Influence of uremia and hemodialysis on circulating interleukin-1 and tumor necrosis factor α

Kidney International, Vol. 37 (1990), pp. 116—125

Influence of uremia and hemodialysis on circulating interleukin-1 and tumor necrosis factor a ANDRE HERBELIN, ANH THU NGUYEN, JOHANNA ZINGRAFF, PABLO UREA, and BEATRICE DESCAMPS-LATSCHA INSERM U 25 and CNRS UA 122, INSERM U 90 Hôpital Necker, Paris, France

pathology symptoms is the tumor necrosis factor a (TNFa) [3, 11]. Although TNFa and IL-i are biochemically and immunologically distinct, they share remarkable similarities in their biological properties [12, 13]. The fact that both monokines exert pleiotropic activities affecting distinct organs [14] along with the observation that they can circulate in the blood, could suggest that they be considered as hormones of inflammation [3, LL-l plasma levels did not differ from those observed in normal 4]. Lastly, TNFa is able to induce the production of IL-i in vivo and in vitro [15], and a synergy between the two monokines has subjects. By contrast, TNFa was found significantly increased although less than in long-term HD patients. During the first dialysis session, no also been shown in vivo during inflammation [16] and septic significant increase was observed in the levels of either monokine, shock [171. To our knowledge, TNFa has not been extensively Lastly, regardless of the group of patients, no significant influence of Influence of uremia and hemodlalysis on circulating interleukin-1 and tumor necrosis factor a. Interleukin-1 (IL-I) and tumor necrosis factor a (TNFa) were determined in the plasma of long-term hemodialysis (HD) patients and uremic (UR) patients undergoing their first dialysis session using either cellulosic (CUP) or synthetic (PAN-AN 69) membrane-equipped dialyzers. In long-term HD patients, plasma IL-I and TNFa levels were significantly increased compared to their levels in normal subjects. During a single dialysis session, a significant increase in IL-I but not in TNFa was observed. In not yet dialyzed UR patients,

the dialysis membrane could be detected, suggesting that the observed changes are not exclusively secondary to the activation of complement. Altogether, these results suggest that the passage of the blood through

the extracorporeal dialysis circuit triggers the secretion of IL-i and further exacerbates that of TNFa by monocytes. The presence of increased TNFa in the plasma of first-dialysis UR patients suggests that factors unrelated to dialysis contribute to the activation of monocytes in these patients. Lastly, the concomitant presence of IL-I and TNFa in the plasma of long-term HD patients could be responsible for some of

investigated in long-term HD patients [18], whereas more consistent studies have been reported in acute clinical situations such as severe septicemia [19] or renal allograft rejection [201.

It has also long been documented that uremia can itself impair

the immune response capacity, as has been shown both in

patients with end-stage renal failure [21] and in experimental uremia models in the rabbit [22, 23]. Most of these studies have the clinical features observed in these patients, and provides strong focused on T cell-mediated reactions [241, or on neutrophil and evidence favoring the concept that HD can be assimilated to a recurrent monocytic functions such as chemotactic, adherence, phagoacute-phase inflammatory response.

cytic or bactericidal activities in order to explain the high

susceptibility of uremic patients to infections [25—27]. However, the possibility that terminal renal failure could also result Growing interest has focused in recent years on the role of in monocyte activation leading to monokine production has not cytokines in relation to the acute phase response of the inflam- yet been investigated. matory process [1—3]. Among these cytokines, interleukin-1 Among the dialysis-related factors which can result in mono(IL-i), which is mainly derived from monocyte-macrophages, cyte alteration and/or activation, the cellulosic nature of the has been shown to exert a broad range of activities associated dialysis membrane has been incriminated [28—30], mainly via its with both acute and chronic inflammatory changes [4, 5], and to capacity to activate complement system. In this regard, we

possess distinct and multiple biological properties recently

previously showed both in vivo and in vitro that cellulosic

confirmed by molecular cloning [6]. As early as 1983, Henderson et al [7] postulated that IL-l may be involved in some of the acute and chronic secondary effects of long-term hemodialysis (HD) treatment. However, the actual immunopathological role of IL-i in HD patients is just begin-

cuprophane (CUP) but not synthetic polyacrilonitrile (PAN-AN 69) dialysis membrane leads to a strong activation of phagocyte oxidative metabolism which is detectable within whole blood and is, at least partly, induced by activated complement com-

ning to be investigated [8—10].

Another monokine that may well be relevant in HD-induced

ponents generated in the plasma following blood interaction with the membrane [31]. In 1986, we first reported a preliminary study showing that plasma IL- 1 is significantly increased in long-term HD patients and that this elevation occurred regardless of the nature of the dialysis membrane [8]. Furthermore, the capacity of monocytes

Received for publication October 25, 1988 and in revised form May 11, 1989 Accepted for publication July 24, 1989

to release IL-i on contact with dialysis membranes has also been observed in vitro and even in the absence of complement

© 1990 by the International Society of Nephrology

[32].

116

117

Herbelin et a!: Cytokines in ure,nic and hemodialyzed patients

The present study attempts to determine the respective influence of hemodialysis and uremia on circulating IL-I and TNFa and to evaluate the influence of the nature of the dialysis membrane on the presence of these monokines. For this purpose, plasma from both first-dialysis UR and long-term HD patients have been investigated at the start and at the end of the dialysis session, using either CUP or PAN-AN 69 membranes. Methods

Patients and control subjects Forty-four patients with end-stage renal failure, including 18 males and 22 females ranging in age from 21 to 69 years (mean

Cytokines

Human IL-i (hu IL-i), purified from monocyte supernatant, was purchased from Genzyme (Suffolk, England, UK). Recombinant human TNFa (hu rTNF), recombinant human IL-1/3 (hu

rIL-1.3) and recombinant human IL-la (hu rIL-la) were from

Dr. F. Floc'h, (Rhône Poulenc, Vitry, France). Based on half-maximal PHA-induced thymocyte stimulation, 1 U/mI of hu rIL-13 preparation corresponds to 100 pg/mi as defined by immunodetection (ELISA, Cistron Biotechnology, Pine Brook, New Jersey, USA). The activity of recombinant human interleukin-2 (hu rIL-2), provided by the Sandoz Forschunginstitut, Vienna, Austria, was calculated based on the NIH standard.

