1 Renal Division, Department of Internal Medicine and 2 Department of Clinical Biology, Microbiology and Immunology, University Hospital, Gent, Belgium and 3 Renal Division, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA
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Abstract |
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Methods. In the present in vitro study, the effect of calcitriol, uraemic ultrafiltrate (UUF) and a combination of both on the human promyelocytic leukaemia cell line, HL-60, was studied by evaluating bromodeoxyuridine (BrdU) incorporation into the DNA, luminol-amplified chemiluminescence (CL) production, expression of CD14, and levels of vitamin D receptor mRNA (VDR mRNA) and CD14 mRNA.
Results. The ability of calcitriol to block cell proliferation (37.4±5.4 to 30.5±5.6% cells incorporating BrdU, P<0.01) was neutralized when UUF was applied together with calcitriol (53.4±21.3% cells incorporating BrdU, P<0.01 vs calcitriol alone). Similarly to what was observed for BrdU incorporation, the CL production of HL-60 cells was enhanced in the presence of calcitriol (20126±10154 to 61528±24021 cpm, P<0.01), and was suppressed again in the presence of calcitriol and UUF (20916±12075 cpm, P<0.01 vs calcitriol alone); finally UUF also inhibited the calcitriol-induced CD14 expression (71.1±11.2 to 54.9±17.7% CD14 positive cells, P<0.05). On the other hand, the calcitriol-induced CD14 mRNA levels were not significantly different in the presence of calcitriol and UUF compared to calcitriol alone. This points to an inhibition by UUF at a post-transcriptional level. Similar data were found for VDR mRNA levels. UUF was fractionated by HPLC in four fractions, hydrophilic uraemic solutes being eluted first (F1) and hydrophobic solutes being eluted last (F4); fractions 1, 2 and 3 simultaneously affected both BrdU incorporation and CL production in a significant way.
Conclusions. It is concluded that UUF contains factors that impair calcitriol-activated function of HL-60 cells. Hence, the differentiation and immune response of these promyelocytic leukaemia cells, as induced by the supplementation of calcitriol, is neutralized in the presence of uraemic biological fluids. This may be of relevance for the propensity to infection and malignancy of the uraemic patient.
Keywords: calcitriol; HL-60 cells; immune system; uraemia; vitamin D
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Introduction |
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Chronic renal failure is associated with both abnormal calcitriol metabolism [12] and functional anomalies of the immune system [13], resulting in an increased incidence of infection and a higher risk of malignancy [14]. It has been demonstrated that uraemic biological fluids inhibit cell-mediated immune responses in vitro [15] and suppress calcitriol synthesis as well as its biological actions. The latter are mediated through regulation of gene expression in a ligand-dependent manner. In addition, CRF is associated with calcitriol deficiency and with resistance to its biological functions [16].
The human promyelocytic leukemia cell line, HL-60, has proved to be an excellent model for studying the differentiation of leukocytes and their capacity to destroy infective agents. Calcitriol has been shown to induce HL-60 cells to differentiate along the monocytic/macrophage pathway [10].
In the present in vitro study, the effect of uraemic ultrafiltrate (UUF) on the response of HL-60 cells to 1,25(OH)2D3 was evaluated. The following parameters were studied: (i) bromodeoxyuridine (BrdU) incorporation into DNA as a parameter of cell proliferation, (ii) luminol-amplified chemiluminescence (CL) response as a measure of free-radical production, (iii) expression of CD14, a lipopolysaccharide receptor, and (iv) levels of CD14 mRNA and VDR mRNA.
