Kidney International, Vol. 35 (1989), pp. 1212—1218

In vivo induction of interleukin-1 during hemodialysis

NICOLE HAEFFNER-CAVAILLON, JEAN-MARC CAVAILLON, CHRISTIAN CIANCI0NI,F1NcoIsE BACLE, SIMONE DELONS, and MICHEL D. KAZATCHKINE

INSERM U28, Unite d'Immunopathologie and Service de Nephrologie, Hôpital Broussais; Unite d'Immunoallergie, Institut Pasteur; andCentre E. Rist, Paris, France

In vivo induction of interleukin-1 during hemodialysis. In vivo induc-tion of interleukin-1 (IL-i) production during hemodialysis was inves-tigated by measuring IL-I activity in monocyte lysates from 59 patientsundergoing long-term maintenance hemodialysis with complement ac-tivating and non-complement activating devices. In patients dialyzedwith new hollow-fiber cuprophane dialyzers, predialytic (TO) monocyte-associated IL-i activity was 12.5 3.0 U/ml (mean SEM), a value thatwas higher than that found in normal individuals (2.85 0.85 U/mi; P<0.0025) and in non-dialyzed patients with chronic renal failure (0.950.85 U/ml, P < 0.0001). Cell-associated IL-i activity was consistentlyincreased after five hours of dialysis with cuprophane membranes (42.4

5.5 U/mI, P < 0.0005). Systemic complement activation was demon-strated by the finding of increased plasma levels of C3adesArg antigenduring dialysis. In patients dialyzed with high permeability polyacrylo-nitrile and polysulfone membranes, no intradialytic change in cell-associated IL-i and no complement activation occurred. However, themean predialytic values of monocyte-associated IL-I in these patients(that is, 32.9 5.6 U/mI and 38 5.65 U/mI for the polyacrylonitrileand the polysulfone groups, respectively) were higher than the predialy-tic levels of cell-associated IL-I in the patients from the cuprophanegroup (P < 0.0025). Monocytes obtained at the beginning and five hoursof dialysis from patients dialyzed with polyacryionitrile devices, andmonocytes obtained at five hours but not at the beginning of dialysisfrom patients dialyzed with cuprophane membranes, spontaneouslyreleased extracellular IL-I after 24 hours of culture in serum freeconditions. Neither polyacrylonitrile nor cuprophane differed in theircapacity to stimulate monocytes that had adhered to the membrane, toproduce IL-l in serum free cultures. These results indicate that IL-I istransiently generated during hemodialysis with complement activatingmembranes when C3aiC3adesArg and CsaiC5adesArg, which areknown inducers of IL-i production, are generated in plasma. Non-complement dependent factors, possibly LPS or fragments of LPS thatoften contaminate bicarbonate dialysates, may be responsible forchronic stimulation of IL- 1 production in patients dialyzed with highpermeability membranes.

lation of the anaphylatoxins C3a and CSa and of their stableplasma desArg derivatives C3adesArg and C5adesArg [3, 7].C5a and C5adesArg are directly involved in the pathogenesis ofthe transient dialysis leukopenia, which is mediated by in-creased expression of the adhesive membrane protein Mol(CD1 ib) on granulocytes [8] and sequestration of activated cellsin the pulmonary vasculature [9, 10]. Little or no complementactivation and no leukopenia is observed in patients dialyzedwith plate polyacrylonitrile devices [3, 11]. Intravascular com-plement activation and/or generation of other inflammatorymediators may also be responsible for certain long-term clinicalsequelae in patients on maintenance hemodialysis [2, 12]. Acritical mediator in this respect could be interleukin-1 (IL-i).IL-i represents a family of polypeptides primarily derived fromstimulated mononuclear phagocytes [13]. Endotoxins (LPS)[14] and their polysaccharidic moieties [15], human C5a,C5adesArg [16, 17], C3a/C3adesArg [171 and a number of othersubstances that could be relevant to hemodialysis [18] inducemonocytes to produce IL-I in vitro after a brief exposureperiod.

The present study demonstrates that IL-i is transiently andconsistently generated in vivo during hemodialysis with com-plement-activating cuprophane membranes; we also report thatmonocytes from patients dialyzed with complement-biocompat-ible polyacrylonitrile and polysuifone membranes are chroni-cally stimulated to produce IL- 1, although in most patients nointradialytic increase in IL-i production is observed.

