Hemodialysis with cuprophane membrane modulates interleukin-2 receptor expression

Kidney International, Vol. 39 (1991), pp. 1020—1026

Hemodialysis with cuprophane membrane modulates interleukin-2 receptor expression PHILIPPE ZA0uI, WAYNE GREEN, and RAYMOND M. HAKIM Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, and Histocompatibility Laboratory, Veterans

Administration Medical Center, Nashville, Tennessee, USA

Hemodialysis with cuprophane membrane modulates interleukin-2 receptor expression. Chronic dialysis patients have several indices of immune deficiency. We examined the hypothesis that the biocompatibility of dialysis membranes may influence the ability of lymphocytes to express interleukin-2 (IL-2) receptors on their surface, a key event in

is an index of lymphocyte activation and the extent of activation is expressed as the percent of circulating IL-2Ra positive cells

[10]. The /3-subunit (IL-2R/3) also known as p70 subunit is expressed constitutively on resting T-cells and is regulated

cellular immune response. We investigated the potential role of the separately [11]. However, increased expression of IL-2R13 is dialysis membrane in eight chronic hemodialysis patients. The study also considered a hallmark of lymphocyte activation [11, 12]. design was a cross-over study using cuprophane and polyrnethyl- These subunits have low affinity for IL-2, but their association methacrylate (PMMA) membranes. Chronic dialysis with new Cuprophane membrane leads to an increase in baseline expression of the two subunits of IL-2 receptors, IL2Ra (p55, CD25) and IL-2R/3 (p7°), in peripheral blood mononuclear cell (PBMC). However, Phytohemagglutinin (PHA) stimulation of PBMC harvested after two weeks of dialysis with cuprophane membrane showed a markedly decreased expression

of high affinity 1L-2 receptors. These findings are reversed when patients were dialyzed with a PMMA membrane which is also associated with minimal complement activation. The increased expression of

IL-2 receptor subunits are reproduced in vitro by direct contact of PBMC with cuprophane membrane and by the addition of the anaphylatoxin C5a. This study confirms the participation of lymphocytes in the complex blood-membrane interactions that occurs during dialysis; the results may be relevant to observations of immune deficiency in dialysis patients.

results in a smaller number of receptor complexes, IL-2R, with high affinity for IL-2 (Kd — 10 pM) [13]. Several studies have indicated that this high affinity receptor is uniquely involved in the rapid binding and internalization of IL-2 and mediates most of its biological activities [13—16]. Defects in the ability of T-lymphocytes to express high-affinity IL-2 receptors are asso-

ciated with impaired lymphocyte responses in a number of diseases [17—21].

Because of the importance of high-affinity IL-2 receptor expression in immune responses, we hypothesized that defects

in cellular immunity seen in hemodialysis patients may be related to defects in the ability of lymphocytes harvested from these patients to express high-affinity IL-2 receptors. We therefore studied the potential role of the dialysis membrane on the

expression of these receptors in eight chronic hemodialysis Cuprophane hemodialysis membranes have been shown to patients in a short term, prospective cross-over design using activate several pathways of the inflammatory response, as well either cuprophane or PMMA membranes for two weeks each. as cellular elements such as neutrophils, monocytes and plate- In parallel, we also measured the precent of lymphocytes which lets [1—4]. These inflammatory responses are less evident with express the subunits of IL-2 receptors, IL-2Ra and IL-2R/3, as non-cellulosic membranes such as the polymethylmethacrylate indices of lymphocyte activation. The results indicate a proba(PMMA) or polyacrylonitrile (PAN) membrane [3]. ble role for dialysis membrane in the ability of lymphocytes to Lymphocytes have been assumed to be passive bystanders in express high-affinity IL-2 receptors and its subunits; these this complex blood-membrane interaction, although minor findings may be relevant to observations of cellular immune changes in lymphocyte count and subsets have been demondeficiency in dialysis patients. strated [1, 5, 6]. Recently however, specific markers of lymphocyte activation such as increased numbers of T-lymphocyte Methods bearing interleukin-2 receptors (TAC antigen) and increased Patients soluble interleukin-2 receptor levels have been demonstrated in Eight hemodialysis patients, on chronic hemodialysis for 44 dialysis patients [7, 81. 24 months were selected for participation in this study. All IL-2 receptors (IL-2R) are complex and made up of at least two subunits: a and /3 [9]. The IL-2Ra subunit (also known as patients were previously dialyzed with reused cuprophane TAC antigen, p55 or CD25) is expressed de novo; its expression membrane involving the use of formaldehyde in the reuse process. This reuse procedure significantly attenuates the complement activating ability of cuprophane membranes [3]. On average, the number of reuses for each dialyzer was 12; thus, Received for publication June 26, 1990 and in revised form December 21, 1990 prior to their participation in the protocol, these patients were Accepted for publication December 22, 1990 exposed to a new cuprophane membrane once every month. The mean age of the patients was 69 years (range 51 to 76). © 1991 by the International Society of Nephrology 1020