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Subjects and methods |
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Preparation of uraemic ultrafiltrate (UUF), normal ultrafiltrate (NUF) and fractions (F) of the uraemic ultrafiltrate
UUF (135.7 mmol/l Na+; 4.9 mmol/l K+; creatinine 10.4 mg/dl; urea 135.0 mg/dl) was pooled from nine chronic haemodialysis patients (four men and five women with a mean age of 58.3±13.8 years). Five patients were treated by low-flux polysulphone dialysers (Rapido BLS 643, Sorin-Bellco, Mirandola, Italy), and the other four patients with high-flux polysulphone dialysers (Rapido BLS 627). Ultrafiltrate was collected at the beginning of the haemodialysis session and filtered through a 0.2 µm filter (Minisart NML, Sartorius GmbH, Göttingen, Germany). Each 5 ml of UUF was separated by semi-preparative, reversed-phase HPLC using a 10 µm Rsil C18 column (250x10 mm) (Bio-Rad Laboratories, Hercules, CA) at a flow rate of 3 ml/min, with a linear gradient of 50 mmol/l ammonium formate pH 4.0 from 100 to 0%, and methanol 0% to 100% over 60 min. This approach resulted in a separation of hydrophilic and hydrophobic compounds respectively eluting early or late during chromatography. HPLC peaks were detected by monitoring of ultraviolet-absorption at 254 nm (Uvicord SII, Pharmacia, Bromma, Sweden). We tested fractions collected from 9 to 16 min (F1), 17 to 24 min (F2), 25 to 40 min (F3) and 41 to 60 min (F4).
NUF (136.4 mmol/l Na+; 3.6 mmol/l K+; creatinine 1.2 mg/dl; urea 25 mg/dl) pooled from three healthy donors (one man and two women with a mean age of 43.0±12.1 years) was filtered through an Amicon Centriflo® ultrafiltration membrane cone (Danvers, MA, USA), and each 5 ml was subjected to the same HPLC treatment as UUF.
UUF and NUF were reconstituted from HPLC fractions collected after the first 8 min of chromatography, omitting the dead volume and the salt-containing fraction, or fractions were kept separately. UUF, NUF, and HPLC fractions were then lyophilized and redissolved in 5 ml of standard culture medium.
Experimental media
HL-60 cells were cultured, up to 7 days, in (i) medium, (ii) medium containing UUF, (iii) medium containing 10 nmol/l calcitriol, and (iv) medium containing 10 nmol/l calcitriol and UUF, NUF or the HPLC fractions of the UUF (F1, F2, F3 and F4). Cell viability exceeded 90% as assessed by propidium iodide exclusion on flow cytometry.
Bromodeoxyuridine incorporation (BrdU)
At day 7 of incubation, cell proliferation by the measurement of the incorporation of BrdU into the DNA was evaluated. Anti-BrdU (Becton Dickinson, San Jose, CA, USA) was used in flow cytometric analysis to identify cells that were synthesizing DNA during exposure to BrdU (Sigma Chemical Co, St Louis, MO, USA).
Cells were incubated for 30 min, with BrdU (10 µmol/l) in the CO2 incubator at 37°C. After two washings with 1% bovine serum albumin/phosphate-buffered saline (BSA/PBS), the pellet was resuspended in 200 µl of PBS on ice, and to fix the cells, this suspension was slowly added to 5 ml of 70% ethanol (-20°C) followed by a 30-min incubation period on ice. After centrifugation and aspiration of the supernatant, to denature the DNA, 1 ml 2N HCl/0.5% Triton X-100 was slowly added to the pellet followed by a 30-min incubation period at room temperature. After centrifugation, 1 ml of 0.1 mol/l Na2B4O4.10H2O, pH 8.5, was added to the pellet to neutralize the acid, followed by a second centrifugation step. Cell concentration was adjusted with 0.5% Tween/ 1.0% BSA/PBS to achieve 106 cells/test. Cells were incubated with fluorescein isothiocyanate (FITC)-conjugated Anti-BrdU for 30 min and washed once in 1 ml 0.5% Tween/ 1.0% BSA/PBS, and finally resuspended in PBS containing 5 µg/ml propidium iodide and analysed on a FACScan (Becton Dickinson).
Chemiluminescence production (CL production)
CL production by HL-60 cells was determined at day 7 of incubation in the different culture media. We added 500 µl of luminol solution (56 µmol/l) and 100 µl of HBBS or stimulus (10 mg/l phorbol 12-myristate 13-acetate (PMA)) to 50 µl of cell suspension. The test tube was immediately processed into a luminescence analyser (Lumicon, Hamilton, Switzerland). The photon-counting value, which is indicated arbitrarily in relative light units (RLU), was registered with a counting interval set at 30 s and a total recycling time of 30 min. Integral of counts over the total cycling time was calculated.
