Polymorphonuclear leukocyte rigidity is defective in patients with chronic renal failure

Athanasios T. Skoutelis1,, Vasilios E. Kaleridis2, Dimitrios S. Goumenos1, George M. Athanassiou2, Yannis F. Missirlis2, Jannis G. Vlachojannis1 and Harry P. Bassaris1

1 Department of Medicine, Patras University Medical School, 2 Department of Mechanical Engineering, Patras University, Patras, Greece



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The purpose of the study was to investigate the rigidity of polymorphonuclear leukocytes (PMNs) in non-dialysed chronic renal failure (CRF) and haemodialysis (HD) patients.

Methods. PMN rigidity as well as tumour necrosis factor {alpha} (TNF-{alpha}) and interleukin 1ß (IL-1ß) plasma levels were assessed in 10 early-stage CRF, 10 late-stage non-HD, and 10 HD patients, before and during dialysis. In HD patients both cellulose acetate and polysulphone membranes were used. Ten healthy subjects served as controls. Rigidity was tested by counting the deformability in morphologically passive PMNs by the micropipette method. Cytokine levels were measured by enzyme-linked immunosorbent assay.

Results. PMN rigidity was significantly increased in end-stage CRF patients regardless of HD but not in early-stage CRF. In HD patients PMN rigidity increased significantly 60 min after initiation of HD. There was an increase of TNF-{alpha} and IL-1ß levels in end-stage non-HD and HD patients and a further increase at 60 min after initiation of HD. The percentage of morphologically activated PMNs was increased only during dialysis. The nature of the HD membrane had no influence on rigidity, PMN activation, or cytokine production.

Conclusions. The results indicate that PMN rigidity is defective in end-stage chronic CRF patients and is further increased 60 min after initiation of HD, regardless of the nature of the HD membrane used. PMN activation, increased TNF-{alpha} and IL-1ß levels, or a direct PMN impairment may cause the observed cell rigidity.

Keywords: chronic renal failure; deformability; haemodialysis; polymorphonuclear leukocytes; rigidity; uraemia



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Blood contact with haemodialysis (HD) membranes has been shown to result in complement activation and leukostasis in the pulmonary capillaries, leading to granulocytopenia, hypoxaemia and pulmonary dysfunction in the initial phase of dialysis [1,2]. A similar mechanism has also been postulated in a number of clinical syndromes such as cardiopulmonary bypass, sepsis and septic shock, and adult respiratory distress syndrome. Polymorphonuclear leukocyte (PMN) rigidity has been shown to be impaired in these situations [35]. Therefore PMNs with an average 6–8-µm diameter lack or decrease their ability to deform in order to pass through average 5–6-µm diameter capillaries [3]. This results in intravascular aggregation and sequestration of PMNs, leading to hypoxaemia and other deleterious sequelae. It has also been documented that uraemia and end-stage renal failure impair host defences, including PMN functions such as adherence, chemotaxis, phagocytosis, and destruction [47]. There is also a substantial amount of evidence indicating that circulating levels of tumour necrosis factor alpha (TNF-{alpha}), interleukin 1ß (IL-1ß), and other cytokines are elevated in undialysed chronic renal failure (CRF) and HD patients and also during dialysis [811]. Cytokines, complement, and cell activation increase PMN rigidity [12,13].

Although cell rigidity may play a role in leukoaggregation or impaired PMN function in uraemic patients, there are no studies addressing this issue. We conducted this study to investigate passive (non-activated) PMN rigidity and its possible relationship to circulating cytokines or other factors that are known to impair the mechanical properties of PMNs in undialysed CRF and HD patients. We also evaluated the effect of the time course of HD and the use of membranes with different biocompatibilities on PMN rigidity.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Study population
The study was approved by the Research and Ethics Committee of the University Hospital, Patras, Greece. Informed consent was obtained from each participant. Thirty patients with CRF were included in the study. Ten of them had early-stage CRF (serum creatinine less than 2.5 mg/dl), 10 had end-stage CRF, but were not being dialysed (estimated GFR <10 ml/min), and 10 were long-term HD patients. The HD patients were being dialysed thrice weekly with bicarbonate dialysate and single-use cellulose acetate membranes (Altra Nova, Althin Medical Inc, Miami Lakes, USA). To investigate the influence of the nature of the HD membrane on PMN rigidity the patients dialysed with cellulose acetate were switched to polysulphone membranes (F7 HPS, Fresenius Medical Care, Frankfurt, Germany) which cause less complement activation [14].

PMN rigidity and cytokine production were assessed once for each patient with both types of membrane.

All HD patients had been on dialysis from 4 to 5 h per session, thrice weekly, for the previous 3 months, with the same type of membrane.