Zaoui et a!: IL-2 receptor modulation by cuprophane membrane

1021

None of the patients had any clinical evidence for an active inflammatory disease process, malignancy and none were diabetic or had IgA nephropathy as the etiology of their chronic renal failure. None of the patients were on steroid or immunosuppressive therapy and none were on aluminum-chelation

Green fluorescence analysis was performed on a flow cytometer EPICS 753 with Argon Laser. Fluorescence was standard-

therapy with desferrioxamine (which is known to inhibit transferrin and IL-2 receptors expression) [22]. During the period of the study, there was no statistical change in the weekly drawn BUN, hematocrit, differential cell counts or albumin. None of the patients had any evidence of intercur-

log fluorescence channel (log units) of positive cells were

ized each day by fluorescent microbeads and voltage, and amplification and compensation was kept identical for all procedures. The percentage of IL-2Rcl positive cells and the mean

analyzed on 5,000 lymphocytes gated by size and granularity and recorded on a Coulter computer using Immuno" (Diagnostics) software. Background autofluorescence with unstained cells from the same experiments and non-specific staining of rent viral infections, and all were tested to be free from cells with irrelevant mouse IgG-FITC were digitally subtracted. 2) IL-2Rf3 (p70) expression. Two times 106 cells were simiHepatitis or HIV infections. Informed consent, approved by the Vanderbilt University Medical Center and the Nashville VA larly incubated with 1 jig anti-IL-2R13 antibody (mouse hybridoma IgG2, supplied by H. Helderman) and revealed with 5 d Hospital IRB was signed by all participants. Methods

fluorescien isothionate conjugated (FITC) goat antimouse F(ab)'2 fragment (Becton Dickinson). Cytofluorometric analysis

Study design. The design of the study was a cross over for IL-2R/3 expression was performed using procedures similar design. During the first phase of the study all patients were to those described for IL-2Ra expression. IL-2Rf3 expression dialyzed for six consecutive times (3 times a week) with a new cuprophane hollow fiber membrane (Gambro, GF- 120) (phase I). After two weeks, the patients were switched to two weeks of PMMA (TORAY, B2-1.2H) membrane (phase II) and then were returned to an additional period of new cuprophane membranes for another period of two weeks (phase III). All other parameters were maintained constant. Mononuclear cells were harvested from blood drawn pre- and post-dialysis at the first and sixth (that is, beginning and end of

was measured only during phase III when the antibody became available to the authors.

PBMNC (5 x l0 cells/ml) were placed in 24 well plates (NUNC) and incubated for four days at 37°C in a 5% CO2 saturated humidity incubator (Forma Scientific). Viability by Trypan blue dye exclusion was >99% at the beginning of the

ml. Such "minidialyzers" were manufactured from cu-

Radioreceptor assay of high affinity IL-2 receptor. Briefly PHA-induced blasts were counted and incubated for one hour at 37°C in fresh RPM! 1640 to remove endogenous IL-2 then resuspended with V25 IL-2 (Dupont NEN, Wilmington, Delaware, USA) in triplicates of 2 x 106 cells in 100 d RPMI 1640 supplemented with 10% FCS in 1.5 ml Eppendorf silanized (SIGMACOTE) microtest tubes. The method used has been the two week period) dialysis with each type of membrane. detailed elsewhere [161. Serial dilutions of 1125 IL-2 were Control samples, obtained from 18 uremic, non-diabetic and assayed in this culture to determine the saturation (plateau) of non-dialyzed patients with serum creatinine greater than 5.0 specific high affinity binding. This was observed at a concenmg/dl (uremic controls) and from 12 normal volunteers (normal tration of IL-2 between 10 and 50 p (data not shown). Further controls) without known medical problems, were assessed in studies were carried out using 50 M of IL-2. The specific radioactivity of the probe (1.7 x 10 dpm/pM) was used to parallel. Peripheral blood mononuclear cell (PBMNC) culture. Thirty convert maximal levels of bound radioactivity to number of cc of blood were mixed with equal amounts of Hank's balanced binding sites per cell, assuming one receptor per bound molesalt solution (HBSS; Gibco, Grand Island, New York, USA) cule of IL-2. Control experiments with unstimulated cells or and carefully layered over Histopaque (Sigma Chemical Co., 100-fold excess cold IL-2 confirmed only minor nonspecific St. Louis, Missouri, USA) media allowing gradient density binding (<10% maximal binding of the I'25-IL-2). centrifugation. The mononuclear cell layer was collected and Direct effects of dialysis membranes. To investigate the washed twice with HBSS and then resuspended in RPMI 1640 direct effects of the dialysis membrane, independent of complesupplemented with 2 mrvi L-glutamine (Gibco), 100 U/mI peni- ment products or cytokines, we prepared small volume dialyzcillin U, 10 sg/ml Streptomycin (Sigma) and 10% de-comple- ers, made of the same hollow fiber membranes as used clinimented, sterile-filtered certified fetal calf serum (FCS; (Gibco). cally, but with a fiber bundle volume of approximately 1.5 to 2.0 prophane, PMMA and PAN (polyacrylonitrile) fibers. These

