Clin. exp. Immunol. (1992) 90, 49-55

Alterations in mononuclear cell tumour necrosis factor-alpha (TNF-a) response in patients on long term cuprophane haemodialysis A. Y. ANNENKOV, A. G. STROKOV* & F. S. BARANOVA Laboratory of Immunology and *Department Of Haemodialysis, Institute of Transplantation and Artificial Organs, Moscow, Russia

(Acceptedfor publication 13 May 1992)

SUMMARY We have investigated TNF-a secretory response of peripheral blood mononuclear cells (PBMC) from 13 uraemic patients undergoing regular haemodialysis with cuprophane membrane (CM). Sixteen healthy subjects and five uraemic patients under conservative therapy were also studied as controls. Cells of haemodialysis patients exhibited increased TNF-ot release in vitro in the absence of activating stimuli other than culture conditions, as compared with normal and uraemic controls. In contrast to normal cells, this spontaneous secretion of TNF-a from dialysis PBMC could not be significantly reduced by addition of polymyxin B to culture medium, thus indicating its independence of trace amount of lipopolysaccharide (LPS) present in the medium as contaminant. Furthermore, predialysis PBMC were considerably more sensitive to stimulation with 107 pg/ml of LPS under in vitro culture conditions than normal and uraemic controls. To elucidate a role of direct contact with CM in stimulation of TNF-a release from monocytes, PBMC were cultured on CM in vitro. Contact with CM stimulated TNF-a secretion from PBMC above the level of cells cultured on tissue culture plastic. This response persisted with time in culture in contrast to a transient LPS-induced TNF-cz release. Furthermore, PBMC stimulated by contact with CM for 2 days did not lose the capacity to secrete TNF-cx in response to a subsequent LPS stimulation, while a 2-day treatment of cells with LPS was followed by LPS refractory state. Therefore, direct contact with CM induces in PBMC a long-lasting TNF-oc response which is not down-regulated by the acquisition of refractoriness in a manner similar to that which occurs in the case of LPS stimulation. These in vitro findings provide a possible explanation of the observation that predialysis PBMC exhibit elevated TNF-a secretory capacity.

Keywords monocyte tumour necrosis factor-alpha cuprophane haemodialysis INTRODUCTION of the acute phase response, such as fever and fall in Symptoms blood pressure [1,2], frequently appear in the course of haemodialysis treatment. Elevated levels of acute phase reactants [3,4] and some complications similar to components of chronic inflammation are characteristic of uraemic patients maintained

activation in the course of haemodialysis treatment. Cuprophane dialysis membrane can exert a direct stimulatory effect on monocytes, since contact of cultured monocytes with cuprophane membrane (CM) stimulates their IL-. production [12. In

addition, monocytes are sensitive to activated complement components [13] which are generated on the surface of CM in

.the course of haemodialysis treatment [14]. Besides dialysis membrane, various bacterial products which may contaminate dialysate are stimulatory for monocytes, lipopolysaccharide yyyppy (LPS) of Gram-negative bacteria being the most potent in this [5], and, along with IL-i, is implicated as mediator of acute and regard. chronic inflammation-related complications in haemodialysis While LPS is a well recognized inducor of monocyte TNF-a TNF-oc 6In the is patients [4, 1]. body primarily produced by secretion, much less is known about their sensitivity to direct monocytes and macrophages activated in response to various immunologic, infectious and inflammatory stimuli. To date, a interaction with CM. Here we report that direct contact with CM stimulates cultured blood monocytes to secrete TNF-ax. lack of perfect compatibility between blood and dialysis memThis response is persistent, in contrast to the transitory LPSbrans hs benfvourd a th majr cuse f bood onoyte induced TNF-a~production. Correspondence: A. Y. Annenkov, Laboratory of Immunology, The host is capable of desensitizing its TNF-a~response, Institute of Transplantation and Artificial Organs, 1, Shchukinskaya since over-production of this cytokine may result in detrimental St., 123436, Moscow, Russia. effects. For example, monocytes of animals repeatedly exposed on

intermittent. These observations

st

y

interttet haemodialysis. sneatons stongly Trhgers ob ers .the suggestNthat hedialysi i nflammatory resp.on mediates many aspects of the inflammatory reaction TNF-a

