Regulation of ß2-microglobulin expression in different human cell lines by proinflammatory cytokines

Thomas Vraetz1,3, Thomas H. Ittel2, Michelle G. van Mackelenbergh1,3, Peter C. Heinrich1, H. G. Sieberth2 and Lutz Graeve1

1 Institut für Biochemie, 2 Medizinische Klinik II and 3 Interdisziplinäres Zentrum für Klinische Forschung BIOMAT, Rheinisch-Westfälische Technische Hochschule Aachen, D-52057 Aachen, Germany

Correspondence and offprint requests to: Thomas H. Ittel, MD, Chief, Department of Internal Medicine, Klinikum der Hansestadt Stralsund, Große Parowerstr. 47–53, D-18435 Stralsund, Germany.



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Proinflammatory monocytic cytokines such as interleukin-1 (IL-1), tumour necrosis factor-{alpha} (TNF-{alpha}) and IL-6 have been incriminated in the pathogenesis of elevated ß2-microglobulin (ß2M) serum concentrations in patients undergoing haemodialysis with so-called bioincompatible dialyser membranes. However, neither the source of the elevated serum ß2M nor the precise role of monocytic cytokines in the expression of the ß2M gene have been elucidated conclusively. The aim of the current study was to evaluate whether monoytic cytokines, and in particular IL-6, are regulators of ß2M gene expression in human hepatoma cells, T-lymphocytes and monocytes.

Methods. HepG2 and HuH7 human hepatoma cells, Jurkat T-cells, monocytic MonoMac6 cells, primary human monocytes and synoviocytes were stimulated with IL-1ß, IL-6, interferon-{alpha} (IFN-{alpha}), IFN-{gamma} or conditioned media from lipopolysaccharide (LPS)-treated monocytes. Expression of ß2M mRNA was analysed by Northern blotting, ß2M protein synthesis was determined by metabolic labelling and immunoprecipitation, and ß2M secretion was measured by an enzyme-linked immunosorbent assay (ELISA).

Results. In all cell types tested, IFN-{gamma} and, to a lesser extent, IFN-{alpha} stimulated gene expression of ß2M resulting in an increased synthesis and secretion of ß2M protein. Neither IL-1ß and IL-6 nor supernatants from LPS-treated monocytes were capable of inducing ß2M gene expression, with the exception of a small increase in HuH7 hepatoma cells upon IL-1ß treatment.

Conclusions. The present study provides evidence that interferons are important regulators of ß2M expression. It also shows that proinflammatory monocytic cytokines do not modulate directly the expression of ß2M in cells of hepatic, monocytic and T-lymphocytic origin. Whether they influence ß2M synthesis and secretion indirectly by modulating interferon synthesis needs to be elucidated.

Keywords: bioincompatibility; cytokines; hepatoma cells; interleukin-6; long-term haemodialysis; ß2-microglobulin amyloidosis



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß2-Microglobulin (ß2M) amyloidosis is a disabling clinical condition that develops in patients undergoing long-term haemodialysis [1]. The disease comprises a wide spectrum of symptoms such as osteoarthritis, osteolytic bone lesions, fractures and carpal tunnel syndrome [2]. Previous work has linked the pathogenesis of ß2M amyloidosis to the bioincompatibility of specific dialyser membranes such as cuprophane materials [3]. However, there is still controversy as to whether there is predominantly an increased synthesis of ß2M or whether the use of membranes with a different clearance capacity for ß2M would contribute to the variable incidence of dialysis-related amyloidosis [4,5]. In addition, although bioincompatible membranes have been shown to elicit a profound inflammatory reaction in human mononuclear blood cells, there is no convincing correlation between the induction of mononuclear proinflammatory cytokines such as tumour necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1 (IL-1) and the enhanced production of ß2M by lymphocytes [3,610]. This suggests that either other proinflammatory cytokines such as IL-6 might be involved in the up-regulated ß2M production or that elevated ß2M serum concentrations in dialysis patients might stem from sources other than blood cells [3,11]. In addition, proinflammatory cytokines may exert noxious effects through other mechanisms such as the enhancement of ß2M amyloid fibril formation or deposition. With respect to the former mechanisms, IL-6 is known to induce several acute phase proteins in the liver by signalling via Jak tyrosine kinases and STAT transcription factors [1214].

