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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Effects of dialyzer membrane on interleukin-1beta (IL-1beta) and IL-1beta-converting enzyme in mononuclear cells
1Division of Nephrology, Medizinische Hochschule Hannover, Hannover, Germany.
Insights
End-stage renal disease patients on Cuprophan hemodialysis show impaired interleukin-1beta (IL-1beta) secretion. Switching to synthetic membranes normalizes this function, reducing inflammation.
Area of Science:
- Immunology
- Nephrology
- Biochemistry
Background:
- End-stage renal disease (ESRD) patients on Cuprophan hemodialysis exhibit elevated cell-associated IL-1beta.
- IL-1beta is synthesized as inactive pro-IL-1beta and requires ICE for activation to mature mIL-1beta.
- This suggests a potential defect in IL-1beta release from activated cells in ESRD patients.
Purpose of the Study:
- To investigate whether ICE-dependent processing or secretion of mature IL-1beta is impaired in ESRD patients.
- To compare IL-1beta processing and secretion in patients undergoing peritoneal dialysis (PD) and hemodialysis (HD) with different membranes versus healthy controls.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from healthy controls, PD patients, and HD patients were stimulated with lipopolysaccharide (LPS).
- Cell-associated and extracellular pro-IL-1beta and mIL-1beta levels were measured using immunoassays.
- HD patients were studied with Cuprophan, low-flux polysulfon, and high-flux polysulfon membranes.
Main Results:
- Total IL-1beta production was similar across all groups.
- ICE-dependent processing of pro-IL-1beta to mIL-1beta was reduced in PD patients but not in HD patients on Cuprophan.
- Secretion of mIL-1beta was impaired in PD and Cuprophan-treated HD patients, but normalized when HD patients switched to polysulfon membranes.
Conclusions:
- PBMC IL-1beta production is normal in PD and HD patients.
- Impaired mIL-1beta secretion in Cuprophan-treated HD patients is normalized by switching to synthetic membranes.
- Reduced secretion of active mIL-1beta contributes to inflammation in ESRD and can be mitigated by using synthetic dialysis membranes.
Background:
In vitro stimulation of mononuclear cells (peripheral blood mononuclear cells; PBMCs) with an endotoxin (lipopolysaccharide; LPS) revealed elevated cell-associated levels of interleukin-1beta (IL-1beta) in end-stage renal disease (ESRD) patients on Cuprophan hemodialysis (HD), suggesting a defect in the process of IL-1beta's release from activated cells. IL-1beta is initially synthesized as an inactive precursor called proIL-1beta. ProIL-1beta is processed into the biologically active mature form of IL-1beta (mIL-1beta) requiring the specific IL-1beta-converting enzyme (ICE).
Methods:
Using specific immunoassays (enzyme-linked immunosorbent assays), we measured cell-associated and extracellular proIL-1beta as well as mIL-1beta in LPS-stimulated PBMCs to test whether ICE-dependent processing of proIL-1beta and/or secretion of mIL-1beta was impaired in ESRD patients compared with healthy controls. PBMCs of healthy controls (N = 9), of ESRD patients on peritoneal dialysis (PD, N = 10), and of those patients on intermittent HD (N = 8) were studied. The same HD patients were studied three times with low-flux Cuprophan (GFS 12), low-flux polysulfon (F6 HPS), and high-flux polysulfon (F60S) in randomized order. PBMCs were separated from whole blood by Ficoll-Hypaque centrifugation and incubated in vitro for 18 hours in the presence LPS (10 ng/mL). Extracellular (PBMC culture supernatants) and cell-associated (cell lysates) levels of proIL-1beta and mIL-1beta were measured.
Results:
The total production (cell-associated plus extracellular) of LPS-induced IL-1beta (proIL-1beta plus mIL-1beta) was similar in healthy controls (25.96 +/- 0.84 ng/2.5 x 10(6) PBMC), PD patients (29.53 +/- 1.31 ng/2.5 x 106 PBMC), and in Cuprophan-treated HD patients (23.28 +/- 1.24 ng/2.5 x 10(6) PBMC). In normal controls, 43.6% of the total IL-1beta was processed into mIL-1beta, which was significantly more than that in PD patients (27.3%, P < 0.02) but was similar to that in Cuprophan-treated HD patients (37.1%). Comparing cell-associated and extracellular concentrations of mIL-1beta, PBMCs of normal controls secreted 82.2% of mIL-1beta; this was significantly more than that in PD patients (59.4%, P < 0.01) and that in Cuprophan HD patients (54.2%, P < 0.01). When HD patients were switched from Cuprophan to F6 HPS or F60S, neither total IL-1beta production nor processing of IL-1beta changed. However, secretion of mIL-1beta increased significantly with F6 HPS (80.6%, P < 0.01) as well as with F60S (76.6%, P < 0.02) compared with Cuprophan.
Conclusion:
We conclude that the ability of PBMCs to produce IL-1beta in response to LPS is normal in PD patients as well as in HD patients. ICE-dependent processing of inactive proIL-1beta into biologically active mIL-1beta is reduced in PD patients, but not in HD patients. Secretion of mIL-1beta is impaired in PD and HD patients treated with Cuprophan. This impaired ability to secrete active mIL-1beta seems to be independent of ICE activity and is normalized when HD-patients are switched from Cuprophan to low- or high-flux polysulfon. Increased cell-associated levels of biologically active mIL-1beta in circulating PBMCs represent a state of inflammation that may contribute to chronic inflammatory diseases such as beta2-microglobulin amyloidosis. Replacement of Cuprophan by synthetic membranes normalizes PBMC function and reduces the state of inflammation in ESRD patients.
