1 Institute of Immunology and Transfusion Medicine and 2 Department of Nephrology, University of Lübeck School of Medicine, Lübeck, Germany
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Abstract |
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Methods. In 11 patients previously treated with polysulphone haemodiafiltration, UFR was reduced from 4046 ml/min to 2428 ml/min, then to 710 ml/min before it was reinstated at 4046 ml/min for periods of 4 weeks each. Monokine secretion into culture supernatants and mRNA expression (assessed using a novel Taqman PCR technique), were determined in a whole blood assay after lipopolysaccharide stimulation.
Results. Reduction of UFR led to a significant increase in IL-10 secretion and mRNA expression (P=0.012, P=0.001). Conversely, a substantial (but not complete) decrease was observed when UFR returned to initial levels. In contrast, supernatant concentrations of IL-1ß (P=0.04) and IL-6 (P=0.003), and mRNA expression of both monokines (P<0.001, P<0.001) decreased significantly when UFR was reduced. Calculation of the IL-1ß/IL-10 ratio also revealed a decrease when UFR was reduced, with an increase again being observed when the initial degree of UFR was reinstated (P<0.001).
Conclusions. These results indicate a significant impact of UFR on the production of monokines at both the transcriptional and the protein level. We suggest that middle molecule removal has to be considered as a possible pathophysiological mechanism to explain our findings. Since monokine production in vitro was shown to be closely correlated with the in vivo immune status in patients on cuprophane haemodialysis, further investigations are necessary to clarify the impact of UFR on the immunocompetence of patients under polysulphone haemodiafiltration.
Keywords: haemodiafiltration; immunocompetence; monokines; ultrafiltration flow rate
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Introduction |
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An impaired activation of T-lymphocytes by accessory cells such as monocytes seems to play a key role in dialysis related immunodeficiency [9]. As alterations in monokine production represent a common finding in haemodialysis patients, it was recently found that the production of regulatory (IL-10) and proinflammatory monokines (IL-1ß, IL-6) in vitro correlates closely with the immune defect and is therefore of high clinical relevance [10,11].
The current study examines the impact of UFR on the monokine response in an intra-individual study design. As the separation of monocytes from a physiological environment is likely to have profound modifying effects on cell function and can cause pre-activation [12], the induction of IL-10, IL-1ß and IL-6 upon lipopolysaccharide (LPS) stimulation was investigated under whole blood conditions in vitro. Monokine secretion into culture supernatants was compared to mRNA expression measured using a novel Taqman PCR-technique [13].
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Subjects and methods |
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We selected patients with a blood flow of 250 ml/min and a minimum withdrawal of 2000 ml, to exclude the possibility of any relevant back-filtration. The dialysate flow was 500 ml/min. The UFR was calculated for each patient considering replacement fluid, withdrawal and dialysis time. All participants had been haemodialysed at 9000 ml replacement fluid (UFR of 4046 ml/min) for a minimum of 2 months before the onset of the study. Subsequently the replacement fluid was reduced to 4500 ml (UFR 2428 ml/min), 0 ml (UFR 710 ml/min) and returned to 9000 ml (UFR 4046 ml/min) at 4-week intervals. The efficiency of haemodiafiltration treatment and UFR reduction was verified by determination of urea and ß2-microglobulin reduction ratio and ß2-microglobulin serum concentrations. Only sterile and pyrogen-free replacement fluid was used containing 34.0 mmol/l lactate (Biosol, Sondalo, Italy). Analysis of the dialysate revealed sterile water (culture test) and endotoxin levels of <0.24 IU/ml (LAL test) throughout the surveillance period. Whole-blood samples were always collected immediately before haemodialysis treatment at the onset of the study and at the end of each of the 4-week periods. The study was approved by the Ethics Commission of the Lübeck University School of Medicine. Written informed consent was provided by all participants.
Whole-blood assay
To approximate the in vivo immune status, the production of monokines was determined in non-separated blood [14]. In brief, 200 ml lithium heparinized blood was mixed with 1600 ml culture medium (RPMI-1640 medium, Biochrom, Berlin, Germany) supplemented with 1% L-glutamine and 1% penicillin/streptomycin. 200 ml of LPS solution was added, so that a final concentration of 1 mg/ml LPS was achieved for inducing IL-10, IL-1ß and IL-6. LPS-free cultures served as negative controls and were treated identically. Cultures were incubated under 5% CO2 in a humidified incubator at 37°C for 24 h.
Quantification of IL-10, IL-1ß, and IL-6 in culture supernatants
Supernatants from whole-blood assays were frozen at -80°C prior to monokine determination. Values of IL-10, IL-1ß and IL-6 were quantified by an enzyme immunoassay (EIA) technique (R&D Systems, Minneapolis, MN, USA). Determinations on all samples, negative controls and standards were performed in duplicate. According to information provided by the manufacturer, no cross-reactivity to any related cytokines was known.