Previous work of Homma et al. has provided preliminary evidence that IL-6 is also an inducer of ß2M in a human hepatoma cell line HuH7 [15]. The aim of the present study was to evaluate whether IL-6 might also be a regulator of ß2M in other hepatoma cells, lymphocytes and monocytes.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Restriction enzymes and the random primed DNA labelling kit were purchased from Boehringer Mannheim (Mannheim, Germany). [{alpha}-32P]dATP (110 TBq/mmol) was obtained from Amersham International. [35S]Cysteine and [35S]methionine were from ICN Biomedicals (Meckenheim, Germany). Dulbecco's modified Eagle's medium (DMEM), DMEM/ F12-Mix, minimum essential medium (MEM) with Earle's salts and RPMI 1640 were from Gibco (Eggenstein, Germany) and fetal calf serum (FCS) was from Seromed (Berlin, Germany). Cell culture supplements were from Gibco (Eggenstein, Germany) and bovine serum albumin (BSA) fraction V was purchased from Serva (Heidelberg, Germany). Recombinant human IFN-{alpha} (RoferonTM) was obtained from Roche (Basel, Switzerland), recombinant human IFN-{gamma} was a gift from Bioferon (Laupheim, Germany), recombinant human IL-6 with a specific activity of 1x106 B-cell stimulatory factor-2 U/mg was prepared as described by Arcone et al. [16] and recombinant human IL-1ß (1.3x107 U/mg) was kindly provided by Dr A. Shaw (Glaxo Institute of Molecular Biology, Geneva, Switzerland). The plasmid pBluescript SK(+/-) containing the human full-length ß2M cDNA was kindly provided by W. Mikultis (Institute of Molecular Biology, University of Vienna, Austria); for hybridization, a 1.4 kb EcoRI–XhoI fragment was used.

Cell culture and stimulation
HepG2 cells and HuH7 cells were grown in DMEM/ F12-Mix, and Jurkat and MonoMac6 cells in RPMI 1640 at 5% CO2 in a water-saturated atmosphere. All cell culture media were supplemented with 10% FCS, streptomycin (100 mg/l) and penicillin (60 mg/l). Cells were grown in 6-well dishes to 80% confluence and stimulated with cytokines in medium containing 0.2% BSA or conditioned monocyte medium (mixed 1:1 with the respective culture medium). The incubation time was 18 h for RNA extraction and immunoprecipitations and 60 h for enzyme-linked immunosorbent assay (ELISA).

Assessment of ß2-microglobulin mRNA levels
ß2M mRNA levels were assessed by Northern blot analysis. Cells were dissolved in 1 ml of 4 M guanidinium thiocyanate, 0.5% sodium N-laurylsarcosine, 25 mM sodium citrate, pH 7.0, and RNA was extracted according to Chomczynski and Sacchi [17]. RNA was quantified by measurement of the absorbance at 260 nm. A 5 mg aliquot of total RNA was mixed with a solution containing 20 mM MOPS, 4 mM sodium acetate, pH 7.0, 1 mM EDTA, 2.2 M formaldehyde, 50% formamide and saturated bromphenol blue, heated to 65°C for 10 min and chilled on ice. RNA was loaded on a 1% agarose gel containing 0.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, pH 7.0 and 1 mM EDTA, separated by electrophoresis and transferred to a nitrocellulose membrane. RNA was immobilized by baking for 2 h at 80°C. The filters were pre-hybridized in 50% formamide, 20 mM sodium phosphate, 5x saline sodium citrate (SSC), 0.2 mg/ml denatured salmon sperm DNA, 0.02% Denhardt's and 1% glycine for at least 2 h at 42°C. The subsequent hybridization was performed in the same buffer except that glycine was replaced by 10% dextran sulfate for 18 h at 42°C with labelled cDNAs. The filters were then washed twice in 2x SSC + 0.2% SDS for 20 min at room temperature followed by one wash in 0.2x SSC + 0.2% SDS for 20 min at 42°C. The filters were dried and subjected to autoradiography using intensifying screens at -80°C. Equal loading of RNA gels was assessed by ethidium bromide staining of 18S and 28S RNA or by hydrization with a cDNA coding for glyceraldehyde-3-phosphate dehydrogenase.