on

49

50

A. Y. Annenkov, A. G. Strokov & F. S. Baranova

to sublethal doses of LPS progressively lose the ability to mount a TNF-a response upon LPS stimulation [15]. Thus, one could expect that, as a result of repeated exposure to various activating stimuli, including LPS, monocytes of haemodialysis patients may lose responsiveness to these activating stimuli. To explore this possibility peripheral blood mononuclear cells (PBMC) from patients maintained on long term haemodialysis with CM were compared with normal controls for their sensitivity to LPS-stimulation. Furthermore, we addressed the question of whether or not stimulation of cultured monocytes by contact with CM is followed by the state of refractoriness in a manner similar to that which occurs in LPS stimulation. Results presented here argue against desensitization of monocyte TNF-a response as a result of haemodialysis treatment.

with supplements (106 cells/ml) with or without LPS from Escherichia coli 055: B5 (Calbiochem, La Jolla, CA) and/or 100 U/ml of polymyxin B (Serva) and 02 ml aliquots of this suspension were added to each well of 96-well flat-bottomed tissue culture plates (Costar, Cambridge, MA). For in vitro experiments designed to investigate the effect of dialysis membrane on PBMC, flat cuprophane sheet removed from a dialyser was cut into circles of the same diameter as the wells of 96-well tissue culture plate (6-4 mm). Before use, these circles were sterilized with 96° ethanol, treated with 105 U/ml of polymyxin B and extensively washed in RPMI 1640 medium with supplements. They were tightly wedged into the culture wells, resulting in a flat, even-bottomed surface. The cell suspension was added as indicated above. Cultures of PBMC generated in membrane-uncovered wells served as negative controls. PBMC were cultured at 370C in a humidified atmosphere of 5% CO2, 95% air.

PATIENTS AND METHODS Patients and controls Thirteen patients with end-stage renal disease undergoing intermittent acetate haemodialysis for 4-5 h, three times per week with dialysers using CM in sheet format (dialyser type DIP-02-02, Dnestrovski Plant of Medical Equipment, Belgorod, Russia) were included in this study. Before the study, patients were dialysed with this type of dialyser for 3-76 months (mean 28 months). Ten patients were male and three female; their ages ranged from 21 to 51 years, with mean age of 31 5 years. The primary kidney disease was chronic glomerulonephritis in all patients. None of them had clinical evidence of acute infection or underlying autoimmune disease. No drugs known to modify the immune response were taken at the time of the study. Mean s.e. serum creatinine level was 1 02 + 0-02 mM/l. To provide control data healthy volunteers (10 male and six female; mean age 35 4 years, range 26-53) and five uraemic nonhaemodialysed patients with chronic glomerulonephritis under conservative therapy (two male and three female; mean age 45 3 years, range 31-58; mean s.e. serum creatinin level 0 95+0-1 mM/l) were studied. Blood samples were drawn into sodium ethylenediaminetetraacetate (Sigma Chemical Co., St Louis, MO), pH 7 2 (final concentration in blood was 0 25%). Dialysed patients were bled before start of haemodialysis from an arteriovenous fistula. The time of blood sampling from normal and uraemic nonhaemodialysed subjects was not defined. Informed consent was obtained. Cell isolation and culture PBMC were prepared from anti-coagulated blood diluted 1:1 in PBS by Ficoll-Hypaque (Pharmacia Fine Chemical, Uppsala, Sweden) sedimentation [16]. The cells were washed twice in PBS and once in RPMI 1640 medium (Serva, Heidelberg, Germany) supplemented with 0-002 M of L-glutamine (Serva), 0 05 mg/ml of gentamycin (Serva) and 10% heat inactivated fetal calf serum (FCS; Flow Laboratories, Irvine, UK). If PBMC were destined for culture in the presence of polymyxin B (Serva), the RPMI 1640 medium used for their third wash, in addition to the conventional supplements, also contained 100 U/ml of this drug. Washed PBMC were resuspended in RPMI 1640 medium