Analysis of membrane-bound CD14 expression on HL-60 cells
Expression of CD14 on HL-60 cells was assessed by direct immunofluorescence after 2 days of culture. HL-60 cells were stimulated for 10 min at 37°C with PMA (2 mg/l). 50 µl cell suspension was incubated with SimultestTM LeucogateTM (Becton Dickinson) at 4°C in the dark. SimultestTM LeucogateTM contains FITC-conjugated CD45 monoclonal antibodies (Anti-HLe-1) and phycoerythrin (PE) conjugated CD14 monoclonal antibodies (LeuTM-M3). After washing procedures the cells were submitted to flow cytometric analysis (FACScan® Becton Dickinson). Fluorescence was standardized by microbeads (CalibriteTM particles, Becton Dickinson) with amplification and voltage kept constant throughout the procedures. Analysis was performed on 10 000 events (detector threshold FSC-H:200, parameter FSC-H:1.00). The cell population was gated according to forward and right-angled light scatter. Background binding was estimated by isotype-matched negative control antibodies (SimultestTM Control, Becton Dickinson).
Measurement of CD14 mRNA and VDR mRNA levels
Cultured HL-60 cells were harvested after 2 days of incubation. Total RNA was isolated with Tri Reagent (Sigma) according to the guidelines of the manufacturer. The quality of the isolated RNA was tested by agarose/ formaldehyde gel electrophoresis, and compared with the migration of 16S and 23S E. coli ribosomal RNA.
Polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA, USA) was used to make CD14 DNA and VDR DNA templates under transcriptional control of a T7 promoter. In the case of CD14 we used human genomic DNA, sense primer 5'-CTA-GAG-CCG-TTT-CTA-AAG-CG-3' and antisense primer 5'-TAA-TAC-GAC-TCA-CTA-TAG-GGA-AGC-TGG-AAA-GTG-CAA-GTC-C-3', a 5x concentrated buffer containing 300 mmol/l TrisHCl (pH 8.5), 75 mmol/l (NH4)2SO4 and 10 mmol/l MgCl2 with an annealing temperature of 60°C yielding a 265-base-pair PCR product; in the case of VDR , plasmid DNA containing the 2.1 kb human VDR cDNA inserted into the Eco RI site of the vector pGEM-7Zf(+) (a gift from Dr J. W. Pike, Houston, TX, USA), sense primer 5'-ATG-CTA-TGA-CCT-GTG-AAG-GC-3' and antisense primer 5'-TAA-TAC-GAC-TCA-CTA-TAG-GGC-CAT-CAT-TCA-CAC-GAA-CTG-G-3' with an annealing temperature of 55°C yielding a 399-base-pair PCR product were applied. For both templates, 35 cycles were performed. PCR products were purified using MicrospinTM S-400 HR columns (Pharmacia, Uppsala, Sweden).
MAXIscriptTM T7, an in vitro transcription kit (Ambion, Austin, TX, USA), was used to incorporate [-32P] UTP (ICN, Costa Mesa, CA, USA) into the RNA probes during transcription of the CD14 and VDR PCR-DNA templates. We used pTRI-cyclophilin (165 base-pair) as control template DNA and CenturyTM marker template (Ambion) for the synthesis of radioisotopically labelled RNA molecular size standards with lengths of 100, 200, 300, 400 and 500 bases.
Before using the RNA probes in the ribonuclease protection assay, their purity was tested by polyacrylamide gel electrophoresis on a 6% trisbase, boric acid, EDTA (TBE)/urea gel (Novex, San Diego, CA, USA).
The RNA probes were hybridized to the RNA isolated from HL-60 cells cultured in the different culture media. The hybridization reactions were treated with ribonuclease to remove free probe. A sample containing yeast tRNA (Boehringer, Mannheim, Germany) was included as control for background hybridization and completion of the ribonuclease digestion.
The RNase-resistant probes annealed to homologous sequences in the sample RNA were analysed by electrophoresis on a 6% TBE/urea gel (Novex). For autoradiographs, the dried gels were exposed to Hyper X-ray films (Amersham, Buckinghamshire, UK).