Assessment of ß2-microglobulin protein synthesis
At 18 h after stimulation, cells were labelled with [35S]methionine and [35S]cysteine for 3 h. After labelling, the cells were incubated for another 3 h with DMEM/F12 + 0.2% BSA containing unlabelled cysteine and methionine. For pulse–chase experiments, cells were labelled for 1 h and chased for the times indicated in Figure 6Go. Supernatants were removed and cells were solubilized for 30 min at 4°C in 1 ml of lysis buffer containing 1% Triton X-100, 0.4% sodium deoxycholate, 66 mM EDTA, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 25 mg/ml aprotinin, 5 mg/ml pepstatin, 5 mg/ml leupeptin and 10 nM phenylmethylsulfonyl fluoride (PMSF). Nuclei were removed by centrifugation for 10 min at 12 000 g. Supernatants were equilibrated to 1% Triton X-100. Cell lysates and supernatants were incubated with 10 mg of anti-ß2M antibody (rabbit, polyclonal; DAKO A 072, Hamburg) for at least 2 h at 4°C. Immunocomplexes were bound to protein A–Sepharose (5 mg/ml lysis buffer) for 1 h at 4°C. After centrifugation, the beads were washed four times with 1% Triton X-100, 0.4% sodium deoxycholate, 0.1% SDS, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl. Proteins were eluted by boiling in gel electrophoresis sample buffer and separated on an SDS–polyacrylamide (17%) gel.



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Fig. 6. Synthesis and secretion of ß2M in the human hepatoma cell line HuH7 after exposure to IFN-{gamma} and IL-6 as followed by a pulse–chase experiment. Cytokine concentrations are as in Figure 2Go.

 
Assessment of ß2-microglobulin protein content
The ß2M content of the culture medium was determined using a commercially available ß2M ELISA (Immuno Biological Laboratories, Hamburg, Germany) according to the manufacturer's instructions except that the samples were diluted 1:10 instead of 1:50. All measurements were performed at least in duplicate.

Production of LPS-conditioned medium of primary monoytes
The peripheral blood monocytes were isolated from buffy coats of healthy donors (kindly provided by the Institute for Transfusion Medicine, RWTH Aachen, Germany) as described by Böyum et al. [18]. Cells were pre-cultured at a density of 2x106 cells/ml in Teflon bags (Biofolie 25, Heraeus, Osterode, Germany) for 5 h at 37°C in RPMI 1640 containing 1% FCS, 2 mM HEPES and 2 mM sodium bicarbonate. Lipopolysaccharides (LPS, 25 ng/ml) was added to the medium and the cells were incubated for up to 21 h at 37°C. The medium was removed and the cells were sedimented by centrifugation (400 g, 10 min).



   Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
When human hepatoma HepG2 cells were stimulated with interferon-{gamma} (IFN-{gamma}), IL-1ß and IL-6, only IFN-{gamma} treatment resulted in a significant increase in ß2M mRNA levels. IL-1ß and IL-6 at any concentration tested had no effect (Figure 1Go). ß2M de novo protein synthesis and secretion were also only increased upon IFN-{alpha} or IFN-{gamma} stimulation (Figure 2Go). These results were corroborated further by ELISA measurements of ß2M protein concentrations in supernatants of HepG2 after 60 h of cytokine treatment. Control cells and cells stimulated with IL-1ß and IL-6 released 190 ng/106 cells within 60 h. In contrast, after IFN-{gamma} treatment, 400 ng/106 cells were detected (Figure 3Go). To exclude that proinflammatory cytokines other than those tested are potent inducers of ß2M expression, we incubated HepG2 cells with conditioned medium from LPS-treated monocytes, which are known to contain numerous mediators. However, no effect on ß2M mRNA expression was observed with monocyte conditioned media (Figure 4Go).