minidialyzers were sterilized before each use with high pressure steam. Role of monocytes. To ascertain the role of monocytes in the culture and >90% at the end of the four-day culture period. Part of the cultures were activated with 1% vol/vol Phytohemagglu- in vitro experiments described above, PBMNC from the same tinin (PHA-M, Gibco) in parallel, to investigate the ability of normal controls were further purified by passage through nylon wool columns and panning to yield mostly T-cells. Purity of the these cells to express high affinity IL-2R [161. Cytofluorometric studies. I) IL-2Ra (p55) expression. Cell yield was confirmed by an increase in the CD3 staining of 74.8 3.2% and a loss of monocyte markers (CD 14 staining less pellets (2 x 106 cells) were harvested on the fourth day of culture and incubated in 500 d phosphate-buffered saline (PBS) than 0.6% and CD35 less than 1.2%). These purified T-cells

with 2% FCS, 0.1% sodium azide (modified PBS) with 5 d were then incubated in the minidialyzers for four hours and fluorescein conjugated anti-IL-2Ra antibody (Becton-Dickin- cultured as described previously at 37°C in 5% CO2 for four son) for 30 minutes at 4°C in the dark. Cells were then washed days. Endotoxin contamination. Supernatants of cultures from in twice with 2 ml of the modified PBS and kept in 500 pi of this solution at 4°C until cytometric analysis, generally less than two vitro dialyzer incubation and from the cross over study were hours after staining. tested by a quantitative LAL assay (LAL, QCL 1000, Whit-

1022

Zaoui et a!: JL-2 receptor modulation by cuprophane membrane A Phase II (pmma) a,

1)

—j

—o 5),')

B Phase III (cupropharie)

15.0

.

0 2 a..-.

10.0

•- L)

15.0

10.0

a.,')

1)

o.c zo

z

5.0

5.0 Ca

.E.

0.0 Control

6th dialysis

1st dialysis

Fig. 1. Percent of PBMC expressing IL-2Ra (p55) subunit during chronic dialysis with new cuprophane membrane (phase I). Symbols are: (U) pre-dialysis, () post-dialysis, (•) uremic and normal controls. After two weeks, the percent of IL-2Ra positive cells was significantly

higher, both pre- and post-dialysis. **p <

0.005

compared to first

dialysis and controls.

0.0 1st dialysis

6th dialysis

1st dialysis

6th dialysis

Fig. 2. During dialysis with the PMMA membrane, the % of PBMC expressing IL-2Ra returns to baseline (A), but is again increased during the third phase of the study using cuprophane membrane (B). Symbols are: (U) pre-dialysis, () post-dialysis. P < 0.05; ** < 0.005 compared to first dialysis of phase I.

taker Bioproducts Inc., Maryland, USA) and were always increased by the chronic use of new cuprophane from 36.3 below 2.5 E.U/ml. Statistical analysis

Comparisons between the different phases of the cross over study was by the use of multivariate analysis (ANOVA) using BMDP software package. Statistical significance between dialyzer phases were analyzed using t-test for paired data, whereas comparison with control groups was done using non-paired t-test, after testing for normality. Results

Spontaneous IL-2Rcs expression during dialysis

Prior to beginning the experimental period with new cu-

2.7 log units at the beginning of the study to 52.3 1.2 log units after the sixth new cuprophane dialysis during phase I (Table 1).

The MLFC decreased during dialysis with the PMMA membrane (phase II) but increased again during phase III when cuprophane membrane was again used for a further two week period (Table 1). Thus, chronic dialysis with new cuprophane

membranes leads to an increase in the percent of IL-2Ra positive cells and these cells express higher density of IL-2Rcs on their surfaces. These effects are reversible with the use of non-cellulosic membranes such as PMMA. Spontaneous IL-2Rf3 expression during dialysis