Recombinant munne interleukin-4 (mu rIL-4), of Dr. F.

Melchers (Basel Institute for Immunology, Basel, Switzerland), 50.1 years), participated in the study upon informed consent. was provided by Dr. E. Ruuth [34], and recombinant human Twenty-six of them were long-term hemodialyzed (HD) pa- IL-4 (hu rIL-4) was provided by Dr. J. Banchereau (Unicet, tients (46 9 months, mean suM), undergoing from four to Dardilly, France). The effects of mu rIL-4 and hu rIL-4 in the five hours of maintenance thrice a week. Eighteen uremic LAF assay were tested by adding twofold serial dilutions of patients (UR) not yet dialyzed were tested just before and IL-4 to the thymocytes starting at concentrations far above the during their first dialysis session. The etiology of renal failure optimal concentration determined in their respective assay. did not differ greatly from that of the general dialysis population Recombinant human interieukin-6 (hu rIL-6, Janssen Biochem(glomerulonephritis, 15; polycystic kidney disease, 8; renal ica, Beerse, Belgium) was from the Institut Henri Beaufour vascular disease, 6; hereditary nephropathy, 5; and unknown (Les Ulis, France). etiology, 10). Inclusion criteria were the absence of intercurrent infection and/or immunosuppressive therapy. Bioassays for cytokines Two types of dialyzers of comparable surface (1 to 1.4 m2) were used: a disposable plate kidney equipped with synthetic The lymphocyte activating factor (LAF) assay. IL-i activity polyacrilonitrile (PAN-AN 69 membrane, Hospal, Meysieu, was determined by measuring the augmentation of phytohemagFrance) and a kidney equipped with cellulosic cuprophane glutinin (PHA)-induced murine thymocyte proliferation, as decapillary membrane (CUP, Hospal). All long-term HD patients scribed [35]. Briefly, io thymocytes obtained from lipopolysacwere dialyzed with the same type of membrane for at least three charide-low responder C3H/HeJ mice, at four to six weeks of months. Acetate baths were used in all patients except two who age, (C.S.E.A.L. Orleans, France) were incubated in RPM! were dialyzed with bicarbonate baths. 1640 medium supplemented with 5% heat-inactivated fetal calf Twenty-five healthy subjects recruited among volunteer serum (FCS, Flow Laboratories Limited, Irvine, Scotland, blood donors served as controls. UK), 25 mi Hepes, 2 mri L-glutamine, 100 lU/mi penicillin (Gibco), 100 sg/mi streptomycin (Gibco), 5 X i0 M 2-ME, 1 pg/ml of PHA (Weilcome Research Laboratories, Beckenham, Plasma preparation England, UK), and plasma samples. Each plasma sample was Patient blood samples of 15 ml were collected in heparinized tested in triplicate. DNA synthesis was assayed for the last tubes (Liquemine Roche, Paris, France, 10 U/mI of blood) from eight hours of a 72 hour incubation at 37°C under 5% CO2 the arterial site of the arteriovenous fistula at the start and at the atmosphere by pulsing the cultures with 1 pCi/well of [3H1end of dialysis sessions. In normal subjects, blood samples thymidine ([3H]-TdR, 5 Ci/mmol, Centre d'Energie Atomique, Saclay, France). The standard deviation of triplicate samples were obtained by cubital venous puncture. Blood samples were centrifuged (600 X g, 10 mm). The was less than 15% of the mean. Since responsiveness to both plasma was separated into small aliquots and frozen at —70°C PHA and IL-i varied among thymocyte preparations, pre- and and all assays were carried out on samples thawed only once. In post-dialysis plasma fractions were assayed on the same thyorder to remove inhibitory factors, plasma were gel filtrated mocyte population. IL-l activity in plasma fractions was exbefore testing for IL-i according to the methodology first pressed as a costimulation index (CI) defined by the mean of proposed by Cannon and Dinareilo [331 and also used by other triplicate determination of [3H]-TdR incorporation in each groups [8, 9]. Briefly, 0.3 ml of plasma was applied to a I x 30 eluted fraction, divided by the mean background incorporation cm sterile column with packed autoclaved Sephadex 0-50 fine in the presence of fractions eluting before the void volume. To discriminate the optimal fractions corresponding to IL-i (Pharmacia Fine Chemicals, Uppsala, Sweden). The column was equilibrated with RPM! 1640 medium (Gibco Laboratories, elution, normal plasma supplemented with various concentraGrand Island, New York, USA) supplemented with 25 mM tions of hu rIL-1/3 was subjected to gel filtration. A single peak Hepes (Gibco), 2 mri L-glutamine (Gibco), 100 lU/mi penicillin of IL-i activity eluting approximately 12,000 to 18,000 daltons (Gibco), 100 pg/ml streptomycin (Gibco), and 5 X iO M was detected. Based on these findings, all plasma from patients 2-mercaptoethanol (2-ME, Merck, Darmstadt, FRG). Thirty- and controls were fractionated and the 12,000 to 18,000 dalton two 0.7 ml fractions were collected in sterile, pyrogen-free range fractions were tested for their LAF(IL- 1) activity. Plasma IL-i level was expressed as the mean activity of these fractions. polypropylene tubes at a flow rate of 1 mI/mm.