The intensities of the specific bands were determined using Graphcompar (Applied Maths, Kortrijk, Belgium). The amount of CD14 mRNA and VDR mRNA was quantified relative to the amount of cyclophilin mRNA. The results were expressed as percentage of the results obtained in the calcitriol-containing medium for CD14 mRNA and as percentage of the results obtained in the medium alone for VDR mRNA.
Statistical analysis
Data are expressed as mean±SD. Statistical analysis was performed using Student's t-test for quantification of mRNA and Wilcoxon signed rank test for the other results. A P value of <0.05 was considered significant.
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Results |
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Chemiluminescence production
The luminol-enhanced CL production was significantly upregulated when cells were cultured in the presence of calcitriol and this pro-differentiating effect of calcitriol was abolished when cells were cultured in the presence of calcitriol and UUF (Figure 1B). No such effect was observed with NUF (n=3) (data not shown). The CL response of cells cultured in the presence of UUF alone was comparable to cells cultured in medium alone.
Membrane-bound CD14 expression
Cells cultured in the absence of calcitriol showed no CD14 expression. Calcitriol markedly induced CD14 expression (Figure 1C), which is compatible with cell differentiation. The fraction of cells expressing CD14 was significantly inhibited when cultured in the presence of calcitriol and UUF (Figure 1C
). Again no such effect was observed with NUF (n=3) (data not shown). There was no difference in mean fluorescence intensity (mean CD14 expresssion per cell) between the different experimental conditions.
Expression of CD14 mRNA and VDR mRNA
Figure 2A shows the unprotected RNA probes for CD14 (1) and VDR (2) which were used in the ribonuclease protection assay. CD14 mRNA was virtually absent when no calcitriol was added to the medium but was induced by the presence of calcitriol; CD14 mRNA levels were similar when HL-60 cells were cultured in the presence of calcitriol and UUF (Figure 2B
).
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The effect of HPLC fractions of UUF on the response to calcitriol
BrdU incorporation.
The antiproliferative effect of calcitriol was significantly abolished by the presence of all four separate HPLC fractions in the calcitriol-containing culture medium (Figure 3A). Although fraction 4 seemed to have the strongest effect, no statistical significance was reached between the different fractions.
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Chemiluminescence production.
The luminol-enhanced CL production was significantly upregulated when cells were cultured in the presence of calcitriol. This pro-differentiating effect of calcitriol was only significantly inhibited when cells were cultured in the presence of calcitriol and fractions 1 and 3 (Figure 3B).
Membrane bound CD14 expression.
None of the four separate HPLC fractions inhibited the calcitriol-induced membrane-bound CD14 expression (Figure 3C).
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Discussion |
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Production of calcitriol is decreased in renal failure [12]. In addition to calcitriol deficiency, there are also several arguments in favour of resistance to the biological functions of calcitriol in renal failure [16].
Patients with end-stage renal disease suffer from immune deficiency [13] and have increased susceptibility to infection [17]. Our study demonstrates that uraemic ultrafiltrate suppresses the calcitriol-induced CL response and CD14 expression (Figures 1B, C
), pointing to a suppression of the response of immune competent cells in uraemia. These results parallel data from a previous study by our group, which demonstrated an inhibitory effect of UUF, both in the absence and the presence of calcitriol, on CD14 expression on the surface of normal human monocytes [18]. UUF also inhibited the luminol-enhanced CL production by normal human monocytes both in the absence and the presence of calcitriol (unpublished data). Therefore the combination of calcitriol deficiency, calcitriol resistance, and uraemia could be involved in the above-mentioned deficient immune response of uraemic patients.
In addition to the evaluation of HL-60 cell functional capacity, the present study was also undertaken to evaluate the effect of uraemic ultrafiltrate on the response to calcitriol of HL-60 cell proliferation. Patients with end-stage renal disease have an increased susceptibility for malignancy [14]. Calcitriol inhibits the expression of specific properties of malignant cells as it induces differentiation and inhibits proliferation in cancer cell lines such as HL-60 and U937 [5,15,19].