IL-2Rf3 is constitutively expressed on cell surfaces of PBMC; in normal unstimulated controls, 24.9 1.2% of PBMC spontaneously express IL-2R/3 on their surfaces (Fig. 3). During phase III, when new cuprophane membrane was again used, the percentage of IL-2R13 positive cells increased from 40.8 5.3% of cells at the beginning of phase III to 58.0 2.3% (P < 0.05 there was an increase in the number of IL-2Ra positive cells vs. control) just before the sixth dialysis with new cuprophane 0.5% (P = NS). However, when membrane and reached 65.4 2.0% post-dialysis (P < 0.05 vs. from 2.6 0.3% to 3.8 sampled at the sixth dialysis, the number of IL-2Rs positive pre-dialysis). cells had increased to 7.5 2.7% pre-dialysis, and 9.9 1.6% The cellular density of IL-2R/3 positive cells, assessed by post-dialysis, both significantly greater (P < 0.005) than con- MLFC of positive cells, was also increased during dialysis with trols or values at the beginning of the first phase (Fig. I). During cuprophane membranes (phase III) from 41 2.1 log units prior the first dialysis with the PMMA membrane, the percent of to the first dialysis to 65 3.1 log units at the end of the sixth IL-2Ra positive cells remained significantly high, (10.7 2.5%, dialysis (P < 0.02 from baseline) 2.6% post-dialysis). However, prior to pre-dialysis and 9.4 the last (6th) dialysis on PMMA membrane, the number of PHA -stimulated high-affinity IL-2 receptors (IL-2R) IL-2Ra positive cells had returned to baseline value, 3.8 0.8% The number of cells expressing IL-2Ra as well as IL-2R/3 (Fig. 2). When the same patients were again dialyzed with new cuprophane membranes (phase III) results similar to phase I were significantly and maximally increased in PHA stimulated were obtained. Thus, the percent of cells expressing IL-2Ra PBMC harvested from patients chronically dialyzed with reincreased from 2.9 0.6% before the first dialysis with cu- used cuprophane membranes, uremic (non-dialyzed) controls prophane membrane to 8.4 1.6% after the sixth dialysis with as well as normal controls. Furthermore, there were no differences in the percent positive cells or the MLFC of these cells this membrane (P < 0.05 compared to baseline) (Fig. 2). Density of IL-2Ra expression per cell was assessed by the between these groups. Thus, the percent of positive cells in mean log fluorescence channel (log units) of IL-2Ra positive these mitogen stimulated cultures was 82.5 7% for IL-2Ra cells. The mean log fluorescence channel (MLFC) was also and greater than 92.7 6.5% for IL-2Rf3 subunits. MLFC was

prophane membranes, the percent IL-2Ra (p55) positive cells in our study population was 2.6 0.3%, which was not significantly different from uremic non-dialyzed or normal controls (2.7 0.6%; Fig. 1). Following the first dialysis with new cuprophane membrane,

1023

Zaoui et a!: IL-2 receptor modulation by cuprophane membrane

Table 1. Mean log fluorescence channel units (MLFC) of IL-2Ra positive cells in unstimulated cultures of PBMC dunn g the cross-over study Phase III Phase II Phase I PMMA cuprophane cuprophane 6th 1st 6th 1st 6th 1st Dialysis Dialysis Dialysis Dialysis Dialysis Dialysis MFLC

48.3a/52.3a 1.4/1.2

36.3/36.3 2.7/1.5

SEM

28.2a/28.5a

41.4/38 1.3/0.7

2.5/1.4

27.5/34.3 3.9/3.6

37.0/43.3a 4.9/3.2

For each dialysis, the values (I) represent pre/post-dialysis MLFC. a P < 0.05 vs. first dialysis B Phase II

A Phase I

Phase Ill

a) csj 80

a)

a

4500

a)

60

3500

2500

Control

1st dialysis

6th dialysis

Fig. 3 The effect of dialysis with cuprophane membrane (phase III),

1500

ctrl

1st dialysis

ili 6th

1st

1 6th

on IL-2Rf3 expression. Prior to the first new cuprophane dialysis, the percent of IL-2R/3 positive cells was 40.8 5.3%. During the sixth dialysis with cuprophane membrane, the percent of PBMC with IL-2R/3 expression increased from 58.0 2.3% pre-dialysis (La) to 65.4 2.0% < 0.01 compared to uremic controls (U). post-dialysis (s). 0.01 compared to pre-dialysis.

Fig. 4. The number of high affinity JL-2R on PHA stimulated PBMC

7.8 log units for IL-2Ra and 75.2 2.1 log units for 71.0 IL-2Rp, respectively. PBMC harvested pre- and post-dialysis at the beginning and end of each of the three phases of the study were then assessed for their capacity to express high-affinity IL-2R receptors. PHA

cant difference in PHA induced IL-2R between the pre- and post-dialysis samples in this phase of the study. The decrease during the second period of cuprophane exposure (phase III)

stimulation induced the expression of high affinity IL-2R in PBMC harvested from dialysis patients during the three experimental phases, but the extent of this expression varied depending on sampling time (Fig. 4). Prior to the initiation of the first cuprophane dialysis (phase I), the number of inducible IL-2R 177 receptors/cell, similar to receptors per cell was 4152 levels seen in cells obtained from normal and uremic controls

