Unidad de Investigación, Servicio de Nefrología, Hospital Universitario Reina Sofía, Córdoba, Spain
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Abstract |
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Methods. The study was performed in vitro examining the effects of CU membrane and LPS on mononuclear cells from normal subjects and from end-stage renal failure patients. Cells were analysed by flow cytometry with fluorescent monoclonal antibodies to determine CD14 expression and with Annexin-V labelling to determine apoptosis.
Results. In mononuclear cells from uraemic patients cultured for 48 h, there was a subset of cells with low CD14 expression; this subset of cells was not observed in normal monocytes cultured for the same period of time. Cells with low CD14 expression were also observed when normal or uraemic mononuclear cells were cultured in the presence of CU membrane. Simultaneous measurement of apoptosis and CD14 expression revealed that cells with low CD14 expression underwent apoptosis. The addition of LPS to the medium markedly reduced the number of mononuclear cells with low CD14 expression and also reduced the rate of apoptosis in these cells.
Conclusion. Our data suggest that mononuclear cell apoptosis induced by uraemia and the CU membrane is associated with low CD14 expression. Furthermore, LPS prevented the decrease in CD14 and reduced the rate of apoptosis.
Keywords: apoptosis; CD14; haemodialysis; LPS; mononuclear cells
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Introduction |
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Studies on normal monocytes have shown that the down-regulation or enzymatic removal of CD14 molecules results in monocyte apoptosis whereas increased expression of CD14 protects these cells from apoptosis [5,6]. The CD14 molecule is the receptor for the LPSlipopolysaccharide-binding-protein complex (LPSLBP complex); it is a 53 kDa glycoprotein anchored to the cell membrane by a phosphatidylinositol linker. CD14 is expressed in pro-monocytes, monocytes, various tissue macrophages, and in activated granulocytes. The stimulation of monocytes by LPS not only induces up-regulation of the CD14 receptor [7], but also reduces apoptosis [6].
A recent report showed a lower CD14 expression in monocytes from uraemic patients [8], and there is additional evidence that during haemodialysis, LPS may enter the blood compartment across the dialyser and induce mononuclear cell activation [912]. Thus, we hypothesized that the increased mononuclear cell apoptosis observed in uraemic patients may be associated with a low expression of the CD14 molecule.
The aim of the present study was to investigate whether increased apoptosis in cultured mononuclear cells from uraemic patients was related to a down-regulation of the CD14 molecule. Furthermore, we designed in vitro experiments to evaluate a possible relationship between the mononuclear cell apoptosis induced by the Cuprophan (CU) membrane and a low CD14 expression. As LPS up-regulates CD14 expression in normal mononuclear cells, we evaluated whether LPS was able to increase CD14 expression and reduce the rate of apoptosis in uraemic mononuclear cells.
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Materials and methods |
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After purification, representative control experiments were performed in cells isolated by flow cytometry and sorted using a mAb against the CD14 molecule (Leu-M3, Becton-Dickinson). In these experiments, the forward and side light scatter analysis in the cytometer allows identification and gating of CD14+ adherent mononuclear cells into a group, and the rest of the leukocytes were gated out [6]. These cytometric parameters were maintained in all subsequent analysis.
Cell culture
Adherent mononuclear cells were cultured at 37°C in complete culture medium containing RPMI 1640 supplemented with L-glutamine (2 mM), HEPES (20 mM), sodium pyruvate (1 mM), streptomycin (50 ng/ml), penicillin (100 UI/ml), and 10% fetal bovine serum (Bio-Whittaker, Walkersville, MD, USA). Serum was heated (56°C during a 60-min period) to inactivate complement in order to evaluate the effect of the CU membrane itself on mononuclear cell apoptosis independently of the complement activation. Cells were seeded in 96-well microtitre plates (Falcon, Becton Dickinson and Company, Paramus, NJ, USA) at 2x105 cells per well and incubated in the presence of 0.6 cm2 of CU plate membrane dialyser (Lundia IC 5N, GAMBRO, Sweden) or AN69 polyacrylonitrile plate membrane dialyser (Biospal 3000S, Hospal, France). In designated experiments, LPS from Escherichia coli strain 0127: B8 (1 ng/ml) (Sigma Chemical Co., Poole, UK), was added to the culture medium. In addition, some experiments were performed using F(ab')2 fragments of anti-CD14 mAb.
Purification of F(ab')2 fragment of CD14 mAb
The CD14 mAb was dialysed with acetate buffer (pH 4.0, during 4 h at 4°C). After dialysis the concentration of mAb was adjusted to 1.5 mg/ml in acetate buffer. Pepsin (100 ml of 0.1 mg/ml) was added to each tube, and incubated during 48 h at 37°C. After incubation, the reaction was stooped by adding 50 ml of 2 M Tris base and was then dialysed against 1 l of PBS during 24 h at 4°C to eliminate acetate buffer. All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).