We found that the addition of UUF abolished the decrease of malignant cell proliferation induced by calcitriol (Figure 1A). In contrast to our data, studies by Inaba et al. [15] and Nathan et al. [20] reported no change or an inhibition of the proliferation of HL-60 cells, cultured in the presence of uraemic serum or uraemic toxins (guanidino compounds). These differences could be due to different experimental conditions but also to UUF obtained from different patients. Our data, which parallel those found for functional capacity, point toward a neutralizing effect of the uraemic milieu on the pro-differentiating impact of calcitriol on the leukaemic HL-60 cell line.
Since CL response and CD14 expression are also markers of cell differentiation and UUF was found to inhibit calcitriol-induced CL response and CD14 expression, one can conclude that UUF inhibits the pro-differentiating effect of calcitriol.
The genomic action of calcitriol is believed to be mediated through a hormone-receptor complex interacting with nuclear chromatin. CD14 protein expression is regulated by a specific and well-defined gene, and this gene contains a vitamin D-responsive element [1]. Consequently the molecular mechanism for the inhibition of CD14 protein expression in the presence of UUF was evaluated by studying levels of CD14 mRNA in HL-60 cells. Together with determinations of CD14 mRNA, levels of VDR mRNA were assayed.
The VDR is known to be upregulated by its own ligand [1]. An inhibited expression of vitamin D receptor protein in HL-60 cells in the presence of uraemic serum was reported by Inaba et al. [15] and in human peripheral blood mononuclear cells from patients with end stage renal failure by Martinez et al. [21]. VDR mRNA has to our knowledge not yet been evaluated using immune-competent cells under uraemic conditions. For both CD14 mRNA and VDR mRNA, no inhibition at this level of the genomic pathway in the presence of UUF was detected (Figure 2B, C
), which points in the direction of an inhibition at a post-transcriptional site.
Likewise, Patel and co-workers studied the regulation of intestinal calcitriol receptor and its mRNA by calcitriol in renal failure rats and concluded that the uraemic status is related to a low VDR and a higher VDR mRNA in renal failure [22]. Other authors have also studied levels of VDR mRNA in renal failure, and in none of these studies could a decrease be demonstrated [23]. Several reports demonstrate that 1,25 (OH)2D3 transcriptional induction of the cyclin-dependent kinase inhibitor p21 mediates the antiproliferative effects of the sterol in cells of the monocyte/macrophage lineage including HL-60 [24]. Direct assessment of the effects of uraemic plasma ultrafiltrate on 1,25(OH)2D3 induction of p21 would clarify the effects of UUF-mediated increase in BrdU incorporation.
In an attempt to further specify the characteristics of the uraemic retention solutes which are responsible for the modification of cellular response to calcitriol, four separate HPLC fractions of the uraemic ultrafiltrate were analysed. Separation by reversed-phase HPLC occurs on the basis of polarity of solutes: hydrophilic compounds elute early during chromatography and hydrophobic compounds elute later. All four HPLC fractions abolished the antiproliferative effect of calcitriol on HL-60 (Figure 3A). The calcitriol-enhanced CL response was only significantly inhibited in the presence of fractions 1 and 3 (Figure 3B
). The latter two fractions correspond to those that inhibited the response to calcitriol of normal human monocytes [18]. The calcitriol-induced CD14 expression was not inhibited in the presence of either of the four separate HPLC fractions (Figure 3C
). A possible explanation for the different impact of fractions on different aspects of functional response of HL-60 could be that these functions are not interrelated, and that among the myriad uraemic solutes, several might exert distinct effects on distinct mechanisms of the response to calcitriol. The fact that HL-60 cell proliferation is not coupled to its differentiation has already been demonstrated [25]; to our knowledge, such an uncoupling has, however, not yet been demonstrated in the uraemic condition.
In conclusion, UUF contains factors that impair calcitriol-activated function of HL-60 cells. The differentiation and immune response of promyelocytic leukaemia cells induced by the supplementation of calcitriol is neutralized in the presence of uraemic biological fluids. This may be of relevance for the propensity to infection and malignancies of the uraemic patient.
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Acknowledgments |
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Notes |
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References |
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