107 receptors/cell). After the first dialysis with cu(4174 prophane membrane, the maximal PHA inducible number of IL-2R expressed was only 2451 200 receptors per cell, significantly less than pre-dialysis (P < 0.01). After two weeks of dialysis with the cuprophane membrane, the ability of PHA to induce high affinity receptors remained significantly attenuated even in pre-dialysis cultures (mean number of receptors 3352 176; P < 0.01). This was further reduced to 2720 267 receptors/cell post-dialysis induced by PHA at the last (6th) dialysis with cuprophane membrane. Although the mitogeninduced expression of high affinity IL-2R was still impaired at the first PMMA dialysis (2768

276 high affinity receptors/cell

pre-dialysis), it improved after two weeks of PMMA dialysis 142 receptors/cell). Furthermore, there was no signifi(3528

during 2 weeks of cuprophane (phase I) and PMMA (phase II) mem-

brane dialysis. The number of IL-2R decreases post-dialysis () compared to predialysis (LI) during the initial dialysis and is further

decreased pre-dialysis during the sixth dialysis of phase I. The findings

< 0.01 compared to pre-dialysis values of the first cuprophane dialysis (phase I) and controls (U).

are reversed during phase II but recur during phase III.

was similar to phase I (data not shown). In four patients studied at the end of a third week of new cuprophane dialysis (phase III), the number of high affinity receptors post-dialysis induced by PHA mitogen remained low (2799 321 receptors/cell) but was not significantly different from its value at the end of two weeks. Thus recurrent dialysis with new cuprophane membrane attenuates the ability of lymphocytes to respond to mitogenic stimulation in terms of expression of high affinity IL-2R; this effect is reversible by the use of non-cellulosic membranes such as PMMA.

In vitro incubation of PBMC with dialysis membranes

We incubated PBMC obtained from normal volunteers in various small volume dialyzers made from different types of fibers currently in use (cuprophane, polymethylmethacrylate, polyacrylonitrile) without further stimulation. As can be seen in Figure 5, whereas incubation with synthetic membrane dialyz-

ers which do not have the polysaccharide structure of the cuprophane membrane, such as the PMMA or PAN (AN69) membranes resulted in no significant increase in the percent of cells expressing IL-2Ra, incubation of the same cells with the cuprophane hollow fiber membrane in vitro for four hours prior to culture resulted in a significant increase both in the percent of

1024

Zaoui et a!: IL-2 receptor modulation by cuprophane membrane 15.0

a: (J

in the patients for at least an interdialytic period, as seen during the cross over period of 72 hours from the sixth dialysis with cuprophane to the first dialysis with PMMA. However, com-

12.5

_J — U) 0) U)

a

a, •

xc, a,,,, z—.

10.0

**

7.5

parison of the changes of IL-2 receptors at two weeks and at three weeks of cuprophane membrane exposure suggests that these defects are not progressive beyond two weeks. Increased IL-2Ra expression, concomitant with decreased

5.0

ability to express high affinity IL-2R by lymphocytes stimulated

2.5

associated with impaired immune responses [17—21]. Although

0.0

Control AN69 PMMA Cuprophane Fig. 5. In vitro incubation of normal PBMC with hollow fiber dialysis

membranes before (U) and after () incubation with C5,. Cuprophane

membrane directly increases the % of PBMC expressing IL-2Ra. Addition of C5a at 50 ng/ml further increases this percentage, but has no appreciable effect on PMMA or PAN non-cellulosic membranes. *D < 0.01 compared to control and non-complement activating membranes; **p < 0.05 compared to unstimulated cells incubated with cuprophane.

with mitogens has been shown in a number of disease states these findings appear at first glance discrepant, recent studies have suggested that T-lymphocytes that have undergone a cycle of proliferative response become refractory to further stimulation by various mitogens and antigens, and have decreased IL-2R response upon successive stimulation [23, 24]. Thus our

hypothesis is that the extent of IL-2Ra and p subunits expressed on lymphocytes harvested following chronic dialysis with cuprophane membrane is an index of their pre-activation and may make them refractory to subsequent mitogenic stimulation by limiting their expression of high affinity IL-2R.