118

Herbelin et al: Cytokines in uremic and hemodialyzed patients

The detection limit of hu rIL-1f3 added to plasma and sub- sponding to a concentration of hu rTNFa of 28 pg/ml. It was jected to gel filtration was 15 pg/mi, corresponding to a CI of controlled that addition of up to 15% plasma had no effect on 1.8. Fifty pg/mi of hu rIL- 113 gave a significant index of 4, and the TNF cytolytic assay. The bioassay for IL-2. The plasma fractionated samples were 1 pg of hu rIL-1/3 corresponds to approximately 0.12 CI. In addition to IL- 1, the possible participation of IL-2, IL-4 or tested for their ability to maintain the growth of an IL-2-

dependent murine T-cell line (CTLL), as described [35]. LAF assay, was investigated. In the presence of 1 tg/ml of Briefly, cells were adjusted to a density of io cells/mi in RPM! IL-6, known to mediate thymocyte proliferation 135—37] in the

PHA (that is, the concentration used for detecting IL-i activity), no significant increase in thymocyte proliferation could be detected with hu rIL-6 up to 12,500 U/mi. By contrast, and as already described by other groups, significant increase in LAF activity was observed upon addition of hu rIL-6 together with 10 jig/mi of PHA. As reported by Ziotnik et al [36], we also

found that murine rIL-4 induced significant LAF activity.

1640 medium containing antibiotics and 5% FCS. A total of i0 cells/well of a 96-well plate (Falcon) were incubated at 37°C in 5% CO2 for 48 hours with various sample concentrations. Cells were pulsed for the final eight hours with [3H]-TdR and harvested as already described above. Recombinant human IL-2, titrated along with the unknown samples, was used as positive control.

However, in our test conditions this LAP activity could not be

induced with human rIL-4, regardless of the concentration used, thus confirming its species-specificity [38]. Lastly, plasma fractions were tested for their IL-2 activity by the CTLL assay. In some experiments, plasma IL-i activity was confirmed by

Immunoassays for cytokines Enzyme-linked immunosorbent assay (ELISA) for IL-I /3. A

commercial, enzyme-linked immunosorbent assay (ELISA, Cistron Biotechnology) was used according to the manufacturneutralization with an antibody against human IL-i (rabbit er's suggestions for measuring IL-i/3 levels in diluted plasma polyclonal antibody, Cistron Biotechnology). In these experi- samples. The antiserum in this assay was shown to be specific ments, plasma fractions with significant IL-i activity were for IL-l /3, and the lowest concentration of hu rIL- 1/3 detectable incubated for six hours at 37°C with 2% to 0.25% (vollvol) rabbit from the zero level was 15 to 20 pg/mI. Samples below the antibody or normal rabbit serum (as control) and then tested in detection level of 15 pg/mi were assumed to have a value of 7.5 the LAF assay. In these conditions, we found that this antipg/mi, that is, the midpoint of the interval between 0 and 15. body, when used at 2% (voL1vol), inhibited 90% of the biological Radioimmunoassays (RIA). A commercial radioimmunoasactivity mediated by 80 pg of hu rIL- 1/3. This neutralization was say (RIA, Cistron Biotechnology), which was standardized with observed as well with chromatographed plasma supplemented hu rIL-1f3, was used for measuring IL-l/3 levels in diluted with hu rIL-la, hu rIL-i/3 or hu IL-i. In addition, this polyplasma samples. Previous experiments to validate the immuclonal anti-IL-i was tested for its effect on hu rIL-6-mediated noassay showed a detection limit of 250 pg/mI for hu rIL- 1/3 in thymocyte proliferation and no significant inhibition could be plasma, less than 10% interassay variation and more than 80% detected. recovery in the detection of recombinant IL-1/3 added to plasma The TNFx activity assay. TNFa was measured by its cyregardless of plasma origin (control subjects, HD or UR patolytic activity on the L929 tumor cell line [39]. L929 cells were tients). maintained in RPM! 1640 medium supplemented with antibiotTNFa, IL-2 and gamma interferon (IFNy) were assessed in ics and 10% FCS. For the assay, cells were treated with trypsin the plasma of patients and controls by means of soluble phase (0.125%) (Gibco), washed twice with RPMI and adjusted to a RIA (Prof. P. Franchimont, University of Liege, Belgium) and density of 2.5 x l05/ml. The cells were seeded in 96-well IRE-MEDGENIX (Fleurus, Belgium), using specific rabbit flat-bottomed culture plates (Falcon 3072, Becton-Dickinson and C°, New Jersey, USA) at a density of 2.5 x 104/well and polyclonal antisera and iodinated human recombinant cytokine incubated 24 hours at 37°C under 5% CO2 atmosphere in 100 jil medium consisting of RPM! 1640 supplemented with antibiotics and 10% FCS. The supernatant was eliminated and replaced by 75 ji! of diluted plasma supplemented with 75 jil of actinomycin

D (1 jig/mi; Behring Diagnostics, La Jolla, California, USA), and the plate was further incubated for 18 hours. Cytotoxicity was measured by the MTT (dimethyl-thiazol2-yl diphenyltetrazolium bromide, Sigma Chemical Co., St. Louis, Missouri, USA) colorimetric assay [401 at 570 nm using a microplate reader (MR 600, Dynatech, Alexandria, Virginia, USA) with a reference filter at 630 nm. Plasma samples were tested in duplicate and results of optical density of the sample well as a percentage of that of the control well was taken as a measure of cell survival. RPM! 1640 supplemented with 10% FCS was used in the control wells. The amount of TNFa of each plasma sample was calculated on the basis of dilution curves plotted against cell survival. One TNFa unit was defined as the amount of TNFa giving 50% cell survival. Before testing,

molecules as described in [41, 42]. The sensitivities of the RIA used, defined as the smallest amount of unlabelled antigen able to significantly reduce the binding of the corresponding cytokine to each given specific antibody, were as follows: 50 pg/mi for TNFa, 0.4 U/mI for IFNy and 0.5 U/mi for IL-2. As already detailed elsewhere [41, 42] the antiserum used in each RIA did not show cross reactivities that could affect the specificity of the assays in the experimental conditions used.