L929 cell bioassay for TNF-Lx activity Cell-free culture supernatants of PBMC were kept frozen at -40'C until assayed for TNF-a activity by titration on the L929 mouse fibroblast cell line. The L929 cells were maintained in RPMI 1640 medium with supplements. Forty thousand cells in 0-1 ml of culture medium were added to each well of a 96-well tissue culture plate and the cells were allowed to establish a confluent monolayer by incubating overnight at 37°C in a humidified 5% C02-air mixture. Samples of culture supernatants to be tested serially diluted in culture medium were added (0 1 ml) to each well. Actinomycin D (Serva) was added to give a final concentration of 1 yug/ml. The plates were incubated for at least 18 h at 37CC. The adherent cell monolayer was stained with crystal violet (0-1 ml, 0-5%) in methanol-water (1:4 by vol.). After a 15 min incubation, the dye solution was removed by inverting and flicking the plates. The plates were then washed five times with tap water, the remaining dye of each well was solubilized with aqueous sodium dodecylsulphate (0 1 ml, I %), and the absorbances were measured with a microplate spectrophotometer (Labsystem, Helsinki, Finland) equipped with 570 nm filter. The number of U/ml of TNF-ax activity was defined as the reciprocal of the dilution giving 50% decrease of absorbance relative to control cells exposed to actinomycin D alone. Under all culture conditions used only TNF-ac was generated by PBMC, since the cytotoxicity of the supernatants could be completely neutralized by anti-TNF-a neutralizing MoAb 1OF (Institute of Haematology and Blood Transfusion, Minsk, Byelorussia) which did not cross-react with TNF-f,. The specific TNF-cx-neutralizing activity of 1OF was 1 6 gug/mg. For monitoring reproducibility of the L929 cell bioassay recombinant human TNF-a (gift from Dr Regina Turetskaya, Institute of Molecular Biology, Moscow, Russia) was titrated in each experiment. Concentrations of this preparation required for a 50% inhibition of target cell growth determined on four separate occasions in the course of the study were 19, 17, 21 and 18 pg/ml. Polymyxin B at a concentration of 100 U/ml was unable to exert any effect on the growth of L929 cells. Statistical analysis Student's t-test was used for comparing data.

51

TNF-c and haemodialysis 1P 005) (Fig. 2). Thus, haemodialysis treatment, but not uraemic state, stimulated TNF-a secretion of PBMC. Experiments comparing sensitivity of normal and patient PBMC to LPS stimulation are summarized in Fig. 2. The increase in average levels of TNF-a secretion upon stimulation of PBMC with 107 pg/ml of LPS was statistically significant in normal and dialysis groups (P 0 05), though PBMC from four of five patients in this group exhibited a statistically significant increase in individual TNF-a levels upon LPS stimulation. LPS-induced TNF-ac content in supernatants of normal cells was 21 5 + 5-7 U/ml (mean + s.e., n = 16). Stimulated PBMC from dialysed patients produced 1 32- 1 + 53 7 U/ml (mean ±s.e., n= 13) of TNF-x and differed significantly from the group of healthy subjects (P 0 05). To explore the possibility that the difference in LPS sensitivity between normal and dialysed PBMC resulted from a non-optimal dose of LPS used for stimulation of the former cells, we investigated the dose-response effect of LPS on TNF-x release from PBMC. Figure 3 shows the TNF-x response of donor and patient PBMC stimulated with LPS at a wide concentration range from 10 to 107 pg/ml. Upon LPS stimulation the cytokine was produced in a dose-related fashion. Doseeffect relations of LPS demonstrated a biphasic pattern for the majority of PBMC preparations studied which is similar to some other biological effects of this substance [17]. The concentration of 107 pg/mI was optimal or at least suboptimal in all cases. Moreover, more frequently it was suboptimal in the haemodialysis group (patients 11, 12, 14 and 15) than for healthy controls (patients 3 and 6). Therefore, if instead of TNF-cz release

A. Y. Annenkov, A. G. Strokoth & F. S. Baranova

52 Donors

40

30

20 10

-

135 7 2

2020-

E10 L-

17 3

1

3

37 1

37 1

5

4

357 6

uraernic

1357

1357

2 468

2 4 68

2 468

24 6 8

7

8

9

10

357

the inhibitory effect of polymyxin B ranged from of-21 'S to 61 ).0 This from the control PBMC three strikingly differed healthy subjects whose very low spontaneous TNF-x activity dropped to undetectable levels as a result of polymyxin B treatment.