The presence of high affinity IL-2R is crucial for T-cell those cells (4.9 1.1%, P < 0.01) as well as the density of activation and differentiation since it is a target of the T-cell receptors as determined by MLFC (from 24.0 5.0 log units to growth factor IL-2 [9, 12, 24, 25]. In association with low 32.0 7.0 log units, P < 0.05). The addition of the anaphyla- bioavailability of IL-2 in supernatants of stimulated uremic toxin C5a to the culture media prior to their incubation with this T-cells [7, 26, 27], the present study may shed some light on hollow fiber dialyzer membrane lead to a further increase in the various observations of defective cell-mediated immune repercent of IL-2Ra positive cells to 8.1 2.5% (P < 0.05); sponse in hemodialysis patients. These include decreased proconcomitantly the MLFC increased to 39.0 5.0 log units (P < liferation and low IL-2 production in response to antigenic and 0.05 compared to unstimulated cultures). mitogenic challenge [7, 27], cutaneous anergy [28, 29], deIL-2R/3 expression was also affected by incubation with the creased antibody response to T-helper dependent antigen proccuprophane minidialyzer. The percentage of mononuclear cells essing such as influenza [30] and hepatitis B vaccine [31], expressing IL-2Rj3 was increased by direct contact with the increased incidence of cell-mediated infections such as mycomembrane (from 24.9 1.2% at baseline to 47.3 4.6%) and bacterium tuberculosis [32, 33] or viral hepatitis B [34]. was further increased when C5a was added prior to incubation, Several observations made in this and other studies suggest to 79,5 4.2% (P < 0.01 vs. baseline). Density of receptors as that the primary defect in the attenuated lymphocyte responses assessed by MLFC of positive cells also increased significantly seen in hemodialysis patients may reside in the monocytes. from 22.0 5.0 to 37.0 8.0 log units after cuprophane Meuer and colleagues have suggested that the decreased proincubation and 49.0 7.0 log units when C, was added to the liferative response of T-lymphocytes in hemodialysis patients is due to defective monocyte activation and in particular their culture. inability to support primary T-cell activation in vitro; when the Role of monocytes in in vitro experiments activation pathway of lymphocyte bypasses monocyte stimulaIncubation of the monocyte-free purified T-cell population tion the T lymphocyte response returns to normal [35]. Tsakowith cuprophane membrane alone or following the addition of los et a! have also shown that the lymphocyte proliferative C5a resulted in no increase in IL-2Ra or f expression and no response is impaired in hemodialysis patients but is corrected change in the MLFC. Thus monocyte free cultures of PBMC do not respond to C5a or to the polysaccharide structure of cuprophane.

by addition of control monocytes [36]. Recent studies on monocyte activation during dialysis with cuprophane mem-

branes [21 as well as our own observations in this study have pointed out that monocyte activation can occur even in the Discussion absence of complement activation, by direct interaction of the The present cross-over study highlights the participation of monocytes with the polysaccharide structure of the cupróphane lymphocytes in the blood-membrane interactions occurring membrane [2, 37]. Thus, our hypothesis is that the immunomodulatory effects of during dialysis with cuprophane membrane. Chronic dialysis with cuprophane membranes, the most widely used dialysis the cuprophane membrane are mediated by the chronic stimumembrane and one which is associated with activation of lation of monocytes either by direct contact with the polysacseveral pathways of the inflammatory response [1—4], leads to chande structure of cuprophane or via their C5a receptors an increase in the expression of IL-2Ra and IL-2R/3 subunits, leading to a persistent up-regulation of IL-2Ra and f3 subunits markers of lymphocyte activation. More importantly, the num- on lymphocytes. This chronic stimulation decreases the reber of high affinity receptors elicited by PHA is significantly sponse to further antigenic or mitogenic stimuli [23, 24]. This is reduced. These findings are seen following as little as two also supported by our observation on the reversibility of these weeks of dialysis with new cuprophane membrane, and persist findings when a non-cellulosic (and non-complement activating)

1025

Zaoui et al. IL-2 receptor modulation by cuprophane membrane

dialysis membrane is utilized. This hypothesis needs to be confirmed by further studies.

While these indices of lymphocyte activation were not present when the study was initiated in patients chronically dialyzed with formaldehyde-reused dialyzers (which have a markedly attenuated complement activation capacity), it is important to point out that chronic dialysis with cuprophane

of preactivated T cells in hemodialyzed patients: Their possible role in altered immunity. Proc NatI Acad Sci USA 83:7457—7461, 1986 8. BEAURAIN G, NARET C, MARCON L, GRATEAU G, DRUEKE T, URENA P, NELSON DL, BACH i-F, CHATENOUD L: In vivo T cell preactivation in chronic uremic hemodialyzed and non-hemodialyzed patients. Kidney mt 366:636—644, 1989 9. TESHIGAWARA K, W0NG HM, KATO K, SMITH KA: Interleukin-2

high affinity receptor expression requires two distinct binding proteins. J Exp Med 165:223—238, 1987 dialyzers reused with bleach (which leads to recurrent comple- 10. UCHIMAYA T, BRODER 5, WALDMAN TA: A monoclonal antibody ment activation [381 and is prevalent in greater than 30% of (anti-TAC) reactive with activated and fucntionally mature T cells. dialysis units) may well result in similar findings. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. Jlmmunol 126:1393—1397, 1981 Finally, although these findings of reduced inducible IL-2 receptors are seen as early as two weeks after initiation of new 11. DUKOVITCH M, WANO Y, THYU L, THI B, KATZ P, CULLEN B, KEHR J, GREEN WC: A second human IL-2 binding protein that cuprophane dialysis, and persist for up to three weeks, longer may be a component of high affinity IL-2 receptors. Nature

term studies are needed to define the persistence of these

327:518—522, 1987

findings during chronic hemodialysis as well as to define the time course of the onset of reduced IL-2 receptor expression