Presentation of results and statistical analysis In long-term HD patients, each post-hemodialysis cytokine plasma level was corrected according to hemoconcentration measurement using the method described by Bergstrom and Wehle [43]. Such correction was not made in nondialyzed UR patients undergoing their first dialysis since no ultrafiltration was performed. All data are given as mean SEM and compared either by the Student's 1-test or by the paired 1-test depending on the sample

plasma samples were heated at 56°C for 30 minutes. The groups (unpaired or paired) to be compared. Differences were detection limit of TNFa in plasma was about 10 U/mI cone-

considered to be significant when P was less than 0.05.

119

Herbelin et al: Cytokines in uremic and hemodialyzed patients

8

Table 1. Inhibition of plasma IL -1 activity by a polyclonal antibody against human IL-l

Plasma ILia 6

Patient

(CI)

1/25

1/50

1/100

1/200

1

4.2 4.5 5.9 6.2

90

82 73 63

89 79 76

75

86

45 60

2 3

4

0 > U

S.

. S

4

% Inhibitionb with anti IL-I dilution of the antibody

hurlL-1/3

18.2

86

79 85 92

85

70 35 21 24

200 pg/ml a Plasma LAF (IL-i) activity expressed as costimulation index (CI). b% inhibition: CI of plasma sample in the presence of the antibody

CI of plasma sample in the presence of control serum

S

xIOO.

I: 2

0

.

.

0

445

8

C

UR

HD

6

Fig. 1. Plasma IL-I activity in 25 healthy control subjects (C), 13

first-dialysis uremic patients (UR) and 25 long-term heinodialysis patients (HD) before a dialysis session. Each point represents the mean

of IL-i activity (CI) in plasma fractions ranging from 12 to 18 kDa molecular mass, obtained in a given patient. Plasma IL-I activity is significantly increased in HD patients, compared both to healthy

0

control subjects and UR patients (P < 0.001).

>-

>

Results

Plasma LAF(IL-I) activity Comparison between first dialysis and long-term dialysis patients. Figure 1 compares the LAF(IL-l) activity observed in plasma fractions from 13 UR patients undergoing their first

2.

dialysis session, 25 long-term HD patients and 25 normal subjects. All patients were tested at the start of the dialysis

session. Compared to normal subjects (CI: 1.05 0.08, mean 5EM), no significant LAF(IL-l) activity was detected in plasma fractions from UR patients (CI: 1.27 0.07, mean SEM). By contrast, and although great variations existed among individI I ual cases, the mean LAF(IL-l) activity was significantly higher To Tf Tf To in HD patients (CI: 2.91 0.30, mean SEM; P <0.001). The UR HD results obtained in control normal subjects show that a significant IL-i activity gives a CI superior to 1.8 (mean + 2 SD of Fig. 2. Plasma IL-i activity in 12 first-dialysis uremic (UR) and 24

0.

controls). According to data obtained with normal plasma long-term dialyzed (HD) patients at the start (T0) and the end (Tf) of a supplemented with hu rIL-1/3, this CI corresponded to approximately 15 pg/mi of IL-i. On this basis, significant LAF(IL- 1) activity was found in 1 out of 25 control subjects and I out of 13 UR first-dialysis patients against 20 out of 25 long-term HD patients (P < 0.001; test). Among the latter, four patients

single dialysis session. Plasma IL-I activity (CI) is measured as

fractions from plasma supplemented with 60 pg/ml of hu rIL-lJ3 (data not shown). In these patients with the most pronounced IL-i activity, we

a similar neutralization pattern was obtained from fractionated normal plasma supplemented with hu rIL-l/3. Effect of the dialysis session. Figure 2 compares the plasma LAF(IL- 1) activity at the start and at the end of a single dialysis session in both first-dialyzed UR and long-term HD patients. In

described in legend of Figure 1. Each point represents the plasma IL-i activity in a given patient. The differences observed between the s iii and the end of the dialysis session in HD patients are significant (P < 0.001).

showed a CI of 5 to 6 corresponding to that obtained with

also verified that this activity was totally neutralized by antiIL-i, and in a dose-dependent manner (Table 1). Interestingly,

Herbelin et at: Cytokines in uremic and hemodialyzed patients

120

8

ity of the assay) in first-dialysis UR patients (P < 0.05) and control subjects (P < 0.03). In addition, this immunoreactive IL- 1 activity increased during the dialysis session (P < 0.05), thus corroborating the LAF assay determination. By contrast, regardless of plasma origin and time of dialysis, no significant IL-l/3 could be detected by RIA in any of the plasma tested. This result confirms our previous observation that the LAF assay is approximately ten times more sensitive than the RIA

r 6

specific for human IL- 1/3 (data not shown). A strong linear correlation was observed (r =0.88, P <0.001,

N 31) when IL-I measured in the above experiments by

(-)

ELISA was compared to that obtained by the LAF assay in the same plasma (Fig. 4).