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Fig. 1. The effect of IFN-{gamma}, IL-1ß and IL-6 on ß2M mRNA expression in HepG2 cells.

 


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Fig. 2. ß2M de novo protein synthesis and secretion in human HepG2 hepatoma cells after exposure to various cytokines. Cells were incubated with IL-1ß (100 U/ml), IFN-{alpha} (500 U/ml), IFN-{gamma} (500 U/ml) and IL-6 (500 U/ml) and metabolically labelled. Cell lysates and supernatants were immunoprecipitated with an anti-ß2M antibody. The fluorogram of the SDS–PAGE is shown. The MHC class I heavy chain (HLA-I) is co-immunoprecipitated with ß2M from cell lysates.

 


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Fig. 3. The effect of IL-1ß, IFN-{gamma} and IL-6 on ß2M protein secretion into HepG2 cell culture supernatants as determined by ELISA.

 


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Fig. 4. The effect of LPS-conditioned monocyte supernatants on the ß2M mRNA levels in HepG2 cells.

 
The study reporting the IL-6 stimulation of ß2M expression was performed with HuH7 hepatoma cells [15]. Therefore, we repeated all experiments with these cells. However, the results were similar to those obtained with HepG2, with the only exception that IL-1ß had a slight stimulatory effect in HuH7 cells (Figure 5Go). A pulse–chase study in HuH7 cells very clearly demonstrated that after IL-6 treatment, there was neither an increased ß2M synthesis nor secretion at any time point (Figure 6Go).



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Fig. 5. The effect of IL-1ß, IFN-{alpha}, IFN-{gamma} and IL-6 on ß2M mRNA levels (a) and protein concentrations in supernatants (b) of HuH7 human hepatoma cells. Cytokine concentrations are as in Figure 2Go.

 
Next, we analysed the regulation of ß2M by cytokines in the human T-cell lymphoma line Jurkat. In these cells also, only IFN-{alpha} and IFN-{gamma} induced ß2M gene expression (Figure 7Go). Finally, we tested the monocytic cell line MonoMac6, as well as primary human monocytes. Neither IL-1ß nor IL-6 were able to stimulate ß2M mRNA levels or ß2M protein synthesis in any of these cells (data not shown).



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Fig. 7. The effect of IL-1ß, IFN-{alpha}, IFN-{gamma} and IL-6 on ß2M mRNA levels (a) and protein concentrations in supernatants (b) of the human T-cell lymphoma cell line Jurkat. Cytokine concentrations are as in Figure 2Go.

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several mechanisms have been implicated in the pathogenesis of ß2M amyloidosis in dialysis patients, such as an increased synthesis and release of ß2M due to bioincompatibility of dialysers, a diminished renal elimination of ß2M, differences in membrane capacities to either clear or absorb ß2M or, alternatively, enhanced polymerization of ß2M into amyloid fibrils due to the release of proteases and reactive oxygen species by activated neutrophils [13,19]. It has been suggested that release of IL-6 due to membrane bioincompatibility or to bioincompatibility of peritoneal solutions might be an important stimulator of ß2M synthesis [20,21]. Furthermore, IL-1 and IL-6 produced from tenosynovial tissues together with dialysis amyloid have been incriminated in causing carpal tunnel syndrome [22]. Since data on the stimulation of ß2M synthesis in uraemia in cells other than lymphocytes are scarce, we investigated whether IL-6, a potent inductor of acute phase proteins in the liver, may induce the synthesis and release of ß2M in hepatocytes, T-lymphocytes and monocytes.

Our findings do not support the notion that IL-6 or other monocytic cytokines are important in the regulation of ß2M expression in various human cell lines. Similar results were obtained in primary cultures of human monocytes with respect to stimulation by LPS and in primary cultures of synoviocytes, studied at the RNA level only, stimulated by IL-1ß and IL-6 plus soluble IL-6 receptor (data not shown).