Quantification of IL-10, IL-1ß, and IL-6 mRNA copies by Taqman PCR
For quantifying monokine mRNA, a novel Taqman technique was applied that was recently established in our laboratory [13]. In brief, total RNA was isolated from the whole-blood cultures immediately after a 24-h incubation using the Purescript RNA isolation kit (Gentra Systems, Minneapolis, USA). Sequence-specific PCR primers and fluorochrome-labelled internal oligonucleotide probes for IL-10, IL-1ß and IL-6 were designed using the Primer express software (Perkin Elmer Cetus, Foster City, CA, USA). In order to use cDNA in the quantitative PCR, 30 µl of total RNA were reverse transcribed using a commercial kit (Ready to Go, Pharmacia, Uppsala, Sweden).
The PCR reaction mixture contained 5 µl 10xTaqman A-buffer (500 mmol/l KCl, 100 mmol/l TrisHCl, 100 mmol/l EDTA, 600 nmol/l passive reference dye ROX at pH 8.3; Perkin Elmer, Foster City, CA, USA), 3.5 mmol/l MgCl2, 300 µmol/l dATP, dCTP, dGTP, 600 µmol/l dUTP, 100 nmol/l of forward and reverse primer, 100 nmol/l fluorogenic probe, U Ampli-Taq Gold-DNA-Polymerase (Perkin Elmer, Foster City, CA, USA), and 10 µl of water control, diluted standards or sample in a total volume of 50 µl. The PCR conditions were 10 min at 95°C for DNA-polymerase activation, followed by 40 cycles of 15 s at 95°C and 1 min 30 s at 60°C with a final 25°C hold.
In order to create standards, cDNA fragments were cloned that encoded ß-actin, IL-10, IL-1ß and IL-6. PCR-products were ligated into vector pCRII (Original TA Cloning Kit, Invitrogen, San Diego, CA), and were used to transform INVaF' competent E. coli cells. The plasmid concentration was determined by measuring the optical density at wavelength 260 nm using a U-3000 spectrophotometer (Hitachi, Tokyo, Japan). All PCRs for monokine mRNA quantification were performed in an ABI PRISM 7700 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). Standardized monokine mRNA quantities (monokine mRNA copies/106 ß-actin copies) were determined by dividing the interpolated values derived from the monokine standard curve by the ß-actin mRNA content (which served as a normalization factor). Analysis was performed in triplicate.
Statistical analysis
Statistical analysis was performed using commercially available software for personal computers (SSPS for Windows 8.0, SPSS GmbH, Munich, Germany). Concentrations of monokines in supernatants and numbers of mRNA copies/106 ß-actin copies were reported as mean±SD. The Friedman test and the appropriate post-hoc analysis (Wilcoxon Wilcox test) were used to compare non-parametrically distributed data. P values <0.05 were considered significant.
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Results |
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Determination of peripheral blood counts and C-reactive protein
Analysis of peripheral blood counts revealed constant monocyte counts throughout the study period (P=0.19), so that a possible influence on the observed results could be excluded: (UFR 4046 ml/min, 0.473±0.129x109/l; UFR 2428 ml/min, 0.541±0.212x109/l; UFR 710 ml/min, 0.486±0.185x109/l, and UFR 4046 ml/min, 0.444±0.151x109/l). Likewise, C-reactive protein levels remained unchanged (P=0.62): (UFR 4046 ml/min, 1.0±0.9 mg/l; UFR 2428 ml/min, 1.0±0.8 mg/l; UFR 710 ml/min, 0.9±0.7 mg/l; and UFR 4046 ml/min, 0.9±0.8 mg/l).
Secretion of IL-10, IL-1ß, and IL-6 in whole-blood culture supernatants
At the start of the study, all patients had been haemodialysed at a UFR of 4046 ml/min for a minimum of 2 months. Before the UFR was reduced to 2428 ml/min, 710 ml/min and returned to 4046 ml/min, secretion of monokines was determined to provide initial values (Figure 1). While the stepwise reduction in UFR to 710 ml/min resulted in a significant increase in IL-10 concentrations (P=0.012), the return to 4046 ml/min resulted in a substantial, although incom plete, reversal of this effect. In contrast, IL-1ß concentrations decreased in a stepwise manner (P=0.04) before they increased only slightly after UFR was reinstated at 4046 ml/min. Reduction of UFR also resulted in a lowering of IL-6 concentrations (P=0.003), but there was no increase in IL-6 levels, when the high UFR was reinstated. Analysis of LPS-free whole-blood cultures and serum samples of the patients revealed that there was no spontaneous monokine secretion throughout the study.