>-

4

Plasma TNFs immunoreactivity

Comparison between first dialysis and long-term dialysis patients. Figure 5 illustrates the results obtained in the determination of plasma TNFa by radioimmunoassay in long-term 2 HD and first-dialysis UR patients before the dialysis session. It is observed that: 1) compared to its level in normal subjects 4.2 pg/mi, mean SuM), plasma TNFa immunoreac(46.7 tivity was significantly higher in both long-term HD and firstdialysis UR patients (P < 0.001); and 2) among the two groups of patients, long-term HD had significantly higher TNFa levels I I 0 (131.8 5 pg/mI, mean SEM) than first-dialysis UR patients To If To Tf (89.7 3.8 pg/ml, mean SEM, P < 0.00 1). CliP PAN Effect of the dialysis session. As shown on Figure 6, RIA Fig. 3. Comparative effect of cellulosic-CUP membrane and syntheticdetermination of plasma TNFa levels before and at the end of PAN-AN 69 ,nembrane equipped dialyzer on plasma IL-I activity the dialysis session showed that TNFa remained constant, that during a single dialysis session in long-term dialyzed patients. Plasma IL-i activity (CI) is measured as described in legend of Figure 1. Plasma is, mean level comprised between 125 and 135 pg/mI in both IL-i activity is significantly elevated in both CUP and PAN-AN 69 groups of patients. Moreover, regardless of the time of samtreated, long-term dialyzed patients compared to healthy control sub- pling, plasma TNFa levels did not significantly differ between jects (P < 0.00 1) and in both groups further increased between the start the patients treated with CUP and those treated with PAN-AN (Io) and the end (Tf) of the dialysis session (respectively P < 0.001 and 69 dialysis membranes (Fig. 7). P < 0.005). —

Plasma TNFa biological activity

first-dialysis UR patients, the mean LAF(IL-l) activity reTo determine whether TNFa immunoreactivity in plasma mained at its initial level, that is, similar to that observed in from dialyzed patients could exert biological activity, we tested normal subjects (Fig. 1). By contrast, in long-term HD patients, the capacity of some of these plasma to induce cytolytic activity the already high LAF(IL-l) activity observed at the start of the on the TNFa-sensitive L929 cell line. dialysis session further increased in 18 out of 24 patients (P < Table 3 compares the TNFa activity measured in diluted 0.00 1). plasma from five long-term HD patients, four first-dialysis UR As shown on Figure 3, which illustrates the influence of the patients and six normal subjects. No significant TNFa activity

membrane equipping the dialyzer, HD patients routinely could be detected in the plasma of normal subjects, except in treated with the cellulosic (CUP) dialysis membrane showed higher predialysis levels of LAF(IL-1) plasma activity than those with the synthetic (PAN-AN 69) membrane, but the observed differences in the mean CI values (respectively 3.31

one case which showed TNFa level close to the detection limit of the assay (10 TNFa U/mi). Compared to normal subjects,

significant TNFa activity was detected before the dialysis

session in three out of four first-dialysis UR patients and four sEM) were not statistically out of five long-term HD patients. Plasma TNFa activity was

0.52 vs. 2.51 0.21, mean significant. At the end of the dialysis session, LAF(IL-l) also significantly higher in long-term HD patients than in activity significantly increased in both CUP- (P < 0.001) and first-dialysis UR patients, although in both groups it remained PAN-AN 69- (P < 0.005) treated patients. constant between the start and the end of the dialysis session.

Plasma IL-I /3 immunoreactivitv IFNy and IL-2 determinations Table 2 shows results obtained when plasma IL-lJ3 level was determined by its immunoreactivity in ELISA and RIA. Using Neither immunoreactive IFNy nor IL-2, determined either by the ELISA, a significant increase in plasmatic IL-113 concentra- the IL-2 CTLL-dependent assay or by RIA, was detected in any

tion was found in long-term HD patients as compared to its almost undetectable level (<15 pglml according to the sensitiv-

of the plasma from UR (N = patients.

14)

or long-term HD (N =

16)

121

Herbeiin et a!: Cytokines in uremic and hemodialyzed patients Table 2. IL-l/3 immunoreactivity in patients before and after hemodialysis

ELISA

5.05

51.15

5.05

(5)

12.50 5.95 (6)

NDb

ND

ND

ND

ND

(5)

(6)

(6)

(7)

(7)

7.60

pg/mi RIA pg/mi

Long-term HD patients Before After

First-dialysis UR patients After Before

Controls 4,82

13.15

31.15

12.70

(7)

(7)

(6)

sEM). Compared to the basal levels in normal subjects (P < 0.03) and Plasma immunoreactive IL-l/3 levels are expressed in pg/mi (mean pre-dialysis levels in first-dialysis UR patients (P < 0.05), pre-dialysis levels are significantly elevated in long-term HD patients and further increased during HD (P < 0.05). a Number of cases in each group tested b Not detectable, that is, below 250 pg/mi

200 -

.. 150 S

—..— 20

40

60

80

•

IL-i 3, pg/mi EL ISA

100

4. Correlation between measurements of IL-I by LAF bioassay (CI) and ELISA (pg/mi) in plasma from 5 control subjects, 6 firstFig.

z

. S.. 50

Discussion

The present study shows that both IL-i and TNFa are

studies showing that increased intracellular IL- 1 activity can be observed in freshly isolated monocytes [44] and in supernatants

+ S. •

•5• S

S... S

£

significantly increased in the plasma of long-term hemodialysis

patients whereas in first-dialysis UR patients, TNFa, but not IL-l, is detected in the plasma. In both first and long-term dialysis patients, neither IFNy nor IL-2 can be detected. Since IL-i and TNFa are mainly of monocyte origin, whereas IFNy and IL-2 are lymphocyte products, these findings strongly support the hypothesis of selective monocyte activation induced in both first- and long-term dialysis patients. Indirect evidence for the presence of circulating IL- 1 in long-term HD patients had also recently been afforded by

-.••.

