Three monocyte-related determinants of atherosclerosis in haemodialysis

Stefan H. Jacobson1,, Pia Thylén1 and Joachim Lundahl2

1 Departments of Nephrology, 2 Clinical Immunology, Karolinska Hospital and Karolinska Institute, Stockholm, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 
Background. It has been suggested that monocyte-related inflammatory mediators play a role in atherosclerosis. Haemodialysis induces phenotypic changes in adhesion molecule expression on monocytes. Soluble vascular cell adhesion molecule-1 (sVCAM-1), an adhesion molecule involved in monocyte recruitment, has been proposed to correlate with the extent of atherosclerosis in humans. Monocyte chemotactic protein-1 (MCP-1) functions as a monocyte-specific chemoattractant.

Methods. We studied monocyte count, CD11b/CD18 expression on monocytes, MCP-1, and sVCAM-1 in nine patients on either cuprophane or polysulphone haemodialysis (n=18 treatments) at times 0 (before haemodialysis), 3 h (end of haemodialysis), 4, 6, 8 and 24 h after start of treatment, as well as in 18 healthy subjects.

Results. Monocyte CD11b/CD18 expression increased with both membranes (P<0.001) during and after dialysis compared to before treatment. The concentrations of sVCAM-1 and MCP-1 were higher in patients compared to those in controls both before, during and after haemodialysis (P<0.001 at all time points). There were correlations between the expression of CD11b/CD18 on monocytes and the interdialytic concentrations of sVCAM-1 (r=0.76, P<0.001) and MCP-1 (r=0.54, P<0.05) and between MCP-1 and sVCAM-1 before and after haemodialysis (P<0.05).

Conclusion. Patients on haemodialysis have an increased systemic chemotactic activity for monocytes, unphysiological phenotypic alterations in CD11b/ CD18 expression during and after dialysis, and increased sVCAM-1 and MCP-1 concentrations. Prospective studies are needed to establish the role of these abnormalities in the pathogenesis of atherosclerosis in haemodialysis patients.

Keywords: adhesion molecules; atherosclerosis; chemotaxis; haemodialysis; inflammation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 
Atherosclerosis is a frequent cause of morbidity and mortality in patients with end-stage renal disease maintained on haemodialysis [1,2]. One early detectable cellular response in the formation of lesions of atherosclerosis is the adherence of leukocytes to the endothelium [3]. As in other inflammatory responses, the adherent leukocytes migrate across the endothelial cell barrier and accumulate in the subendothelial space, where some of the monocytes ingest lipids and become foam cells. The adhesion of leukocytes on endothelial cells and their transendothelial migration are mediated by adhesion molecules on the endothelial cell membrane that mainly belong to two protein families, namely the selectins and adhesion molecules of the immunoglobulin superfamily. Two members of the latter group, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mediate firm adhesion of leukocytes by interaction with monocyte and granulocyte integrins, preferentially CD11b/CD18 [4].

VCAM-1 has been found to be expressed on endothelial cells before monocytes/macrophages appear in the subendothelium in experimental atherosclerosis and it is also present in advanced human plaques [6]. Circulating, shed forms of adhesion molecules have been described and are probably generated by cleavage of the molecule at a site close to the endothelial cell membrane insertion. Soluble VCAM-1 (sVCAM-1) has been detected in the supernatant of cytokine-stimulated cultured endothelial cells and a correlation of circulating sVCAM-1 with VCAM-1 mRNA expression has been reported in human atherosclerotic aorta [6,7].

Chemoattractants stimulate leukocyte transmigration through the endothelium and may induce directional locomotion to atherosclerotic lesions. Monocyte chemotactic protein-1 (MCP-1) functions as a chemoattractant specific for monocytes and may promote migration of monocytes into the plaque after their initial adhesion to the endothelium [8].

