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      Ex vivo Flow Cytometry Determination of Intracytoplasmic Expression of IL-2, IL-6, IFN-γ, and TNF-α in Monocytes and T Lymphocytes, in Chronic Hemodialysis Patients

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          Abstract

          Aims: To determine the intracytoplasmic expression of TNF-α, IL-2, IL-6 and IFN-γ, ex vivo and in vitro, in both monocytes and T lymphocytes by flow cytometry after appropriate stimulation using phorbol myristate acetate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensin, in order to assess the bio(in)compatibility of different dialysis membranes. Methods: We examined monocytes and T lymphocytes taken from chronic hemodialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitrile (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-stage chronic renal failure patients (n = 3) and healthy volunteers (n = 11). Results: Before any stimulation there was a statistically significant difference in the percentages of TNF-α, IL-6, and IFN-γ- expressing monocytes with respect to the dialysis membrane used. The highest percentages were observed for CUP and AN69 patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy volunteers. One hour after LPS stimulation the patterns remained unchanged for TNF-α and IFN-γ, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in the other groups. When we examined the percentage of IFN-γ-, TNF-α- and IL-6-expressing monocytes in patients before and after a dialysis session, before any stimulation, we found that the results were significantly different for the three membranes (p = 0.01). Thus, a dialysis session with polysulfone membranes had no significant effect on the precentages of IFN-γ-, TNF-α-, and IL-6-expressing monocytes, whereas percentages were significantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-γ- and IL-2-expressing T lymphocytes were only detected after 18 hours of PMA/ionomycin stimulation. The percentages of IFN-γ-expressing T cells recorded for the different membranes were not statistically different from those recorded for healthy subjects or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly different between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7.6%, respectively, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseline, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis session had no impact on the percentages of IL-2-expressing T lymphocytes, whereas it was associated with a significant decrease in the percentage of IFN-γ-expressing cells, but only when cuprophane membranes were used. Conclusion: Cytokine flow cytometry enables one to study, ex vivo, i.e., without any stimulation of the cells, and in vitro after appropriate stimulation, the bio(in)compatibility of dialysis membranes assessed by the intracytoplasmic cytokine profiles of TNF-α, IFN-γ, IL-6 and IL-2, evaluated at the single cell level.

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          Most cited references 2

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          Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies

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            Measurement of intracellular cytokines

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              Author and article information

              Journal
              AJN
              Am J Nephrol
              10.1159/issn.0250-8095
              American Journal of Nephrology
              S. Karger AG
              0250-8095
              1421-9670
              2000
              February 2000
              13 January 2000
              : 20
              : 1
              : 18-26
              Affiliations
              aDepartment of Nephrology, bLaboratory of Immunology, cBiostatistics Department, dUnité INSERM U 395, Toulouse University Hospital, France
              Article
              13550 Am J Nephrol 2000;20:18–26
              10.1159/000013550
              10644863
              © 2000 S. Karger AG, Basel

              Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

              Page count
              Figures: 6, References: 35, Pages: 9
              Product
              Self URI (application/pdf): https://www.karger.com/Article/Pdf/13550
              Categories
              Clinical Study

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