12. ROBB Ri, GREENE WC: Internalization of interleukin-2 is mediated by the /3 chain of the high affinity interleukin-2 receptor. J Exp Med

pre-dialysis. In summary, dialysis with cuprophane membranes leads to a

165:1201—1206, 1987 13. GREENE WC, BOHALEIM E, SIEKAVITZ M, BALLARD DW, FRANZA

low grade stimulation of lymphocytes as expressed by increased IL-2Ra and f3 subunits but impairs their expression of high affinity IL-2R, while dialysis with non-cellulosic activating membranes reverses many of these abnormalities. While it is difficult to extrapolate these findings to clinical observations of immune deficiency in dialysis patients, the present cross-over

study highlights the potential deleterious role of cellulosic membranes on the immune response of chronic hemodialysis patients. A potential pathogenesis of these defects may be in the

chronic activation of complement and monocytes by these membranes.

BR, LOwENTHAL JW: Structure and regulation of the human IL-2 receptor. Adv Exp Med Biol 254:55—60, 1989 14. YAGITA H, NAKATA M, AZUMA A, NITTA T, TAKESHITA T, SUGAMURA K, OKUMURA K: Activation of peripheral blood T cells

via the p75 interleukin 2 receptor. J Exp Med 170:1445—1450, 1989 15. REED JC, Roas Ri, GREENE WC, NOWELL PC: Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation. J Immunol 134:314—323, 1985 16. PETERSEN CM, CHRISTIANSEN BS, KALTOFT K: Binding, uptake

and degradation of human recombinant interleukin-2 (125-ala) in activated human T- and B-lymphocytes and in monocyte-macrophages. Tissue Antigens 29:257—272, 1987 17. K0NIsHI K, MOLLER DR, SALTINI C, KIRBY M, CRYSTAL RG:

Spontaneous expression of the interleukin 2 receptor gene and Acknowledgments

Antibody against IL-2RJ3 receptor was kindly supplied by Dr. H. Helderman and is gratefully acknowledged. This work was supported. by NIH grant #5-R0l-HL-3605. The authors would like to express their appreciation to the staff and the patients of the Veterans Administration Medical Center and of the Dialysis Clinic Inc. facility in Nashville, Tennessee, for their help and participation.

presence of functional interleukin 2 receptors on T lymphocytes in the blood of individuals with active pulmonary sarcoidosis. J Clin Invest 82:775—781, 1988 18. HAYWARD AR, HERBERGER M:

Culture and phenotype of activated T-cells from patients with type I diabetes mellitus. Diabetes 33:319— 323, 1984

19. SCHENA FP, MASTROLITTI G, JIRILLO E, MUNNO 1, PELLEGRINO

N, FRACASSO AR, AVENTAGGIATO L: Increased production of interleukin-2 and IL-2 receptor in primary IgA nephropathy. Kidney mt 35:875—879, 1989

Reprint requests to Raymond M. Hakim, M.D., Ph.D., Clinical Services, Division of Nephrology, Vanderbilt University, B-2214 Medical Center North, Nashville, Tennessee 37232-2373, USA.

References 1. HAKIM RM, L0wRIE EG: Hemodialysis-associated neutropenia and hypoxemia: The effect of dialyzer membrane materials. Neph-

20. GUPTA 5: Interleukin-2 receptor and transferring receptor expres-

sion in T cells and production of interleukin-2 in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. Clin Immunol Immunopathol 38:93—100, 1986 21. GHIO R, HAUPT E, PIsT0IA V, PERATA A, MINALE P: Impaired in-vitro growth of PHA induced T lymphocyte colonies in hemodialyzed renal failure patients. Blut 50:135—140, 1985 22. CAROTENUTO P, PONTESILLI 0, CAMBIER JC, HAYWARD AR:

Desferoxamine blocks IL 2 receptor expression on human T lymphocytes. J Immunol

ron 32:32—39, 1982

2. ROCCATELLO D, MAZZUCCO G, Coo R, PiccoL! G, R0LLIN0 C, SCALZO B, GUERRA MG, CAVALLI G, GIAcHIN0 0, AM0RE A, MALAVASI F, SENA LM: Functional changes of monocytes due to dialysis membranes. Kidney mt 35:622—631, 1989 3. HAKIM RM, FEARON DT, LAZARUS JM: Biocompatibility of dialysis membranes: Effects of chronic complement activation. Kidney mt 26:194—200, 1984 4. HAKIM RM, SCHAFFER A: Hemodialysis associated platelet activation and thrombocytopenia. Am J Med 78:575—580, 1985 5. CHANDY KG, PAHL M, VAZIRI ND, GUPTA S: Acute effects of dialysis on T lymphocytes in patients with end-stage renal disease.

