls and patients with CRF on conservative therapy were very low compared with basal plasma levels in HD patients (P<0.0001). IL-1ß, TNF-
and IL-6 in short-term HD patients were significantly higher than those in medium- and long-term HD patients, as well as in medium-term compared with long-term HD patients (P values ranged from 0.05 to 0.0001), supporting the results obtained in vitro. Moreover, plasma levels of cytokines at Te were always higher than those at Ts (P values ranged from 0.05 to 0.0001).

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Fig. 4. Plasma levels of IL-1ß (A), TNF- (B) and IL-6 (C) in healthy subjects (CON), patients with chronic renal failure on conservative treatment (CRF), and short-, medium- and long-term HD patients before (Ts) and after (Te) a dialytic session. Values are expressed as means±SEM.
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Correlations between levels of IL-1ß, TNF-
and IL-6 in plasma from short-, medium- and long-term HD patients, and levels of the same cytokines released from purified monocytes in supernatants of cultured cells are shown in Table 2
; they appear to be significant.
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Discussion
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This study confirmed that, in HD patients, the mechanisms that regulate the secretion of inflammation-inducing cytokines are active and are probably responsible for the imbalance in the immune system reported in these patients [2,3]. Purified monocytes from HD patients spontaneously secreted amounts of IL-1ß, TNF-
and IL-6 that were significantly higher than those secreted by monocytes from healthy subjects and from patients with CRF on conservative therapy, as reported in several other studies [8,9]. The increased spontaneous in vitro release of cytokines from purified monocytes clearly reflects the chronic activation of monocytes as demonstrated by the correlation with plasma cytokine levels.
Moreover, we also found increased levels of inflammatory cytokines in plasma from HD patients, which was dependent on dialytic age.
In purified monocytes from HD patients, increased basal levels of cytokine secretion appear to be related to the mRNAs levels evaluated, as compared with controls and patients with CRF; moreover, all three cytokines showed a down-regulation that was inversely correlated to dialytic age, with the expression levels being lower in long-term compared with medium- and short-term HD patients. These findings suggest that cytokine expression is regulated also at the transcriptional level. Our results are in accordance with previous studies [8,10,11]. In contrast, Lonnemann et al. [12] found no differences in total production of IL-1ß between Cuprophan-HD and normal controls, demonstrating also that reduced endotoxin-induced extracellular levels of IL-1ß depend on an impaired secretion but not on reduced production [13]. These controversial results could be explained by the different experimental systems (purified monocytes vs PBMC) or culture techniques [6,9].
The elevated levels of cytokines in HD patients are probably due to the stress of uraemia [11], and the dialytic treatment itself acts as a pro-inflammatory stimulus contributing to a further increase in cytokine secretion at the end of each session. In fact, the dialysis membrane itself can induce marked increases in the release of cytokines [1416]. The significant correlation between the plasma cytokine levels and those secreted in the supernatants of purified monocytes adds further weight to our argument. Direct contact of blood cells with dialysis membranes, complement activation and microbial contamination of the dialysate might be responsible for the activation that occurs during the course of a dialytic session [1719]. In our study we utilized a Cuprophan membrane that (even if it is still in use in several dialysis centres) is one of the most bio-incompatible dialytic membranes and may contribute to emphasizing the release of pro-inflammatory cytokines.
The extent of the LPS-induced release of IL-1ß, TNF-
and IL-6 from purified monocytes of HD patients was slightly lower than that of healthy subjects and of patients with CRF. In short-term HD patients, the cytokine release was significantly greater than that in medium- and long-term HD patients both before and after a dialytic session. These results suggest that IL-1ß, TNF-
and IL-6, acting synergistically, contribute to a monocyte activation that is also inversely correlated to dialytic age.
It is likely that the capacity of purified monocytes from long-term HD patients to release cytokines is impaired because of the chronic exposure to exogenous stimuli, leading to chronic in vivo stimulation [6,20].
In the light of recent studies [21], however, to clarify monocyte response to the pro-inflammatory stimuli from dialysis procedures, intracellular and secreted levels of the various cytokines must be evaluated.
In conclusion, we confirmed the enhanced basal secretion of cytokines and the impaired ability of monocytes from HD patients to respond to stimulation. The progressive impairment of monocytes increased with dialytic age, supporting the fact that prolonged haemodialytic treatment with cuprophane might be responsible for the poor immune response and may contribute to the deterioration of the immunodeficient status of HD patients.
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Acknowledgments
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The authors thank Professor Dr Claudio Franceschi and Dr Max Bonafè for kindly reviewing this text. This work was supported in part by funds MURST 60% from Dr G. Malaponte.
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Notes
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Correspondence and offprint requests to: Dr Grazia Malaponte, Department of Biomedical Sciences, Section of General Pathology, University of Catania, Via Androne 83, 95124, Catania, Italy. Email: g.malaponte{at}unict.it 
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Received for publication: 23. 7.01
Accepted in revised form: 7. 5.02