Determination of CD14 expression and LPSFITC binding
Cells (105/ml) were incubated for 30 min at 4°C with 20 µl of mAb phycoerytrhin conjugated against the CD14 molecule (Becton Dickinson). Thereafter, cells were washed with PBS-0.1% azide and resuspended in 0.5 ml of 1% paraformaldehide. Background fluorescence was determined by PE-conjugated mouse immunoglobulins. Cytofluorometric analysis was performed with a FACScan cytometer (Becton Dickinson). To study LPS binding, cells were incubated during 30 min with 1 ng/ml of LPSfluorescein isothiocyanate (FITC). Background fluorescence was determined using FITC-conjugated mouse immunoglobulins. After incubation, cells were washed, resuspended in PBS, and immediately analysed by flow cytometry.
Evaluation of cell apoptosis
One of the characteristics of cells undergoing apoptosis is the translocation of phosphatidal serine from the inner to the outer side of the cell membrane. This translocation occurs during the early-intermediate stages of apoptosis, and it may be detected using Annexin-V, a Ca2+-dependent phospholipid-binding protein. Thus, an FITCAnnexin-V probe was used to determine the percentage of apoptosis [13]. This method has been widely used to quantify the percentage of cells undergoing apoptosis. Cells were washed in PBS and adjusted to 5x105/ml in binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; Bender MedSystems, Vienna, Austria) and filtered through 0.2 µm filter. Five microlitres of Annexin-VFITC (Bender MedSystems) were added to 195 µl cell suspension, incubated for 10 min in dark, and the cells were washed and resuspended in 190 µl binding buffer with 10 µl propidium iodide (PI) stock solution (20 µg/ml). The degree of apoptosis was assessed by flow cytometry. Live cells were negative for both dyes, necrotic cells or late apoptotic cells were positive for both fluorochomes and were not considered apoptotic, and apoptotic cells were positive for Annexin-V but negative for PI. Background fluorescence was determined using FITC-conjugated mouse immunoglobulins.
Simultaneous detection of CD14 expression and apoptosis by Annexin-V binding
Monocytes were double labelled with PE-conjugated Leu M3 mAb and Annexin-VFITC in HEPES buffer (Bio-Whittaker) during 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as control. Cells that showed staining were then washed and fixed in 4% paraformaldehyde and studied by flow cytometry.
Statistical analysis
Results are presented as mean±SD. Non-parametric data were compared by the KruskalWallis test. Comparisons between two means were analysed by the MannWhitney test for unpaired data and the Wilcoxon signed rank test for paired data. Differences were considered significant at the P<0.05 level.
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Results |
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The percentage of apoptosis was similar in freshly isolated uraemic and normal mononuclear cells (5±2 and 5±1%, respectively). In both uraemic and normal mononuclear cells, viability was >90% as assessed by trypan blue exclusion. In uraemic cells, the percentage of apoptosis increased to 19±6 (P<0.05) and 28±7% (P<0.01) after 24 and 48 h of culture, respectively (Figure 3). However, in normal cells, the percentage of apoptosis did not increase during culture (6±1 and 8±2% at 24 and 48 h of culture, respectively) (Figure 3
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CD14 expression and apoptosis in uraemic and normal mononuclear cells cultured with the CU membrane: the effect of LPS
After 4 h of culture with the CU membrane, the expression of CD14 increased to similar levels in normal and uraemic mononuclear cells (752±57 and 774±86, respectively) (Figure 4). At 24 h, the expression of CD14 decreased to similar levels in both normal and uraemic cells (705±54 and 708±65, respectively). However, these values were still significantly increased when compared with pre-culture values (P<0.05). At 48 h, the values of CD14 expression in normal and uraemic cells were unchanged compared with the values at 24 h (702±37 and 706±29, respectively) (Figure 4
). The changes in LPSFITC binding in uraemic and normal cells during 48 h culture with CU membrane were similar to the CD14 expression changes (data not shown). The in vitro effect of CU membrane on mononuclear cell apoptosis is shown in Figure 3
. After 24-h culture with CU membrane, apoptosis was observed in 26±5% of normal and 32±8% of uraemic cells (P<0.05); after 48 h, apoptosis significantly increased (P<0.05) to 32±6 and 47±11% in normal and uraemic cells, respectively, and was greater in uraemic than in normal cells (P<0.02). In mononuclear cells from uraemic patients and from controls cultured with AN69 membrane, the CD14 expression and the rate of apoptosis were similar to the values observed without membrane controls.
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In cells cultured with the CU membrane, the addition of LPS produced a decrease in apoptosis from 32±6 to 6±2% (P<0.02) in normal cells and from 47±11 to 8±4% (P<0.02) in uraemic cells. Furthermore, the simultaneous evaluations of apoptosis and CD14 expression in normal and uraemic individual cells given LPS revealed that the subset of cells with low CD14 expression was not present (Figure 6B).