PMN rigidity, TNF-{alpha}, and IL-1ß were tested pre-dialysis, and at 1, 30, 120 and 240 min after initiation of dialysis. The same tests were performed on undialysed CRF patients. Routine biochemical and haematological blood tests were performed for each patient at the time of the experiment. Forty healthy, non-smoking subjects recruited from volunteer blood donors served as controls. Subjects or patients with diabetes, a history of malignancy and other immune suppression, active or recent infections, and those receiving corticosteroids, non-steroidal anti-inflammatory drugs, or agents known to influence PMN rigidity were excluded.

Cell preparation
All blood samples were anticoagulated with EDTA (1 mg/ml) because EDTA results in reduced leukocyte activation [15]. The blood was centrifuged at 200 g for 30 min. Leukocytes were taken without separation medium from the buffy coat and washed twice at 200 g for 5 min with phosphate-buffered saline (PBS) enriched with 0.1% albumin, and resuspended in their own plasma.

Rigidity studies
Rigidity studies were performed blindly by calculating PMN deformability using micropipettes to aspirate neutrophils under microscopic observation at rates comparable to those observed in the microcirculation as previously described [16,17]. Briefly, a few drops of appropriate leukocyte suspension were observed using an inverted light microscope (Olympus IM, Japan) with an additional electronic magnification. The overall magnification was approximately x8000. The cell suspension was placed into a reservoir made of two cover slips and a parafilm gasket. A glass micropipette, internal diameter 4.0–5.0 µm, was staged on a 3-dimension micromanipulator and was connected to a hydrostatic pressure measuring system with a resolution of 0.01 mm H2O. The micropipette was manipulated to enter the open side of the reservoir and directed to a selected leukocyte. A constant negative pressure of 8 mm H2O was applied on the selected leukocyte from the pressure system and the micropipette via a pressure transducer (Validyne DP/ 103, USA). The tongue length of PMNs was measured with a video microscaler (FOR A IV 550, Japan). The above experiment is a creep experiment where the tongue length of PMN into the micropipette under the steep negative pressure of 8 mm H2O was calculated over time. The rigidity as a function of the apparent viscosity of the PMN was estimated by calculation of the bulk apparent viscosity as

(expressed in dyn.s/cm2), where {Delta}P-Pcr is the excess suction pressure above the critical pressure Pcr, which is the suction pressure corresponding to the initial hemispherical projection length tongue, dl/dt is the rate of entry into the pipette and Rp/Rout is the ratio of pipette radius Rp to the outside cell radius Rout. Between 3 and 8 neutrophils were studied from each sample. It has been previously reported that different types of leukocytes as well as passive and activated neutrophils can be distinguished by light microscopy via electronic magnification, without staining and without separation medium [1820]. Neutrophils were distinguished from other leukocytes and their precursors by the shape of the nucleus. In mature PMNs, the nucleus consists of two to five lobes. Although sometimes the granules interfere with the view of the nucleus, it always becomes clearly visible upon cell aspiration [18,20]. In this study we measured cell rigidity on passive PMNs only. Passive PMNs were considered to possess the following characteristics: regular cell membrane appearance, spherical shape, and no pseudopod formation. Activated PMNs were assumed to be the cells with visible pseudopod projection greater than 0.5 µm, irregular cell membrane appearance, tongue growth in the pipette abruptly slowed or stopped, or activated upon contact with the micropipette surface [19,20]. Therefore the terms passive and active imply morphological and not functional status of activation. In each experiment the distribution of passive and active PMNs was counted by light microscopy. The experiments were conducted at a controlled temperature of 20–25°C. The duration of each experiment was no longer than 3 h.

Cytokine measurements
Blood obtained from the arterial site of the arteriovenous fistula (HD patients) or by phlebotomy (CRF patients and healthy subjects) was collected in sterile vacuum tubes containing EDTA (1 mg/ml) and centrifuged immediately at 400 g at 4°C for 10 min. Plasma was removed without disturbing the buffy coat and small aliquots were frozen at -70°C. Samples were thawed only once. TNF-{alpha} and IL-1ß were detected by enzyme-linked immunosorbent assay (ELISA) (Endogen Inc, Woburn MA, USA) in duplicate. The method detects <5 pg/ml of human TNF-{alpha} and <1 pg/ml of human IL-1ß.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Some characteristics of CRF patients with are presented in Table 1Go. Table 2Go illustrates the differences in PMN rigidity between study groups and controls. PMNs from end-stage non-dialysed CRF patients, HD patients dialysed with cellulose acetate membranes, and HD patients dialysed with polysulphone membranes showed significantly increased rigidity compared with controls (9306±1742 vs 3970±1104, P<0.05; 8878±2122 vs 3979±1349, P<0.05 and 8662±1869 vs 3979±1349, P<0.05 respectively). No differences were found between early-stage CRF patients and controls, nor among end-stage CRF patient groups. TNF-{alpha} levels were significantly higher in end-stage non-dialysed CRF patients, HD patients with cellulose acetate membranes and HD patients with polysulphone membranes compared with controls (809±197, 820±340, and 734±313 vs 15.5±3.1, P<0.05 respectively) but not in early-stage CRF patients. IL-1ß levels were significantly higher in end-stage non-dialysed CRF patients, HD patients with cellulose acetate membranes, and HD patients with polysulphone membranes compared with controls (319.5±232, 535.2±317.2, and 508.5±297.6 vs 6.5±4.9, P<0.05 respectively). Table 2Go also shows that rigidity, TNF-{alpha}, and IL-1ß levels in early-stage CRF patients differ significantly from all end-stage CRF patient groups.