Methods

Study population

An increasing interest is devoted to the understanding of thedeleterious consequences of blood/membrane interactions inextracorporeal devices [1]. Complement activation which oc-curs during hemodialysis [2] is determined by the type ofdialysis membrane [3—5], whether it is new or re-used [6], andby unidentified individual factors [7]. Complement activationconsistently occurs in patients dialyzed with new cuprophanehollow-fiber dialyzers, which results in generation in the circu-

Received for publication June 20, 1988and in revised form November 18, 1988Accepted for publication November 29, 1988

© 1989 by the International Society of Nephrology

Fifty-nine long-term chronic hemodialysis patients undergo-ing five hours of maintenance hemodialysis three times a weekin the same outpatient dialysis center were chosen for thestudy. Informed consent was obtained. Patients included 31men and 28 women ranging in age from 16 to 72 years (meanage: 37.3). The original nephropathy was chronic glomerulone-phritis (31 patients), interstitial nephritis (17 patients), nephro-angiosclerosis (2 patients), polycystic kidney disease (2 pa-tients) or undetermined (7 patients). All patients had been onmaintenance hemodialysis for more than 12 months and hadbeen dialyzed using the same type of membrane for over twomonths at the time of the study. Twenty-one patients wereundergoing hemodialysis with new cuprophane hollow-fiberdialyzers (Hemofrance, l'Arbresle, France; Gambro, Lund,Sweden; Fresenius, Bad Homburg, FRG); 22 patients were

1212

Haeffner-Cavaillon et a!: Interleukin-1 production during hemodialysis 1213

dialyzed with polyacrylonitrile sheet membrane devices (AN69,Hospal, Lyon, France); 16 patients were dialyzed with polysul-fone fiber dialyzers (Fresenius F50 and F60). Bicarbonatedialysate (Pharmacie Centrale des Hôpitaux de Paris, Paris,France) was used in all patients. The endotoxin content of thedialysates used at the time of the study has not been deter-mined. However, the same batches of concentrate were used inpatients dialyzed with Cuprophane and in patients dialyzed withhigh permeability membranes. When tested at monthly inter-vals for one year, the endotoxin content in bicarbonate concen-trates ranged from 2.5 to 270.0 EU/mI and that in the treatedwater ranged from 0—10 EU/mi, as assessed using a commercialcolorimetric Limuius Amebocyte Lysate assay (Wittaber,Walkersville, Massachusetts, USA).

Control subjects were 14 healthy members of the medicalstaff (age 20 to 50) and 10 non-hemodialyzed patients withchronic renal failure (age 39 to 75; serum creatinine 440 to 1200jmol/liter) including six patients with chronic glomerulonephri-tis, three patients with nephroangiosclerosis and one patientwith interstitial nephritis.

Preparation of peripheral blood monocytes and IL-I assays

Heparinized blood was obtained predialysis from the pa-tients' arteriovenous fistula and subsequent samples were ob-tained from the efferent line of the dialyzer during the dialysissession. Peripheral blood mononuclear cells were separatedfrom whole blood by centrifugation on MSL (Eurobio, Paris,France). Monocytes were identified by staining with non-specific esterase (NSE) using alphanaphtyl acetate as substrate[19]. Mononuclear cells were enriched in monocytes by allow-ing the cells to adhere to 24-well plastic cultured dishes (Nunc,Röskilde, Denmark) in the absence of serum for 45 minutes.Five hundred microliters of RPM! 1640 (MA Bioproduct) con-taining 5 x iO NSE-positive cells were plated in each well.After two vigorous washes with RPM! 1640 containing 100 IU/ml penicillin and 100 g/ml streptomycin, mononuclear adher-ent cells were lyzed by three freeze-thaw cycles at —80°C in 0.5ml RPM! containing antibiotics to extract cell-associated (mono-cyte-associated) IL-i, as described [20—22]. Under these con-ditions, the adherent cells contained more than 85% monocytes,as assessed by morphological analysis by phase contrast mi-croscopy, staining with NSE and indirect immunofluorescenceusing anti-lymphocyte antibodies OKT1 1 (Ortho Diagnostics,Raritan, New Jersey, USA) and IOBI (Immunotech, Marseille,France). After centrifugation of the monocyte lysates at 2000 xg for 10 minutes at 4°C, IL-I activity was measured in thesupernatants by means of a conventional ConA comitogenicassay using C3H/HeJ thymocytes [15]. The freeze-thawingprocedure yielded reproducible results since the SD of the meancell-associated IL-i activity measured in eight lysates of normalLPS-stimulated monocytes (10 tg of Neisseria meningitidis[Nm] LPS/5 x 1O5 cells) did not exceed 10% (795 U/mI 61).Units of IL-i activity/mi of culture supernatant were calculatedas follows:

