Transfer of cytokine-inducing bacterial products across hemodialyzer membranes in the presence of plasma or whole blood.

Cytokine production by peripheral blood mononuclear cells (PBMC) is a sensitive indicator of cytokine-inducing substances which may cross from contaminated dialysate into the blood compartment. The objective of this study was to compare the transfer of cytokine-inducing substances from dialysate contaminated with a culture filtrate from Pseudomonas aeruginosa across dialyzers with low (hemophan) or intermediate ultrafiltration coefficients (modified cellulose triacetate, CTA), under conditions where either 10% plasma or whole blood was circulated in the blood compartment. Eight paired experiments of in vitro dialysis were carried out at 37 degrees C using a countercurrent recirculating loop dialysis circuit with either a new CTA or hemophan dialyzer. 10% plasma in standard tissue culture medium was circulated through the blood compartment and bicarbonate dialysate was circulated in the dialysate compartment. The dialysate was challenged sequentially by log-fold dilutions (10(2), 10(3) or 10(4)) of a Ps. aeruginosa culture filtrate. Samples were drawn from the blood compartment 5 and 15 minutes after each challenge and incubated with suspensions of PBMC in the absence or presence of polymyxin B, in order to block endotoxin. After 24 h at 37 degrees C, total interleukin-1 alpha (IL-1 alpha) was measured by RIA. Although the dialysate contained potent cytokine-inducing substances, there was no significant IL-1 alpha production by PBMC incubated with the plasma mixture from the blood compartment in the majority of experiments with both dialyzers and with each of the three dilutions of the bacterial challenge. Eight experiments were also performed with CTA dialzyers using heparinized whole blood in the blood compartment. Samples of whole blood and dialysate were drawn at baseline, after one hour of dialysis with uncontaminated dialysate and 15 minutes and three hours after dialysis with dialysate contaminated with Ps. aeruginosa filtrate. There was no significant IL-1 alpha production by PBMC isolated from the whole blood 1 h after dialysis with uncontaminated dialysate, and 15 min and 2 h after adding the Ps. aeruginosa filtrate to the dialysate side. In contrast, production of IL-1 alpha by PBMC from the same donors incubated with samples from the dialysate were 263 +/- 50, 1074 +/- 306, 2333 +/- 774 and 2602 +/- 702 pg/2.5 x 10(6) PBMC, respectively at the same four time points. These data suggest that although the Ps. aeruginosa culture filtrate present in the dialysate was a potent inducer of IL-1 alpha, neither dialyzer permitted transfer of cytokine inducing substances from the dialysate into the blood compartment.

Cytokine production by human peripheral blood mononuclear cells stimulated by a Pseudomonas aeruginosa culture filtrate: role of plasma and polymyxin B.

The lack of consensus regarding the significance of transmembrane passage of bacterial products across hemodialysis membranes can be related to several methodological differences in the various studies, including the choice of circulating fluid in the blood compartment of the model, nature and concentration of the bacterial products employed to challenge the dialysate compartment and whether cytokine production by PMBC or the limulus amebocyte lysate (LAL) assay was used as the index of transfer and the cytokine used as the read-out. In this study, we examined the production of interleukin-1 alpha (IL-1 alpha), interleukin-1 receptor antagonist (IL-1Ra) and interleukin-8 (IL-8) by peripheral blood mononuclear cells (PBMC) incubated with a Pseudomonas aeruginosa culture filtrate. Further, the effects of 10% autologous human plasma and Polymyxin B sulfate (PmB) on cytokine production by PBMC were also characterized. The results of our study indicate that the Ps. aeruginosa culture filtrate had both PmB suppressible and PmB non-suppressible components and that the addition of 10% human plasma significantly enhanced cytokine production by both PmB suppressible and PmB non-suppressible components. The enhancing effect of plasma was most evident at low concentrations of the filtrate. The inhibitory effect of PmB was most evident in samples cultured in the presence of 10% plasma. There was a direct correlation between the production of IL-1 alpha and IL-1Ra suggesting that both pro-inflammatory cytokines and cytokine-specific inhibitory proteins are concurrently produced. There results have direct relevance to selection of study conditions for in vitro models used to study the transmembrane passage of bacterial products across hemodialysis membranes.