J Clin

Lab Immunol 17:119—124, 1985

6. CHIDA Y, SAKURAI S, YOSHIYAMA N: The effect of hemodialysis

on lymphocyte subsets during dialysis. Clin Nephrol 25:159—163, 1986. 7. CHATENOUD L, DUGAS B, BEAURAIN G, TOUAM M, DRUEKE T, VASQUEZ A, GALANAUD P, BACH i-F, DELFRAISSY i-F: Presence

136:2342—2347, 1986

23. CANTRELL DA, SMITH KA: The transient expression of interleukin 2 receptors. Consequences for T cell growth. J Exp Med 158:1895— 24.

1922, 1983 CHURILLA AM, BRACIALE TJ, BRACIALE VL: Regulation of T lymphocyte

proliferation. Interleukin 2-mediated induction of

c-myb gene expression is dependent on T lymphocyte activation state. JExp Med 170:105—121, 1989 25. WALDMANN TA, T5UD0 M: Interleukin-2 receptors: Biology and therapeutic potentials. Hosp Pract 22:77—94,

1987

26. LOWENTHAL JW, CEROTTINI JC, MACDONALD HR:

Interleukin

induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells. J Immunol 137:1226— I-dependent

1231, 1986 27. KuRZ P, KOHLER H, MEUER 5, HUTTEROTH T, BUSCHENFELDE

K-H: Impaired cellular immune responses in chronic renal failure: Evidence for a T-cell defect. Kidney mt 29:1209—1214, 1986 28. GOLDBLUM S, REED WP: Host defenses and immunologic alter-

1026

Zaoui et a!: IL-2 receptor modulation by cuprophane membrane

ations associated with chronic haemodialysis. Ann Intern Med 93:597—613, 1980

K-HM, KOHLER H: Selective blockade of the antigen-receptormediated pathway of T cell activation in patients with impaired

29. MoRRIsoN AB, MANESS K, TAWES R: Skin homograft survival in chronic renal failure. Arch Pathol 75:139—147, 1963

primary immune responses, J Clin Invest 80:743—749, 1987 36. TSAKOLOS ND, THEOI-IARIDES TC, HENDLER ED, GOFFINET J,

30. CAPPEL R, VANBEERS D, LIESNARD C, DRATWA M: Impaired

DWYER JM, WISLER RL, ASKENASE PW: Immune defects in

humoral and cell mediated immune responses in dialyzed patients after influenza vaccination. Nephron 33:21—25, 1983 31. STEVENS CE, ALTER MD, TAYLOR PE, ZANG E, HARLEY EJ, SZMUNESS W: Hepatitis B vaccine in patients receiving hemodialysis. Immunogenicity and efficacy. N Eng! J Med 311 :496—501, 1984

chronic renal impairment: Evidence for defective regulation of

32. ANDREW OT, SCHOENFELD PY, HOPEWELL PC, HUMPHREYS MH:

Tuberculosis in patients with end-stage renal disease. Am J Med 68:59—65, 1980

33. LUNDIN AP, ADLER AJ, BERLYNE GM: Tuberculosis in patients undergoing maintenance hemodialysis. Am J Med 67:597—603, 1979 34. CI-JATENOUD L, HEBERLIN A, BEAURAIN G, DESCAMPS-LATSCHA

B: Immune deficiency of the uremic patient. Adv Nephrol 19:259— 274, 1990 35. MEUER SC, HAUER M, Kuz P, MEYER ZUM BUSCI-1ENFELDE

lymphocyte response by macrophages from patients with chronic renal impairment on haemodialysis. Clin Exp Immunol 63:218—227, 1986

37. BETZ M, HAENSCH G, RAUTERBERG E, BOMMER J, RITZ E:

Cuprammonium membranes stimulate interleukin- I release and

arachidonic acid metabolism in monocytes in the absence of complement. Kidney mt

34:67—73, 1988

38. HOENICH NA, JOHNSTON SRD, BUCKLEY P, HARDEN J, WARD

MK, KERR DNS: Hemodialyzer reuse: Impact on function and biocompatibility. mt JArtif Organs 6:261—266, 1983

39. LANGHOFF E, LADEFOGED J: Cellular immunity in renal failure. Depression of lymphocyte transformation by uraemia and methylprednisolone. liii Arch Allergy App! Immun 74:241—245, 1984