We and others have previously shown that haemodialysis induces phenotypic changes in adhesion molecule expression on peripheral leukocytes, which persist in the interdialytic period, and influence the adherence to human endothelial cells [911]. The present study focuses on the intra- and interdialytic alterations in CD11b/CD18 expression on monocytes, as a marker of cellular activation, as well as changes in sVCAM-1 and MCP-1. The question of whether these inflammatory mediators contribute to the pathogenesis of atherosclerosis in this group of patients was, however, not specifically addressed.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 
Study population
Nine haemodialysis patients (four men, five women, mean age 60±6 years, range 26–82 years) undergoing maintenance haemodialysis with polysulphone dialysers thrice weekly participated in a study in which the patients were treated with a low-flux polysulphone dialyser and a cuprophane dialyser in randomized order (18 treatments). All patients had been on haemodialysis for more than 3 months and the dialyses were part of the routine dialysis programme. All patients suffering from infectious diseases, diabetes mellitus, chronic lung disease, or inflammatory diseases, as well as patients receiving antibiotics, corticosteroids, or non-steroidal anti-inflammatory agents were excluded. Informed consent was obtained and the study was approved by the Ethics Committee of the Karolinska Hospital.

Two of the patients had a history peripheral atherosclerotic vascular disease, none had had myocardial infarction, and two had congestive heart failure. Eighteen healthy subjects (seven men and 11 women) with the mean age of 50±2 years, range 43–59 years, without any clinical signs or symptoms of atherosclerotic or other disease and who were not on any medication, were included as healthy controls.

Haemodialysis
The nine patients were treated with a low-flux polysulphone hollow-fibre dialyser (F7HPS, Fresenius AG, Germany) with an area of 1.6 m2 and with a cuprophane hollow fibre dialyser (GFE18, Gambro, Lund, Sweden) with an area of 1.8 m2 in randomized order. Dialysers were not reused. On the days of investigation haemodialyses were performed for 3 h with a blood flow of 250–300 ml/min. Heparin doses were individualized and given as an initial bolus dose followed by a continuous i.v. infusion until 1 h before termination of treatment.

Blood samples were collected prior to dialysis, at 3 h (end of dialysis), 4, 6, 8 and 24 h after start of each haemodialysis treatment. Blood samples from the healthy subjects were collected in the morning.

Preparation of peripheral leukocytes
Blood samples were collected in ethylene diamine tetra-acetate (EDTA) tubes and was kept on ice to prevent further complement activation and receptor modulation. EDTA blood was haemolysed in 100 µl portions by dilution in 2 ml +4°C isotonic NH4Cl-EDTA ‘lysing solution’. After 5 min incubation at +4°C the cells were centrifuged at 300 g for 5 min at +4°C. The leukocytes were washed once and resuspended in 100 ml cold 0.15 mol/l phosphate-buffered saline supplemented with 0.1 mmol/l EDTA (PBS–EDTA) before immunostaining.

Cell surface receptor mobilization
Leukocyte pellets were incubated at 37°C for 15 min in buffer (Roswell Park Memorial Institute 1640 with 5% fetal calf serum) supplemented with 5x10-7 mol/l N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Sigma Chemicals, St Louis, MO, USA) to obtain mobilization of intracellularly stored CD11b/CD18 receptors to the cell surface. After incubation, the leukocytes were washed once and resuspended in 100 µl PBS–EDTA before immunostaining.

Cell count of monocytes, flow cytometric analysis, and monoclonal antibodies
Monocytes were counted using a flow cytometer (Epics XL, Coulter Inc., Hialeah, FL, USA). This instrument gives the actual number of cells and the mean fluorescence intensity (MFI), which represents the antigen density of the cell population within a chosen field. CD11b/CD18 expression on monocytes was analysed by adding 5 µl of phycoerythrin-conjugated monoclonal anti-CD11b (Dako AS, Glostrup, Denmark). Appropriate concentrations of isotype matched control antibodies were used to define the cut-off for positive fluorescence, which was the 99th percentile of the distribution of the cells labelled with the control antibody PE-conjugated IgG2a.