U/mi = cpm of test sample — background cpm

x dilution of test sample

where cpm of test sample is the mean of (3H)-thymidineincorporated in triplicate cultures of thymocytes performed inthe presence of supernatants from monocyte cultures. Back-ground cpm is (3H)-thymidine incorporated in thymocytescultured in RPM! alone.

Each assay of IL-i activity included cultures performed inthe presence of a constant source of monocyte-associated IL-Iobtained by stimulation of monocytes of one individual with 10p.g/5 X i05 cells of Nm LPS as the internal standard. Theinternal standard contained 1000 U/mi of thymocyte- stimulatingactivity and 2.2 ng/ml of IL-i beta antigen as determined usinga commercial radioimmunoassay.

In additional experiments, monocytes from five patientsdialyzed with cuprophane membranes and five patients dialyzedwith a polyacrylonitrile membrane were cultured in the absenceof serum (5 x i05 NSE cells in 0.5 ml RPM! containingantibiotics/well) for 24 hours at 37°C in the presence of 5% CO2.After centrifugation, IL-i activity released in the supernatantswas assayed on inurine thymocytes. RPM! contained no detect-able LPS as assessed using the Limulus assay.

Complement assaysFor measurement of plasma concentrations of C3adesArg

antigen, blood was obtained at times 0, 15 minutes and 5 hoursof dialysis in tubes containing ethylenediamine tetraacetate(EDTA, 5 mii final). Samples were immediately centrifuged at4°C and the plasma was stored at —80°C until processing.C3adesArg antigen levels were determined using the radioim-munoassay developed by Upjohn (Upjohn Co, Kalamazoo,Michigan, USA) [231.

StatisticsAll grouped data are reported as means SEM. Statistical

analysis was performed with Student's 1-test.

MaterialsLPS from Neisseria meningitidis was from Dr. D. Schultz,

Institut Mérieux, Lyon, France. Rabbit anti-human IL-i anti-serum was obtained from Genzyme (Genzyme Corp., Boston,Massachusetts, USA). The radioimmunoassay for IL-I betawas from Cistron Biotechnology, Pine Brook, New Jersey,USA. For in vitro experiments investigating the ability ofdialysis membranes to trigger monocyte production of IL-i,small sterile dishes of Cuprophane and polyacrylonitrile (fromC. Pusineri, Hospal, Lyon, France) were used. The diameter ofthe disc was that of a culture well of 24-well plastic culturesdishes.

Results

Monocyte-associated IL-i activity was measured at the be-ginning and after five hours of hemodialysis in patients under-going hemodialysis with first use cuprophane, polyacrylonitrileand polysuifone membranes. At the beginning of the session(TO), the mean monocyte-associated IL-i activity in 21 patientsundergoing dialysis with cuprophane membranes was 12.53.0 U/mI, a number which was higher than that measured in 14healthy individuals (2.85 0.85 U/mI; P < 0,0025) and in 10non-dialyzed patients with chronic renal failure (0.95 0.85; P< 0.0001). After five hours of hemodialysis, monocyte-asso-ciate IL-i activity increased in 16 of2l patients to reach a mean

background cpm

1214 Haeffner-Ca vail/on et a!: lnterleukjn-J production during hemodialysis

Fig. 1. Cell-associated IL-i activity inmonocytes from chronic hemodialysis patientsundergoing hemodialysis with first use,cuprophane hollow-fiber dialyzers (A),poivacrylonitrile plate dialyzers (B) andpolysulfone hollow-fiber dialyzers (C). Resultsare expressed in units of IL-l activity/mi asdefined in Methods.