Fscts 0/ ('011tact with(cuprophlane n01 TNF-Y response oftnornal PBMC

60 50

0

-

-

13

3_

68a2 4668

4 also demonstrates that spontaneous

moderately affected by polymyxin B treatment. In these cases

Haemrodialysis patients

H 80 r0 70

40 30 20

TNF-x production by PBMC from the other four patients, who were responsive to stimulation with 107 pg/ml of LPS, was only Figure

patients Non-haemodialysis urem ic patIents Non-h2emodialysis

d 1 357

2

-J

d

135 2

spontaneous TNF-Y secretion. This finding supports the notion that, at least in some cases, the 'unresponsiveness' we describe is an artifact from an inappropriate negative control.

We further addressed the question whether or not a direct contact of PBMC with dialysis membrane can result in the induction of TNF-a response. When normal PBMC were cultured in ritro on the surface of CM for various times, an increase in TNF-Y release into the supernatant with time relative to values in controls was observed (Fig. 5). Maximum TNF-x levels in cell-free culture supernatants of CM-stimulated PBMC were obtained at 18-20 h. No significant loss of activity was observed until the end of the culture period (48 h). Levels of TNF-a released from PBMC of six different donors after 18-20 h of culture with CM were averaged. The mean + s.c. of these averaged values was 17 3 + 48 U/'ml. This

_* 3

2 4 668

1 2 4 68

1

246 8

14 15 13 Fig. 3. Dose-response effect of lipopolysaccharide (LPS) o TNFrelease of donor and patient peripheral blood mononuclear cells (PBMC) of healthy subjects (donors) or patients with end-stage renal failure under conservative therapy (nion-haemodialysis uraeemic patients) or patients on haemnodialvsis with cuprophale membrane (haiemodialysis patients) were cultured with LPS at concentrations of 10 (2). 100 (3), l0-(4), 0 (5). 10 (6), 10" (7), or 10 (8) pg ml, or without LPS [1] for 18 20 h. Each cindividual is indicated by a number. I?

Controls

LPS-stimulated 16

between normal and dialysis PBMC. C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1

In a considerable number of PBMC preparations in the o haemodialysis group (38-,,) TNF-x release was not increased 9)19 upon stimulation with 10 pg ml of LPS (Fig. 2). The average I stimulation index (relative degree of stimulation) shown by this subgroup was I + 0-3 (mean + s.c.s n = 5). At the same time, a lack of response to stimulation with 107 pg ml of LPS was exhibited by only one individual in each of the two control groups (6%/0 donors and 20"', uraemic patients). One could suspect that the 'non-responsive' PBMC were in fact cells maximally stimulated by trace amounts of LPS contaminating culture medium, because spontaneous TNF-2. levels of non-responsive' PBMC exceeded those of responders. Thus, in the haemodialysis group, the difference in average values of spontaneous TNF-)c release between the five 'non-

responders' and the eight responders comprised respectively 39-9 + 8-7 and I 1 2 + 6 3 Uml (mean + s.e.) and was statistically significant (P < 0(05). To investigate this possibility the advantage of LPS neutralizing activity of polymyxin B was taken. The effect of polymyxin B on TNF-z release from PBMC of five different dialysed patients and

three.normal PBMC prepara-

tions was studied (Fig. 4). Of the five haemodialysis patients one turned out to be unable to respond to stimulation with 10 pg/ ml Of LPS (patient 19). However, in the presence of polymyxin B,? PBMC of this patient exhibited a potent TNF-a response to this concentration of LPS on account of a 97"< decrease in

20 21

*

22

o

23 I 7

60

20

10

0

40

I 1

I

[

I

I

80 120 160 200 240 280 320 360 400 700

TNF-a

(U/ml)

Fig. 4. EfTect of polymnyxin B on spontaneous and lipopolysaccharide (LPS)-induced TNF-s release of normal and dialysed peripheral blood mononuclear cells (PBMC) of healthy subjects (donors) or patients with end-stage renal failure on haemodialysis with cuprophane membrane (haemodialysis patients) were cultured with (LPS-stimulated) or without (controls) 107 pg ml of LPS, in the presence (-) or in the asne()o oyyi o 8-0h ahidvda sidctdb a number. Each column represents the average of triplicate val}ues + s.c. The difference in TNF-y activity between PBMC cultured in the presence and in the absence of polIymyxinl B is statistically significant (P