Determination of serum factors
Serum levels of MCP-1, sVCAM-1, sICAM-1, soluble E-selectin (sELAM-1, sCD62E) and sL-selectin (sCD62L), were measured with commercially available immunoassays, purchased from R&D Systems Inc. (Minneapolis, MN, USA). The cut-off for respective analysis was as follows: 5.0 ng/ml (MCP-1), 100 ng/ml (sVCAM-1), 7.0 ng/ml (sICAM-1), 2 ng/ml (sELAM-1), 30 ng/ml (sCD62L).

Statistical analysis
Results are expressed as means±SEM. Figures are presented as box plots showing the 10th, 25th, 50th (median), 75th, and 90th percentiles of a variable. Statistical comparisons were made using Student's t-test for paired and unpaired observations and regression analysis.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 
CD11b/CD18 expression on monocytes and monocyte count
The cell surface expression of CD11b/CD18 on monocytes was significantly lower before haemodialysis (6.3±0.5 MFI) compared to that in healthy controls (8.8±1.2 MFI, P<0.05). The expression of CD11b was significantly higher after haemodialysis with both membranes (cuprophane P<0.01, polysulphone P<0.05) compared to before treatment (Table 1Go). At 4 h (1 h after the end of dialysis) the expression of CD11b was significantly higher when the patients had a cuprophane dialyser (10.0±0.8 MFI) as compared to a polysulphone membrane (7.3±0.9, P<0.05). CD11b/CD18 expression on monocytes was significantly higher at 6 h (P<0.001) and 8 h (P<0.01) in comparison to before dialysis when all patients were evaluated together (Figure 1Go).


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Table 1. Expression of CD11b/CD18 on monocytes, sVCAM-1 and MCP-1 before haemodialysis (0 h), and at 3, 8, and 24 h after start of treatment with cuprophane and polysulphone membranes

 


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Fig. 1. Cell surface expression of CD11b/CD18 on monocytes (MFI) during and after haemodialysis in nine patients on cuprophane or polysulphone membranes (n=18 treatments). **P<0.01 vs before haemodialysis=baseline. ***P<0.001 vs before haemodialysis.

 
There were no significant differences in the number of monocytes in the peripheral circulation at the end of haemodialysis, or at time-points 4, 6, 8, and 24 h compared with before dialysis for the treatments with the cuprophane or the polysulphone dialysers. Furthermore, no differences were observed in comparison to healthy subjects.

Concentration of sVCAM-1 before and after haemodialysis
sVCAM-1 was significantly higher in patients on haemodialysis before treatment (1750±131 ng/ml) as compared to healthy subjects (668±64 ng/ml, P<0.001, Figure 2Go). The concentration of sVCAM-1 increased significantly (P<0.001) during haemodialysis with both types of membranes. No significant differences were observed between treatments with cuprophane or polysulphone dialysers (Table 1Go). The mean concentration of sVCAM-1 was above baseline up to 8 h after start of treatment (Figure 3Go). We also analysed the systemic concentration of sICAM-1, sL-selectin and sELAM-1. None of these was higher in patients on haemodialysis before or after treatment as compared to concentrations in healthy subjects, and did not significantly vary during the study period. This indicates that the increase of sVCAM-1 was not primarily caused by retention of the molecule secondary to renal insufficiency.



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Fig. 2. Concentration of sVCAM-1 in nine patients (before the first dialysis in this investigation) on maintenance polysulphone haemodialysis (before treatment) and in 18 healthy subjects.

 


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Fig. 3. Concentration of sVCAM-1 in nine patients before and after cuprophane and polysulphone haemodialysis (n=18). *P<0.05 vs before haemodialysis=baseline. ***P<0.001 vs before haemodialysis.