value of 42.4 5.53 U/mi (P < 0.0005, Fig. 1). The meanmonocyte count in these patients decreased by 21.3 4%(mean SEM) at the end of dialysis as compared with predial-ysis counts. In 22 patients undergoing hemodialysis with poiy-acrylonitrile membranes, a high value of monocyte-associatedIL-I activity of 32.9 5.6 U/mI was found at TO. The lattervalue was significantly different from that observed at TO in thegroup of patients dialyzed with cuprophane dialyzers (P <0.0025) and from that measured in healthy individuals (P <0.0005) and in non-dialyzed patients with chronic renal failure(P < 0.0005). After five hours of dialysis (T5h) with polyacry-lonitrile membranes, monocyte-associated IL-I activity in-creased in 5 of 22 patients, whereas it decreased or remained atpredialysis levels in the majority of the patients. Mean mono-cyte-associated IL-l activity at T5h was 59.2 7.0 U/mi, whichrepresented an increase over predialysis values (P < 0.01). In16 patients dialyzed with polysulfone fibers, the mean value ofmonocyte-associated IL-i activity was 38 5.65 U/mI at TO,which was significantly higher than in the cuprophane group atTO (P < 0.0025). It was 36.5 6.7 U/mI after T5h of hemodi-alysis, a value that was not different from that measured at TOin this group of patients.

Monocyte-associated IL-i activity that was detected in celllysates using the thymocyte proliferation assay was inhibited ina dose-dependent manner by polyclonal anti-IL-i antibodieswhen used at a 1/50 to a 1/500 dilution. The ratio between IL-iactivity and concentrations of IL-i beta antigen in cell lysatesfrom patients' monocytes was similar to that between IL-Iactivity and IL-l beta antigen in cell lysates from normalmonocytes stimulated with LPS.

To examine whether low predialytic levels of cell-associatedIL- 1 in patients dialyzed with cupropharie membranes were dueto the presence of (a) circulating IL-I inhibitor(s) that would not

be dialyzed with low molecular weight cut-off membranes,various amounts of lyzates from monocytes obtained at TO fromthree patients in the cuprophane group were mixed with dilu-tions of lyzates from normal LPS-stimulated monocytes thatyielded half maximal values for IL-I activity in the thymocyteproliferation assay. No inhibition of IL-i activity was observed.

The kinetics of increase in monocyte-associated IL-i activityin patients dialyzed with cuprophane dialyzers were studied infive individuals. Two patients demonstrated increased values ofcell-associated IL-i as soon as two hours after the beginning ofdialysis. In four of five patients, monocyte-associated IL-iincreased by at least twofold at the end of dialysis (Fig. 2). Asshown in Table 1, the relative intradialytic increase in mono-cyte-associated IL-I was similar when tested during repeateddialysis sessions in a given patient.

Measurements of the plasma concentration of C3adesArgantigen could be obtained in 13 of the patients undergoinghemodialysis with cuprophane membranes and 10 of the pa-tients dialyzed with polyacrylonitrile membranes. In patientsfrom the cuprophane group, C3adesArg levels increased from amean predialysis value of 170 S ng/ml to 1154 76 ng/ml inthe efferent sample at 15 minutes after dialysis (T15 mm; that is,a 6.8-fold increase). C3adesArg levels decreased to 453 45 ng/ml at the end of dialysis. In five patients in this group in whommonocyte-associated IL-I was below 10 U/mI at TO, the relativeintradialytic increase in cell-associated IL-i was correlated withthe relative increase in C3adesArg levels between TO and T15mm (r = 0.75). In patients dialyzed with polyacryionitriledialyzers, C3adesArg levels were 206 20 ng/ml at TO, 3367.3 ng/ml at Tl5 mm (a 1.6-fold increase) and 258 26 nglml atT5h.

In order to assess whether increased values of monocyte-associated IL-I activity that were observed in hemodialyzed

A B

175

150

C

125

> 100>

75

50

25

To T0 TDialysis time, hours

T0

Haeffner-Cavailion et a!: Interieukin-1 production during hemodialysis 1215

Time, hoursFig. 2. Kinetics of increase in cell-associated IL-i activity in mono-cytes from patients undergoing hemodialysis with first use, hollow-fibercuprophane dialyzers. Results are expressed as mean 3(H)-thymidinecpm SD incorporated in triplicate thymocyte cultures. The dashedline depicts 3(H)-thymidine incorporated by thymocytes cultured in theabsence of monocyte supernatants.