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Discussion |
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In the present study there were two main findings: (i) the spontaneous apoptosis observed in cultured uraemic cells or in normal and uraemic cells exposed to the CU membrane was associated with a decrease in CD14 expression, and (ii) this decrease in CD14 expression was prevented by LPS. Thus, the subset of cells having low CD14 expression and a high rate of apoptosis was no longer observed when LPS was added to the culture medium.
The observed decrease in CD14 expression in uraemic mononuclear cells may be directly related to uraemia itself. For example, Glorieux et al. [8] reported that uraemic ultrafiltrate suppressed basal CD14 expression of normal mononuclear cells and inhibited the stimulation of CD14 expression induced by calcitriol. In addition, we found that CU exposure membrane decreases the expression of CD14 in a large number of cells from uraemic patients and normal subjects, suggesting that exposure to CU may cause a decrease in CD14 expression that is independent of uraemia. An effect of CU membrane on CD14 expression was suggested by Kaupke et al. [14] who showed that mononuclear cells from patients haemodialysed with the CU membrane had decreased CD14 expression. However, other authors have reported that CU membranes induce the up-regulation of mononuclear cell CD14 expression [15]. These discrepancies may be attributed to the action of other factors such as cytokines and complement fractions [16], which may modulate the expression of CD14 in mononuclear cells from haemodialysed patients. Nevertheless, the results from the present study and reports by others suggest that uraemia and CU membranes induce a decrease in mononuclear cell CD14 expression. The underlying mechanism for this decrease in CD14 expression is unknown and cannot be further clarified by the present findings.
In the present study, simultaneous measurements of apoptosis and CD14 expression revealed that cells with low CD14 expression underwent apoptosis. These results are in agreement with a previous report by Heidenreich et al. [6] who showed that apoptosis of normal mononuclear cells is associated with the down-regulation of CD14, suggesting that the CD14 molecule plays a role in monocyte survival. Furthermore, we observed that addition of LPS to the culture medium failed to produce a population of cells with low CD14 expression and a high rate of apoptosis. Other authors have shown that LPS up-regulates CD14 expression in mononuclear cells cultured in vitro, and that this is associated with a decrease in apoptosis [5,6]. Kanatani et al. [17] reported that CD14 expression was associated with apoptosis induced by transforming growth factor-beta plus dexamethasone. These findings in normal mononuclear cells are in agreement with our observations in normal and in uraemic cells.
We found that when LPS binding to CD14 was blocked by the addition of F(ab')2 fragments of mAb anti-CD14, LPS failed to prevent apoptosis. These results suggest that intracellular signals transduced after the interaction between LPS and CD14 participate in the anti-apoptotic effect of LPS. Also, these results support previous findings by others [18,19], demonstrating that LPS may prevent apoptosis by inducing mononuclear cell activation and differentiation. In support of this, LPS induces synthesis and release of TNF- and IL-1ß, two cytokines that inhibit mononuclear cell apoptosis. In addition, LPS has been shown to inhibit the formation of oxidative radicals that may trigger apoptosis in mononuclear cells [18]. Finally, the induction of anti-apoptotic molecules, A1 and A20, by LPS may also inhibit apoptosis [19].
We observed a clear association between low CD14 expression and apoptosis. However, it is difficult to determine whether the low CD14 expression was a cause or an epiphenomenon of the apoptotic process. Nevertheless, if a decrease was in CD14 expression was prevented by the action of LPS, apoptosis did not occur. This suggested that apoptosis and CD14 expression were not independent events.
Our findings regarding the effect of LPS on apoptosis may have some clinical relevance. Apoptosis is part of a complex mechanism during which activated mononuclear cells are removed from the circulation. During haemodialysis, blood contamination with LPS may interfere with the normal mechanism of mononuclear cell deletion and by doing so it may impede a normal inflammatory response. Nockher and Scherberich [11] have found an enhanced expression of CD14 in monocytes after haemodialysis, which was likely due to chronic exposure to trace amounts of endotoxins. The clinical relevance of the effect of LPS on apoptosis deserves some consideration. Blood contamination with LPS during haemodialysis may interfere with the normal mechanism of mononuclear cell deletion and may thereby affect control of inflammation and the normal immune response to infection. The presence of endotoxins may produce a chronic pro-inflammatory state, but this is stated with caution, as it is difficult to extrapolate in vitro findings to clinical situations. In fact, the effect of LPS on apoptosis is not uniform in all cells. For example, Frey and Finlay [20] found that LPS induced apoptosis of bovine endothelial cells through a CD14-dependent pathway.
In conclusion, our results suggest that mononuclear cell apoptosis induced by uraemia and CU membrane exposure is associated with low CD14 expression. Furthermore, LPS prevented the decrease in CD14 and reduced the rate of apoptosis.
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Acknowledgments |
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Notes |
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References |
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