U-

dialysis UR and 7 long-term HD patients before and after the dialysis session (r 0.88, P < 0.001, N = 31).

$

C

UR

HO

5. Plasma immunoreactive TNFa levels (pg/mi) in 21 healthy control subjects (C), 18 first-dialysis uremic (UR) and 16 long-term Fig.

dialyzed (HD) patients before a dialysis session. Each point represents

the mean of duplicate determination. TNFa levels are significantly elevated in both UR and long-term HD patients compared to C subjects

(P < 0.001), and significantly elevated in long-term HD patients compared to first-dialysis UR patients (P < 0.001).

of unstimulated peripheral blood mononuclear cells isolated from long-term HD patients [10]. Previous studies of plasma known molecular wt of IL- 1; it is neutralized by a polyclonal LAF(IL-l) activity, recently reported by several groups includ- anti-IL-l antibody; lastly, a close correlation exists between ing ours [8—10] involved a limited number of long-term HD LAF activity and the level of immunoreactive IL- 1 as measured patients and did not investigate the presence of this monokine in UR nondialyzed patients.

by ELISA. The presence of circulating IL-i measured by

immunoreactivity has not yet been reported in long-term HD The present study, based on a large series of 44 patients, patients. Although immunoreactive IL-i was detected by provides strong evidence that significant LAF(IL- 1) plasma ELISA, it could not be detected by RIA, which is ten times less activity is detectable in long-term HD patients and also first sensitive. Other cytokines can likewise be present in the plasma of shows that it is not exclusively related to uremia. Moreover, we formally established that IL-i is responsible for the observed these patients and more particularly IL-6, which is active in the LAF activity: indeed, this activity elutes predominantly in the PHA-induced thymocyte assay [37]. Although IL-6 was not 12,000 to 18,000 molecular wt range, corresponding to the specifically tested in the present study, the fact that hu rIL-6

Herbelin et a!: Cytokines in uremic and hemodialyzed patients

122

200

200

150

150

S.-

' 100 a U-

a

U-

100

z

zI-

I—

50

50

0 To

0

I

L

Tf

To PAN

CUP

Tf

To UR

Tf

To HD

Fig. 6. Plasma immunoreactive TNFa levels (pg/mi) in 14 first-dialysis

uremic (UR) and 14 long-term dialyzed (HD) patients at the start (T0) and the end (1') of dialysis session. Each point represents the mean of duplicate determination, TNFa levels are not significantly different at the end of dialysis compared to its level at the start in both UR and HD patients.

induced no significant thymocyte proliferation at the PHA concentration used in our LAF assay argues against the possibility that it could interfere in the IL-i bioassay. Moreover, we could exclude the presence of IL-2, which is also known to interfere in the LAF assay, and the possibility that human IL-4 could induce significant proliferation of murine thymocytes. The presence of plasma TNFa, a cytokine intimately associated with the acute phase response and sharing almost all of its properties with IL-i, has recently been reported in one longterm HD patient by Lonnemann et al [18]. The present study shows that TNFa is considerably increased in plasma of longterm HD patients compared to normal subjects. Unlike IL-l, TNFa is also significantly increased in UR patients before their first dialysis, although not as much as in long-term HD patients.

Tf

Fig. 7. Comparative effect of celiulosic-CUP and synthetic-PAN-AN

69 membrane-equipped dialyzers on plasma immunoreactive TNFa levels (pg/mi) during a single dialysis session in long-term dialyzed (HD) patients. Each point represents the mean of duplicate determinations. TNFa levels are not significantly different in CUP compared to PAN-AN 69 treated HD patients. No significant change in TNFa levels is observed at the end (Tf) compared to the start (T0) of dialysis session regardless of the type of membrane equipping the dialyzer.

Table 3. TNFa activity in patients before and after hemodialysis

Controls 7.05

3.58

(6)

First-dialysis UR patients Before After 39

14.8 (4)

35

12.6

(4)

Long-term HD

patients Before

After

60 28.3

76 24

(5)

(5)

Plasma TNFa activity is expressed in units/mi (mean

5EM).

Compared to the basal levels in normal subjects, pre-dialysis levels are significantly elevated both in first-dialysis UR (P < 0.02) and long-term

HD (P < 0.05) patients, but not further increased during HD in both groups of patients. a Number of cases in each group tested.

This apparent discrepancy between IL-i and TNFa plasma evidence for cell-associated TNFcr has been reported; in addition, we failed to detect its presence in LPS stimulated human monocytes (unpublished observations). Increased TNFa levels in the plasma of not yet dialyzed UR propose that these factors induce the production of both patients could be induced by damaged cells. Baud et al [45] levels observed in uremic patients suggests that uremia-dependent factors may selectively influence the secretion of TNFa without interfering with that of IL-i. Alternatively, one may

monokines but that while the former is secreted, the latter remains intracellular and/or membrane-associated. Previous studies have reported the presence of such a form of IL-i, especially in long-term HD patients [44]. By contrast, no

recently showed that rat mesangial cells produce cytokines related to TNFa. Similar observations were also reported by other groups with rat [46] or rabbit [47] glomeruli. These findings suggest that mesangial cells could be responsible for