Table 1. Measurements of monocyte-associated IL-i activity in apatient dialyzed with first use, cuprophane hollow-fiber devices

during 3 different hemodialysis sessions

Monocyte-associated IL-i activityU/mi

TO T5h SI.Dialysis on day 1 2.2 29 13.1Dialysis on day 15 0 18.6 18.6Dialysis on day 44 0 22 22.0

patients may reflect IL-I activity that could be further releasedin plasma in vivo, monocytes were isolated from blood samplesobtained at TO and T5h of hemodialysis, and cultured for 24hours in the absence of serum. Spontaneous release of IL-iactivity in the culture supernatant was then assessed. Little orno IL-i activity (mean = 0.12 0.11 U/mI) was released bycultured monocytes that had been obtained at TO from fivepatients dialyzed with cuprophane membranes who weretested. A mean of 9.9 2.51 U/mI of IL-i activity was released

by cultured monocytes that had been obtained from thesepatients after five hours of hemodialysis (Table 2). In contrast,high amounts (mean = 25.3 11.61 U/mi) of spontaneouslyreleased IL-l were found in supernatants of cultured monocytesthat had been obtained at TO from five patients dialyzed withpolyacrylonitrile devices. Spontaneously released IL- 1 in-creased only twofold after five hours of hemodialysis in thesepatients (mean = 22.1 U/ml). Monocytes from 14 normal donorscultured for 24 hours under the same conditions in the presenceof 1 g/ml of Nm LPS, released a mean IL-i activity of 155 U/ml (30 to 330 U/mI).

To investigate whether the dialysis membranes may directlytrigger synthesis of IL-I in monocytes, monocytes (5 x l0/well) from four normal individuals were allowed to adhere for45 minutes to plastic culture dishes or to wells that had beenpre-coated with cuprophane or polyacrylonitrile, and furthercultured for five hours in serum-free conditions. The number ofNSE trypsin-detachable cells that had adhered to the surfaceswere 6.09 x lO, 5.10 x iO and 5.64 X iO cells/well for controlplastic culture dishes, cuprophane and polyacrylonitrile, re-spectively. The 45-minute adherence period was insufficient totrigger IL-i synthesis by cells in contact with any of thesurfaces, as assessed by measuring cell-associated IL- 1 activity(Fig. 3). During the five-hour culture period, a time-dependentstimulation of IL-i synthesis was observed on monocytescultured on plastic dishes in the absence of serum. No differ-ence in the amount of cell-associated IL-i activity that wasproduced was found between cells cultured on plastic and cellscultured either on cuprophane or on polyacrylonitrile. Nosignificant adsorption capacity for IL-I of cuprophane andpolyacrylonitrile membranes was found in control experimentsin which a source of cell-associated IL-i was incubated in wellsthat had been precoated with the membranes for five hours at22°C.

Discussion

Interleukin-i (IL-i) is a critical mediator of the inflammatoryreaction that induces a wide range of biological effects at thesite of inflammation and in distant organ targets [13, 18]. LPS[14, 181 and the complement-derived fragments C3adesArg andC5adesArg [16, 171 induce production of IL-l and IL-l releasefrom human monocytes. IL-i is not preformed in unstimulatedcells [21, 241. After stimulation with LPS, cytoplasmic pro IL- 1mRNA can be detected within two hours in vitro [25]. Intracel-lular protein and IL-i activity parallel the IL-l message level[25]. Cell-associated IL-i activity preceeds by at least fourhours the release of IL-I in culture supernatants [26, 271. Someof the adverse effects that may follow exposure of whole bloodto dialysis membranes, including fever, hypotension [i2] and,possibly, the late occurrence of amyloidosis have been sug-gested to be mediated by IL-l. The present study providesdirect evidence for enhanced production of IL-i in hemo-dialyzed patients.

We investigated monocyte-associated IL-l activity ratherthan circulating IL-i for the following reasons: 1) the presencein human plasma of IL-i inhibitors [28] and of factors that mayinterfere with the thymocyte proliferative assay for IL-i activ-ity (29); the unlikelihood of generation of detectable amounts ofreleased IL-l in plasma during the five hours of hemodialysis.Thus, IL-l activity is found in cell lysates after five hours of

20

15 I

10

5

><

Ea(V

0E>-

0

I0 2 5

Abbreviations are: TO, beginning of dialysis; TSh, five hours ofdialysis, S.I., stimulation index. The units for TO were standardized to1.0.