 

Concentration of MCP-1 before and after haemodialysis
The concentrations of MCP-1 were significantly higher in patients on haemodialysis before treatment (586±33 ng/l) as compared to levels in healthy controls (225±72 ng/l, P<0.001, Figure 4Go). The level of MCP-1 decreased during polysulphone dialysis, but no changes between the two types of dialysers were observed (Table 1Go). MCP-1 was significantly lower 8 h after the start of dialysis as compared to before treatment. However, also at that time-point and 24 h after start of treatment the concentrations were significantly higher in patients as compared to concentrations in healthy controls (P<0.001 for both comparisons).



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Fig. 4. Concentration of MCP-1 in nine patients (before the first dialysis in this investigation) on maintenance polysulphone haemodialysis before treatment and in nine healthy subjects.

 



   Correlations
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 
The concentration of sVCAM-1 at 6 h after the start of haemodialysis (3 h after termination of treatment) correlated significantly to the expression of CD11b/CD18 on monocytes in the peripheral circulation at the same time-point (r=0.76, P<0.001, Figure 5Go). There was a significant correlation between the concentration of MCP-1 at 8 h after the start of treatment and the concentration of sVCAM-1 at the same time-point (r=0.53, P<0.05, Figure 6Go). Furthermore, the concentration of MCP-1 before dialysis correlated to sVCAM-1 before treatment (r=0.48, P<0.05) and also 24 h after dialysis (r=0.55, P<0.05). The expression of CD11b on monocytes in the peripheral circulation by the end of dialysis correlated significantly with the concentration of MCP-1 at the same time-point (r=0.57, P=0.01) and also at 8 and 24 h (r=0.54, P<0.05 and r=0.52, P<0.05 respectively).



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Fig. 5. Correlation between the expression of CD11b/CD18 on monocytes at 6 h after start of dialysis (3 h after termination of treatment) and the concentration of sVCAM-1 at the same time-point.

 


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Fig. 6. Correlation between the concentration of MCP-1 at 8 h after start of haemodialysis (5 h after termination of treatment) and the concentration of sVCAM-1 at the same time-point.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 
There is little information in the literature on the relationship between three factors involved in the development of atherosclerosis, namely circulating activated monocytes, and the levels of MCP-1 and sVCAM-1 in haemodialysis patients. Herein we report on a combination of higher concentrations of MCP-1 and sVCAM-1 and the presence of activated circulating monocytes, both during haemodialysis and in the interdialytic period. It is, however, important to point out that this study was not designed to determine the role of these aberrations in the development of atherosclerosis in haemodialysis patients.

In this study we used CD11b expression as a marker for monocyte activation. The rationale for this approach is based on previous reports, both from our group and from others, which describe CD11b up-regulation in monocytes both during and after completion of a haemodialysis session [911]. Besides being a marker for activated monocytes, CD11b is also, together with VLA-4 (very late antigen -4) and -5, involved in the endothelial cell adhesion process [12]. As opposed to CD11b, VLA-4 is not rapidly up-regulated upon cell activation, but its avidity is changed in response to ligand binding [13,14]. VCAM-1 is a natural ligand for VLA-4 on endothelial cells and the VLA-4/VCAM-1 adhesion pathway has been proposed to promote transendothelial migration of monocytes [15].

In patients with atherosclerotic aortic disease, the concentration of sVCAM-1 correlated to the expression of VCAM-1 mRNA expression, which suggests that the circulating VCAM-1 level can be used as an indicator of VCAM expression in atherosclerotic plaques [7]. In a recent study of patients with essential hypertension and peripheral vascular disease, the sVCAM-1 concentration was higher in those patients compared to controls, and the concentration of sVCAM-1 correlated to an index of atherosclerosis in the carotids [16]. In another study of patients with peripheral arterial vascular disease the concentration of sVCAM-1 correlated with the extent of atherosclerosis determined by angiograms [17]. Taken together, these data, and the finding that VCAM-1 also is present on the surface of advanced atherosclerotic plaques, suggest a pathogenic role for VCAM-1 in this disease [5,18].