Herbelin et a!: Cytokines in uremic and hemodialyzed patients

123

the increased TNFa production observed in patients with tients regardless of the type of the membrane equipping the end-stage renal failure. Alternatively, a possible role of in- dialyzer, and given the above-mentioned in vitro studies, we creased TNFa levels in the development of renal impairment before dialysis should be considered. In fact, some authors

have recently shown that TNFa infused in rats can induce metabolic acidosis and acute renal failure [3]. Moreover, Rhee, McGoldnck and Meuwissen [271 reported the presence of a neutrophil stimulatory reduced oxygen species (ROS) factor in

serum from nondialyzed UR patients and suggested that it is specifically associated with renal dysfunction. Although this serum factor was found in the range of 1,000 dalton molecular mass, the role of TNFa, known to induce ROS production by

conclude that the type of membrane does not exert a pivotal role on the presence of these circulating cytokines. Among other factors which may contribute to triggering the secretion of these monokines in hemodialysis patients and consequently to masking complement-dependent monokine production, the microbial contamination of the dialysate should be considered. Although all the evidence is not yet in hand, it is

highly probable that repeated exposure of blood to minimal

doses of endotoxin may lead to chronic monocyte activation. In addition, according to a recent report from Nisbeth et a! [55], phagocytic cells [48], remains to be investigated in nondialyzed significant amounts of circulating endotoxins can be present in UR patients. Progressive renal damage may indeed result from not yet dialyzed UR patients, thus suggesting a possible role of TNFa-induced production of ROS by neutrophils in the vascu- LPS in the increased levels of TNFa observed at the beginning lar membrane of the kidney. of the first dialysis. The fact that IL-i was not detectable in Interestingly, during the first dialysis session, IL- 1 remained these patients could be related to a recent report [49] showing undetectable and TNFa maintained its initial level. These increased plasma TNFa but not IL-i in normal subjects treated findings may suggest that repeated dialysis sessions and/or with endotoxins. accumulation of dialysis-related factors are necessary to induce Even though we show that IL- 1 and TNFa can be detected in

or to amplify the secretion of these cytokines. Comparative plasma of long-term HD patients, it remains to be verified investigation of TNFa and IL-i plasma levels at the beginning whether these two monokines are present in sufficient amounts and the end of a single dialysis session in long-term HD patients to be responsible for the clinical manifestations seen in longshowed a significant increase in IL-l but not in TNFa. This term dialysis patients. Concerning IL-i, this study provides discrepancy between the evolution of IL-i and TNFa levels evidence for the presence of circulating immunoreactive IL-i in could be explained by differences in the kinetics of secretion of long-term HD patients, thus corroborating previous [8] and the two monokines. In this regard, Michie et a! [49] have present findings obtained by the LAF assay. However, the IL-i reported that TNFa response following injection of endotoxin levels observed, ranging between 30 and 60 pg/mI (except in one in human volunteers occurs between the 90th and 120th minute and is no longer detected at the 4th hour. In contrast to our previous report showing that CUP but not

case in which it rose over 100 pg/mi), are much lower than those reported by Maury and Teppo [56] in renal allograft recipients

undergoing acute rejection. Concerning TNFa, recent studies

PAN-AN 69 membrane induces a significant activation of have demonstrated elevated levels of plasma TNFa in infecphagocyte oxidative metabolism [311, we did not detect a major influence of CUP membrane on IL-l or TNFa secretion. Since

CUP but not PAN-AN 69 membrane has the potential of

tious diseases. Scuderi et al [57], using an enzyme immunoassay, found a mean level of TNFa reaching 119 pg/ml in patients with malaria, while Waage, Halstensen and Espevik [19], using a bioassay, found raised TNFa levels (440 U/mI corresponding to 100 pg/mI) in septicemic patients. The fact that in our study,

activating the complement system, our observations suggest that IL-i and TNFa production in long-term HD patients is not exclusively caused by the activation complement fragments on the one hand, similar immunoreactive TNFc levels are which are well known to be potent inducers of both IL-i and found in the plasma from long-term HD patients and, on the TNFa secretion [50—52]. Interestingly, Binge! et al [531 recently other hand, that this TNFs is biologically active, favors the reported that long-term HD patients treated with CUP mem- hypothesis that TNFa could be at least partly implicated in the branes display high plasma levels of IL-l activity but that this clinical manifestations observed in these patients. was markedly decreased upon a subsequent dialysis with PAN Moreover, particular attention must be paid to synergistic membrane, thus suggesting that this membrane might favor the properties of TNFa and IL-l monokines. Recent studies supbinding or the clearance of IL- 1. The discrepancy between that port the concept that IL-! and TNFa act synergistically in a study and ours remains to be explained. However, the fact that variety of biological processes such as fibroblast prostaglandin our patients were treated for at least three months with CUP or E2 production [58] and induction of polymorphonuclear leukoPAN membrane, allows a more accurate appreciation of the cyte migration [161. Furthermore, the possibility that plasma proper effect of each of the two membranes. Lastly, Haeffner- TNFa when present at sufficient levels could itself induce IL-l Cavaillon et a! [44], measuring intramonocytic IL-! activity, secretion by monocytes [15], must also be considered in longalso found that both CUP- and PAN-treated patients display term HD patients. In this regard, recent results in the study of increased IL-l activity and that no significant difference could the hypotensive effect of IL-l and TNFa in rabbits are relevant to the synergy resulting from the combination of TNFa itself be detected between the two groups of patients. In vitro, Betz et al [32] have shown that a complement plus IL-i induced by TNFa in these rabbits [12]. Similarly, the independent stimulation of IL-i release by human monocytes participation of IL-6 which has been shown to be involved in may occur following a brief exposure to cuprammonium dialy- the acute phase response [59] and to be produced by monocytes sis membranes. Concerning the synthetic (PAN) dialysis mem- in response to TNFa or IL-I [60, 61], requires further study. brane, Lonnemann et al [54] have reported its intrinsic property Furthermore, endotoxin contaminants entering the blood comto stimulate IL-i production. Since circulating IL-i and/or partment from the dialysate may not only induce monokine TNFa are present in first-dialysis UR and long-term HD pa- production but also act as a potentiator of several monokine