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Quantification of IL-10, IL-1ß, and IL-6 mRNA in whole-blood cultures
The content of monokine mRNA was quantified by Taqman PCR. Copy numbers of monokine mRNA were calculated per 106 ß-actin copies, whereby the latter served as a normalization factor to compensate for cell number variability. With reduction of the UFR, IL-10 mRNA copy numbers increased in a stepwise manner, and decreased again when the UFR was returned to 4046 ml/min (P=0.001) (Figure 2). In contrast, IL-1ß and IL-6 mRNA decreased (P<0.001, P<0.001) with reduction of the UFR. When the original UFR was reinstated, IL-1ß mRNA slightly increased again but there was no reversal of this effect for IL-6 mRNA.
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Discussion |
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Our results indicate that UFR has significant long-term effects on the production of monokines at both the transcriptional and the protein level within the same individual. Thus an independent influence on the production of the regulatory monokine IL-10 upon LPS stimulation but not spontaneous IL-10 secretion can be postulated. Subsequent reduction of UFR led to increasing concentrations of IL-10 in culture supernatants, whereas reinstatement of UFR at 4046 ml/min resulted in a subsequent decrease in the values. This result was confirmed by a parallel increase and subsequent decrease in IL-10 mRNA copy numbers. Moreover, it proves that UFR influences the IL-10 synthesis at the transcriptional level. Since monocyte counts remained unchanged throughout the study and copy numbers of mRNA were divided by 106 ß-actin copies for normalizing the data, it can be concluded that the increased IL-10 production is due to an increased synthesis rather than an increase in monocyte counts.
This finding could be of great relevance, since IL-10 exerts important regulatory effects on monocytes. It strongly reduces antigen-specific T-lymphocyte proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of MHC class II expression and inhibits the production of proinflammatory monokines, such as IL-1ß and IL-6 [1820]. Accordingly, with reduction of the UFR, we found a stepwise reduction of IL-1ß and IL-6 in our patients at both the protein and the mRNA level. Thus we suggest a dose-dependent relationship between the UFR and the production of IL-10 leading to a counter-regulation of the pro-inflammatory monokines IL-1ß and IL-6. Moreover, since the production of proinflammatory monokines upon LPS-stimulation in vitro was shown to correlate closely with clinical parameters [11], we postulate a direct impact of the UFR on the clinical immune status.
Comparison with healthy controls that were previously investigated in our laboratory under the same conditions [21] (data not shown), revealed identical levels of IL-6 in our patients prior to UFR reduction. Thus our data would be in accordance with recent findings that described comparable levels of proinflammatory monokines in patients treated with polysulphone haemodialysis and healthy controls [22].
The elimination of middle molecules must be regarded as a potential pathophysiological mechanism to explain our results, since immunomodulating effects of middle molecules have already been postulated [6]. In a recent study, a direct inhibition of lymphocyte proliferation and IL-2 production was demonstrated [7], and it remains unclear whether this inhibition is restricted to lymphocytes or whether middle molecules affect immune functions in a rather unspecific fashion. Determination of ß2-microglobulin reduction ratio confirmed a direct correlation between the UFR and middle-molecule elimination in our patients [23], and reduction of UFR resulted in an increase of ß2-microglobulin serum levels and vice versa (Table 1). In contrast, the urea reduction ratio remained unchanged throughout the study. Therefore it should be clarified whether an accumulation of middle molecules in patients under polysulphone haemodiafiltration enhances the production of IL-10 leading to an inhibition of IL-1ß and IL-6. Interestingly, when a UFR of 4046 ml/min was reinstated, the ß2-microglobulin reduction ratio and ß2-microglobulin serum levels were still elevated compared with the onset of the study. This could indicate either an increased ß2-microglobulin production, or that steady-state conditions were not achieved again within the observation period. Furthermore, it could explain why IL-1ß and IL-6 levels were still reduced. Since C-reactive protein levels remained constant and spontaneous monokine secretion into unstimulated controls was not detectable throughout the study, a direct influence of any undiscovered cytokine-inducing substances in the replacement fluid seems rather unlikely. Moreover, our findings indicate that the UFR could have an independent impact on key immune functions. This could be highly relevant, since the lower mortality in patients treated with haemodiafiltration has not been fully understood and the relative importance of middle-molecule removal vs membrane biocompatibility currently remains a matter of discussion [24,25]. Thus, additional investigations are required to clarify the impact of UFR on the immunocompetence of patients under haemodiafiltration.
In conclusion, the current study indicates a significant impact of UFR on the production of regulatory and proinflammatory monokines in patients under polysulphone haemodiafiltration. UFR reduction resulted in increased IL-10, but decreased IL-1ß and IL-6 production upon LPS stimulation. Since it was shown that monokine production correlates closely with the in vivo immune status in cuprophane haemodialysis, further studies are necessary to understand fully the pathophysiological mechanism and investigate the clinical relevance for patients under polysulphone haemodiafiltration.
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Acknowledgments |
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Notes |
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References |
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