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Table 1. Characteristics of patients with chronic renal failure

 

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Table 2. PMN rigidity and TNF-{alpha} and IL-1ß levels in early-stage, late-stage non-haemodialysis (HD) and late-stage HD chronic renal failure (CRF) patients, before initiation of dialysis

 
Figure 1Go illustrates rigidity, TNF-{alpha} and IL-1ß levels during dialysis with cellulose acetate membrane. In the first minute after dialysis PMN rigidity decreased slightly, but it increased significantly at 60 min compared to pre-dialysis values (12 796±2550 vs 8878±2122, P<0.05). After this peak, rigidity decreased significantly at 120 and 240 min (12 796±2550 vs 10 933±1777, P<0.05 and 10 933±1777 vs 9551±1388, P<0.05 respectively) when it reached the pre-dialysis values. TNF-{alpha} levels increased at 60 min after dialysis (1404±490 vs 812±340, P<0.05) and at 120 and 240 min returned to levels that were not significantly different from the pre-dialysis levels. Although IL-1ß levels showed an increase at 60 min of HD, the values did not reach significance.



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Fig. 1. PMN rigidity and TNF-{alpha}, and IL-1ß plasma concentrations in CRF patients haemodialysed with cellulose acetate membranes before (time 0) and during dialysis. Each dot represents mean±SD of 10 patients. Rigidity is expressed in 1000xdyn.s/cm2.

 
Figure 2Go illustrates rigidity, TNF-{alpha}, and IL-1ß levels during dialysis with polysulphone membranes. A significant increase in PMN rigidity was noted at 60 min after dialysis as compared to pre-dialysis values (11 417±1866 vs 8662±1869, P<0.05). At 120 min rigidity decreased but not significantly and at 240 min rigidity decreased significantly as compared to 60-min nadir values, but it still increased significantly as compared to pre-dialysis values (10 083±1362 vs 11 417, P<0.05 and 10 083±1362 vs 8662, P<0.05 respectively). TNF-{alpha} levels increased at 60min after dialysis (747±283 vs 1276±430, P<0.05) and returned to pre-dialysis levels at 120 and 240 min. Although IL-1ß levels showed an increase at 60 min of HD, the values did not reach significance. There were no differences in rigidity and TNF-{alpha} and IL-1ß levels before and during dialysis between patients dialysed with cellulose acetate and polysulphone membranes. Table 3Go illustrates the percentage of morphologically activated PMNs in control and study patients. There was a significant increase in activated PMNs at 60, 120 and 240 min after HD as compared to pre-dialysis and control values. No differences were found in activation between early-stage CRF, late-stage non-HD CRF, and controls. There were no significant changes in neutrophil count before and during dialysis irrespective of the nature of dialysis membrane. Serum calcium, phosphate, sodium, potassium, magnesium, ferritin, iron, transferrin, PTH and haematocrit were not significantly different between the patients of each group. In all HD patients the urea clearance (residual renal function) was <1 ml/min. The erythropoietin dose in end-stage and HD CRF patients was 2000–4000 IU thrice weekly. None of the patients was an alcohol abuser. All HD patients except two (10–20 cigarettes per day) denied cigarette smoking.



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Fig. 2. PMN rigidity and TNF-{alpha}, and IL-1ß plasma levels in CRF patients haemodialysed with polysulphone membranes before (time 0) and during dialysis. Each dot represents mean±SD of 10 patients. Rigidity is expressed in 1000xdyn.s/cm2.

 

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Table 3. Percentage of morphologically activated PMNs in controls, early-stage, late-stage non-HD and late-stage HD before and during HD with different types of membranes in CRF patients

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Neutrophil sequestration in the microvasculature following intravascular injection of inflammatory mediators is known to involve two margination processes: a rapid sequestration of PMNs that mainly involves an increase in PMN rigidity, and a prolonged sequestration of intracapillary neutrophils that involves cell adhesion via integrins and selectins [14]. While many data have been reported on the expression of leukocyte adhesion molecules during HD, changes in PMN rigidity had not been previously analysed in HD and CRF.