Herbelin et a!: Cytokines in uremic and hemodialyzed patients

124

TA: Blood-membrane interaction in hemodialysis leads to infunctions. Likewise, strong synergy between TNFa and bactecreased cytokine production. Kidney mt 32:84—88, 1988 ria and their products, causing hemorrhagic necrosis and lethal 11. ZIEGLER EJ: Tumor necrosis factor in humans. N Engi J Med shock in normal mice, has been recently described [62]. 318:1533—1535, 1988 In conclusion, the present study supports the hypothesis that 12. DINARELLO CA: The biology of interleukin 1 and comparison to both dialysis and uremia related factors contribute to monocyte tumor necrosis factor. Immunol Lett 16:227—232, 1987 activation and consequent IL-i and/or TNFa production. The 13. LE J, VILCEK J: Tumor necrosis factor and interleukin 1: Cytokines with multiple overlapping biological activities. Lab Invest 56: multifaceted biological effects of monokines, which we showed 234—248, 1987 to be produced in large amounts during HD treatment, may 14. OPPENHEIM JJ, MATSUSHIMA K, Y0sHIMuRA T, LEONARD EJ: The explain some of the pathological findings observed in these activities of cytokines are pleiotropic and interdependent. Immunol patients. In addition, our findings demonstrate that the release Lett 16:179—184, 1987 of IL-i and TNFa into the circulation in man is not confined to 15. DINARELLO CA, CANNON JG, WOLFF SM, BERNHEIM HA, BEUTLER B, CEIM1 A, FIGARI IS, PALLADINO MA, O'CONNOR JV severe bacterial infections, but also occurs in the immunologiTumor necrosis factor (cachectin) is an endogenous pyrogen and cal tissue injury caused by the inflammatory response. Lastly, it induces production of interleukin 1. J Exp Med 163:1433-1450, 1986 is tempting to propose that long-term production of these two 16. WANKOWICZ Z, MEGYERI P, I5SEKUTZ A: Synergy between tumour necrosis factor a and interleukin-1 in the induction of polymorphomonokines, which are known for their synergistic effects, may

account for the occurrence of acute phase changes in HD

nuclear leucocyte migration during inflammation. J Leukocyte

patients.

43:349—356,

17.

Biol

1988

OKUSAWA S, GELFAND JA, IKEJIMA T, CONNOLLY RJ, DINARELLO CA: Interleukin 1 induces a shock-like state in rabbits. J C/in Invest

Acknowledgments The authors gratefully thank: Dr. B. Zins for her help in patient

81:1162—1172, 1988 18. LONNEMANN G, VAN DER MEER JWM, CANNON JG, DINARELLO

management; Prof. P. Franchimont and Dr. M. Baudrihaye for the gift of TNFa, IL-2 and INFyRIA; Dr. L. Chatenoud for her contribution in

of tumor necrosis factor during extracorporeal blood N Engi J Med 317:963—964, 1987 19. WAAGE A, HALSTENSEN A, ESPEVIK T: Association between

these RIA determinations; Dr. B. Dugas for the gift of hu rIL-6; F.

CA, KOCH KM, GRANOLLERAS C, DESCHODT G, SHALDON 5: Induction

purification.

Tresset for her technical assistance; Dr. F. Praz for her contribution in preparing the manuscript; Dr. F. Zavala, and Dr. C. Michel-Herbelin for valuable discussions and suggestions; and D. Broneer for revising the manuscript. Recombinant human TNFa and recombinant human IL-l/3 and IL-la were a gift from Dr. F. Floc'h, Rhóne Poulenc, Vitry,

20. MAURY CPJ, TEPPO AM: Raised serum levels of cachectin/tumor

France. Recombinant human interleukin-2 used in this study was

21. HANICKI Z, CICHOCKI T, KoMoRowsRA Z, SULOWICZ W, SM0-

provided by the Sandoz Forschunginstitut, Vienna, Austria. Recombi-

tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1:355—357, 1987

necrosis factor a in renal allograft rejection. J

Exp Med 166:

1132—1137, 1987

LENSKI 0: Some aspects of cellular immunity in untreated and nant murine interleukin-4 used in this study was a gift from Dr. F. maintenance hemodialysis patients. Nephron 23:273—275, 1979 Melchers, Basel Institute for Immunology, Basel, Switzerland and 22. SILVERBLATT FJ, BEATY HN, PETERSDORF RG: The effect of recombinant human IL-4 was provided by Dr. J. Banchereau, Unicet, uremia on the pyrogenic response of rabbits. Proc Soc Exp Biol Dardilly, France. Recombinant human interleukin-6 used in this study was donated by Institut Henri Beaufour, Les Ulis, France.

Med 132:492—496, 1969

23. TURNBULL AJ, KLUGER Mi, CRANSTON WI, TOWNSEND Y: Are

the kidneys involved in the removal of endogenous pyrogens in the

Reprint requests to Dr. Beatrice Descamps-Lwscha, INSERM U25, Hópita! Necker, 161 rue de Sèvres, 75743 Paris Cedex 15, France.

rabbit?. J Therm Biol 7:95—98, 1982 24. BEAURAIN G, NARET C, MARCON L, GRATEAU G, DRUEKE T, URENA F, NELSON DL, BACH iF, CHATENOUD L: In vivo T cell

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