Our data support the notion that VCAM-1 is up-regulated in dialysis patients, and that the upregulation is further pronounced during a haemodialysis session. Increased levels of sVCAM-1 in dialysis patients have been reported by other groups [1921]. Bonomini et al. [19] found increased sVCAM-1 levels in haemodialysis patients and, in agreement with us, equivalent predialysis values in patients treated with cellulosic and synthetic dialysers. In the present study the concentrations of sICAM-1, sELAM-1 and sL- selectin were also determined. These soluble adhesion molecules were not significantly higher as compared to levels in controls, and did not significantly vary during and after haemodialysis. This indicates that the increased concentration of sVCAM-1 was not simply due to retention of the molecule secondary to renal insufficiency, but mirrors a selective event in adhesion molecule up-regulation. The factors responsible for this selective up-regulation are currently not known, but proinflammatory cytokines, such as interleukin (IL)-4, IL-13, and tumour necrosis factor (TNF)-{alpha} have been reported to be involved in VCAM-1 up-regulation [2124]. Indeed, some of these have been reported to be increased in haemodialysis patients [25,26].

MCP-1 is a chemokine linked to the atherosclerotic process [27]. Haemodialysis patients in the present study had significantly higher serum concentrations of MCP-1 than healthy controls both before and after dialysis. There were, however, no significant differences in MCP-1 concentrations for treatments with cuprophane or the polysulphone membrane. After 8 h, the concentration of MCP-1 was significantly lower as compared to before treatment with both membranes. It is not clear whether this decrease was due to intradialytic elimination and post-dialytic redistribution of the molecule or whether it was caused by immunological mechanisms, e.g. decreased synthesis.

Different approaches have been used to address the question of whether the uraemic state influences MCP-1 concentrations. Recently, Pertosa et al. by use of an in vitro method, reported significantly lower spontaneous production of MCP-1 from mononuclear cells of uraemic patients and patients on cuprophane haemodialysis, compared to healthy subjects [28]. They also reported that haemodialysis with polymethylmethacrylate and polyamide normalized the MCP-1 protein release from mononuclear cells which had been reduced by cuprophane treatment. Another group observed, in accordance with our study, increased serum levels of MCP-1 in dialysis patients treated with cellulosic and synthetic membranes [29]. Thus, when interpreting MCP-1 data in patients with uraemia the methods used for chemokine analysis have to be taken into consideration.

We observed a significant correlation between the concentration of MCP-1 and sVCAM-1 after dialysis, which further emphasizes the interaction between chemotactic stimuli, concentrations of sVCAM-1, and monocyte activation in this group of patients. Moreover, this notion is important in the context of factors involved in MCP-1 generation and VCAM-1 up-regulation. Factors such as IL-1 and TNF-{alpha} have the dual capacity to up-regulate VCAM-1 and concomitantly to generate MCP-1, which, as mentioned above, are intimately involved in monocyte accumulation at the atherosclerotic plaque [30].

In conclusion, haemodialysis induces an acute and prolonged increase in the expression of CD11b on monocytes and an increase in the concentration of sVCAM-1, which is elevated predialysis as compared to controls. This, in combination with an increased concentration of MCP-1, may have an impact on the development of atherosclerosis in this group of patients. Prospective studies, however, are needed to confirm this relationship.



   Acknowledgments
 
The authors wish to thank Anette Bygden RN and Titti Nieminen for skilful technical assistance. The study was supported by grants from Lisa and Johan Grönbergs Stiftelse, Terumo Europe, and the Karolinska Institute.



   Notes
 
Correspondence and offprint requests to: Dr Stefan H. Jacobson MD PhD, Director, Department of Nephrology, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Correlations
 Discussion
 References
 

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Received for publication: 15. 7.99
Revision received 20. 4.00.