1216 Haeffner-Cavaillon et a!: Interleukin-I production during he,nodialysis

Table 2. Spontaneous IL-i release by cultured monocytes from patients undergoing hemodialysis

Patientnumber

Dialysismembrane

Released IL-i activity U/mi

TO T5h

I Cuprophane 0 13.4 2.O2 Cuprophane 0 15.6 2.63 Cuprophane 0.6 9.1 2.64 Cuprophane 0 05 Cuprophane 0 11.4 0.8

Mean SEM 0.12 0.11 9.9 2.51 P = 1 x iO6 Polyacrylonitrile 25 2.7 34.2 4.87 Polyacrylonitrile 1.9 0.2 34.3 4.28 Polyacrylonitrile 69 10.3 71 9.69 Polyacrylonitrile 11.5 1.3 41 5.6

10 Polyacrylonitrile 19.1 2.6 30.8 4.6Mean SEM 25.3 11.61 42.2 5.20 NS

Monocytes isolated from blood samples obtained at TO and T5h of hemodialysis were cultured (5 x iO NSE cells in 0.5 ml RPMiJwell) for 24hours in the absence of serum. IL-l activity spontaneously released in the supernatant of cell cultures was measured as described in Methods.Results are expressed in units of IL-i activity/mi of culture supernatant.

a Mean SD lfl triplicate cultures.b Student's t-test. NS is non-significant.

012345

50

25]

B

01012345

Time, hours

C

012345

Fig. 3. Kinetics of increase in cell-associated IL-Iactivity in monocytes from 4 norma! individuals culturedin the absence of serum in plastic culture dishes (A), inwells coated with cuprophane (B) and wells coated withpolyacrylonitrile (C). Results are expressed as mean IL-1 U/mI SEM.

culture of monocytes in the presence of low amounts of LPS orC3adesArg. Within the same period of culture, less IL-i activityis released extracellularly from monocytes stimulated with LPSand no IL-i activity is detected in supernatants of culturescontaining C3adesArg [17].

At the beginning of dialysis (TO), low levels of monocyte-associated IL-I activity were found in all patients undergoingmaintenance hemodialysis with new hollow-fiber cuprophanedialyzers. The mean value of cell-associated IL-i in thesepatients was higher than that which was measured in monocytesfrom 14 normal individuals and from 10 non-dialyzed patientswith chronic renal failure. High amounts of monocyte-associ-ated IL- 1, equivalent to 20% of the amount that is found innormal monocytes cultured for five hours in the presence of 1p.g/ml Nm LPS, were found at TO in patients dialyzed withpolyacrylonitrile and with polysulfone devices. Monocytes atTO of polyacrylonitrile dialysis were not maximally stimulatedsince the cells could further be triggered in vitro if cultured inthe presence of exogenous LPS (data not shown). Takentogether, these observations suggest that, three days after adialysis session, monocytes from patients dialyzed with highpermeability membranes were stimulated in vivo to produceIL-i. Furthermore, no IL-i was found to be released frommonocytes obtained at TO from five patients dialyzed withcuprophane membranes and cultured for 24 hours in the ab-sence of serum and exogenous stimulus, whereas monocytesobtained at TO from five patients in the polyacrylonitrile groupreleased significant amounts of IL-i under similar culture

conditions. In both groups of patients, plasma concentrations ofC3adesArg antigen at TO were within the limit of normals. Thenature of the complement-independent IL-i inducer that stim-ulates monocytes in vivo in patients undergoing hemodialysiswith high permeability non-complement activating membranesis unknown at the present time. One possible explanation wouldbe the passage of rough LPS and/or fragments of LPS withmolecular weights between 2800 and 4800 daltons [30] throughpolyacrylonitrile and polysulfone membranes. Preliminary re-sults from recently performed experiments indicate that radio-labeled Nm LPS may cross polyacrylonitrile membranes invitro [31]. Cultured monocytes that have been in contact withLPS for five hours continue to produce cell-associated IL-i 72hours after removal of the stimulus (unpublished observations).Intravenously injected LPS may persist for several days in thecirculation in experimental animals [32]. Low amounts ofcell-associated IL-i found at TO in the cuprophane group couldrepresent residual IL-I from the previous dialysis session orIL-i induced by other factors than intact LPS, of which nopassage has been observed through cuprophane membranes[33—35]. Neither polyacrylonitrile nor cuprophane were foundto differ in their capacity to directly stimulate monocytes thathad adhered to these membranes to produce IL-i serum freecultures in vitro.