In the present study we clearly demonstrated that morphologically passive (non-activated) PMN rigidity increased significantly in both dialysed and non-dialysed end-stage CRF patients, but it was unaffected in early-stage CRF patients. Rigidity increased further at 60 min after initiation of dialysis. It has been previously reported that endotoxin, TNF-{alpha}, IL-1ß, and IL-6 are increased in plasma of end-stage CRF patients and that some of the cytokines are increased further during HD [811]. In this study both TNF-{alpha} and IL-1ß were significantly increased in end-stage CRF patients and increased further during HD. These results concur in part with those of previous studies. It is well established that cell activation [12,13], endotoxin, and cytokines [12,13] increase PMN rigidity. Complement activation that occurs upon plasma membrane contact subsequently activates PMNs and increases rigidity. It is therefore likely that activation and cytokines increase PMN rigidity in end-stage CRF patients. It has been previously reported that during PMN activation G to F cytoskeleton actin polymerization leads to increased cytoplasmic viscosity and increased rigidity [4,5,10]. In this study, however, we demonstrated that passive (non-activated) PMN rigidity is increased in uraemic patients, which suggests that some factors or substances found in these patients depress the ability of PMNs to deform. This may differ from the effect of PMN activation on rigidity.

The micropipette system used in the present study is relatively cumbersome to carry out, but has the advantage that the actual process of deformation can be observed and calculated for each individual cell. It is also possible to distinguish between passive and active cells, and to calculate the percentage of activated cells. These characteristics, however, represent the morphological and not the functional status of activation and may sometimes be an insufficient evaluation of the PMN resting stage. However, this methodology has been successfully applied in many experiments by several field experts. Deformability was tested only on passive PMNs because cell activation plays an important role in rigidity. Our finding that the percentage of activated PMNs was significantly higher during HD but not in end-stage non-HD CRF patients favours the hypothesis that activation is not the only factor that influences PMN rigidity in uraemic patients. The influence of activation or the presence of cytokines, however, may explain the increase of PMN rigidity during HD. No difference in rigidity was noted between dialysed (before dialysis) and non-dialysed patients with end-stage renal disease. This indicates that accumulation of uraemic metabolites and the presence of significant amounts of circulating endotoxins and cytokines (because of their defective clearance from the plasma) may directly increase PMN rigidity in non-HD patients and in HD patients between sessions. Increased cytokine production may superimpose and further augment this phenomenon during dialysis. Elevated IL-6 levels have also been found in long-term HD and non-HD patients along with elevated IL-1ß and TNF-{alpha} levels [10]. The effect of IL-6 on PMN rigidity has not been assessed. A synergistic or additive effect of several cytokines, endotoxin, or complement fragments cannot be excluded. It has been reported that IL-1ß, TNF-{alpha}, and IL-6 act synergistically in some biological processes [21,22]. However, a mixture of TNF-{alpha}, IL-1ß, and IL-8 had no synergistic effect on PMN rigidity in one study [13].

Although polysulphone HD membranes cause less complement activation as compared to cellulose acetate membranes [14] we did not find any differences in rigidity, cytokine production, or percentage of activated PMNs between the two types of membrane.

Serum electrolytes, ferritin, iron, and transferrin saturation did not differ among end-stage CRF patients. Although these factors can impair PMN function [23,24], it is not known whether or not they can alter PMN mechanical properties. All end-stage CRF patients were receiving erythropoietin. This substance improves PMN function [25] but its action on PMN rigidity has not been determined.

The defective PMN rigidity in CRF patients may have potential pathophysiological importance. Neutropenia and pulmonary vascular leukostasis during the early phase of HD may be due to increased PMN stiffness which, under specific circumstances, may become critical, inducing PMN retention in capillaries. Furthermore, chemotaxis and phagocytosis are major components of PMN function and require intact cytoskeleton properties. Defective PMN rigidity may, to some extent, explain the impaired PMN function in the susceptibility to infections observed in uraemic and HD patients.

In conclusion, PMN rigidity is defective in end-stage CRF patients and the rigidity increases further at 1 h after initiation of dialysis. Cell activation and circulating cytokines, as well as direct PMN impairment, may be related to increased PMN rigidity. This may explain in part the increased susceptibility to infections in uraemic patients and the sequestration of PMNs observed during HD in some patients.



   Notes
 
Correspondence and offprint requests to: Athanasios T. Skoutelis MD, Section of Infectious Diseases, University Hospital, 26500 Rio Patras, Greece. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 8. 9.99
Revision received 19. 5.00.



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