Thus, in line with other observations [23], our data would strongly argue against a direct stimulatory effect of monocytic cytokines on ß2M serum concentrations in patients dialysed with bioincompatible membranes. Previous work has suggested that uraemia per se may increase the plasma levels of IL-6 and that bioincompatible dialyser membranes or penetration of dialysis membranes by endotoxins will further augment serum concentrations of IL-6, IL-1ß, TNF-{alpha} and other monocytic cytokines [11,20,2431]. Elevated levels of these cytokines have been linked to the reduced immune response in haemodialysis patients and to elevated ß2M serum concentrations [3,7,32]. However, in view of our data, their role may be more complex, and interactions of cytokines with specific inhibitors, antagonists and soluble cytokine receptors may complicate further the interpretation of an inflammatory response [19,28]. Several investigators find either no induction of monocytic cytokines by dialysis membranes or even depressed cytokine secretion [3336]. Furthermore, kinetic aspects such as the length of the period of repetitive exposure to a given type of membrane need to be considered, since cytokine production appears to change during the course of treatments [19,37,38]. Finally, most investigators describe just a correlation between an inflammatory response during haemodialysis and elevated serum ß2M levels but fail to elucidate a clear cause and effect relationship [3,610]. Our data do not corroborate an important part of the cytokine hypothesis of the pathogenesis of ß2M amyloidosis as regards ß2M production, even though they do not exclude a role for cytokines in the later steps of amyloidogenesis. In particular, our findings dispute earlier claims that IL-6 induces the production of ß2M as an acute phase protein in the liver [15].

The present study points to the important aspect of the regulation of ß2M by interferons that needs to be considered in dialysis patients. Expression of ß2M and major histocompatibility complex (MHC) class I genes is known to be regulated coordinately [39]. Interferons have been shown to up-regulate the expression of MHC proteins, and the present study consistently confirms stimulation of ß2M gene transcription as well as ß2M protein synthesis and release by IFN-{alpha} and IFN-{gamma} in hepatocytes and in T-lymphocytes [8,9,40,41]. Increased production of IFN-{gamma} is a feature of Th1 CD4+ T-cell differentiation [42]. If IFN-{gamma}-induced expression of ß2M is important in the pathogenesis of elevated serum concentrations of ß2M, then polarization of CD4+ cells to the Th1 phenotype should be expected in patients treated with bioincompatible membranes. Evidence for this phenomenon in dialysis patients is lacking, and stimulation of proinflammatory monocytic cytokines, in particular IL-6, by cuprophane® membranes should switch the T-cell differentiation to the Th2 phenotype rather than augmenting IFN-{gamma} production [42]. Clearly this issue needs further studies in vivo and in vitro. Our findings provide a strong argument for a broader approach to the issue of bioincompatibility of dialysis procedures with respect to increased expression of ß2M. So far, data on the effect of dialysis membranes on lymphocyte subsets and on the production of interferons are scarce, and one study found no change in IFN-stimulated synthesis of ß2M following haemodialysis [4345].

In conclusion, the present study provides evidence against a role for monocytic cytokines as a direct cause of increased expression of ß2M in cells of hepatic, monocytic, T-lymphocytic and synovial origin. Instead, interferons are important regulators of ß2M expression, and further studies need to address to what degree monocytic cytokines are involved in an up-regulation of interferons as a sequela of bioincompatible dialysis procedures. Preliminary evidence from an in vitro haemoperfusion system using cuprophane® and polyamide dialysers suggests that no IFN-{gamma} is released during experimental perfusion of blood from healthy donors (Nimmesgern and Graeve, unpublished observation).



   Acknowledgments
 
We thank Wiltrud Frisch for excellent technical assistance. This work was supported by grants from the Interdisziplinäres Zentrum für Klinische Forschung BIOMAT, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.



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 Introduction
 Materials and methods
 Results
 Discussion
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Received for publication: 6. 7.98
Accepted in revised form: 21. 4.99