In patients undergoing hemodialysis with new cuprophanehollow-fiber dialyzers, monocyte-associated IL-i was signifi-cantly increased at the end of dialysis as compared with valuesobtained at TO. Similar results were obtained in five patients

Haeffner-Cavaillon et al: Interleukin-1 production during hemodialysis 1217

dialyzed with cuprophane flat sheet devices (data not shown).The half life of monocytes exceeds 24 hours [36], suggestingthat monocytes which were found to produce IL-l at the end ofdialysis were representative of the monocyte population circu-lating at the beginning of the session. Significant amounts ofIL-i were spontaneously released in supernatants of serum-free24 hour cultures of monocytes obtained from cuprophanepatients at the end of the dialysis session, although the cellscould further be stimulated by addition of LPS to the cultures(data not shown). The amount of IL-i that was spontaneouslyreleased by monocytes under these conditions representedapproximately 10 to 20% of that which would be released bymonocytes from normal individuals in the presence of 1 gImlNm LPS in serum free conditions. Release of IL-i by unstim-ulated cultured monocytes from the patients may be an indirectindication that IL-i may be released in vivo in the hoursfollowing the end of the dialysis procedure, the time at whichfever occurs in hemodialyzed patients. Complement activationmay be directly involved in triggering the intradialytic increaseof IL-i production that was observed with cuprophane mem-branes. C3a was generated during dialysis in all patients fromthe cuprophane group. A correlation was found between intra-dialytic increases in IL-l production and C3adesArg levels inpatients in whom amounts of cell-associated IL-i were low atTO. The latter correlation was not found if all patients from thecuprophane group were considered, which may be due to thedifferences that exist between individuals in their ability toproduce IL-i upon stimulation with anaphylatoxins [16, 17].Multiple factors may determine the actual extent of comple-ment-dependent IL- 1 production during cuprophane hemodial-ysis, including: the amount of C3alC3adesArg produced, whichdepends on individual responses to cellulose [7, 371; the relativeefficiency of C5a versus C3a production during complementactivation [38]; the individual monocytic response to C3a1C3adesArg [17] and C3a/C3adesArg [161; and the putativepresence of IL-i inhibitors of monocytic origin [29] released inresponse to activated complement. In contrast, no intradialyticincrease in IL-l production was found in most patients under-going dialysis with polyacrylonitrile and with polysulfone mem-branes in whom no complement activation occurred duringdialysis. C3a'C3adesArg induces IL-l production from mono-cytes in vitro at concentrations equivalent to those found in theplasma of patients dialyzed with cuprophane membranes [171.In addition, elevated levels of C3a may be an indirect indicationof in vivo generation of C5a, which also triggers monocytes toproduce IL-i [16, 17]. Thus, transient activation of complementin the cuprophane group may provide a mechanism for initiatingthe transient stimulation of IL-l production in the patients.

The demonstration of increased IL-i production is anotherexample of the adverse effects that may occur during hemodi-alysis 139]. Transient IL-l production occurs in patients dia-lyzed with complement activating membranes. In patients dia-lyzed with high-permeability polyacrylonitrile and polysulfonedevices, biocompatibility of the membrane for the complementsystem appears to be counter-balanced, at least in some pa-tients, by chronic stimulation of IL-I production. The long-termeffects of intermittent and chronic stimulation of IL-i produc-tion in hemodialyzed patients remain to be established.

Acknowledgments

This work was supported by Institut National de Ia Sante et de IaRecherche Médicale (INSERM), France. LPS from Neisseria menin-gitidis was a gift from Dr. D. Schultz, Institut MCrieux, Lyon, France.The small sterile dishes of Cuprophane and polyacrylonitrile used inthis study were provided by C. Pusineri, Hospal, Lyon, France. Thetechnical help of C. Fitting and the secretarial assistance of A. Viouxare gratefully acknowledged.

Reprint requests to Dr. Michael D. Kazatchkine, Hôpital Broussais,96, rue Didot, 75674 Paris CEDEX 14, France.

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