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Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects

Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble fact...

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Autores principales: Brencicova, E., Diebold, S.S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333792/
https://www.ncbi.nlm.nih.gov/pubmed/28159551
http://dx.doi.org/10.1016/j.jim.2017.01.008
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author Brencicova, E.
Diebold, S.S.
author_facet Brencicova, E.
Diebold, S.S.
author_sort Brencicova, E.
collection PubMed
description Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble factors such as cytokines by the cultured cells is common and can have an impact via autocrine mechanisms. The addition of cytokine-specific neutralizing antibodies leads to neutralization of the targeted factors irrespective of their source and affects paracrine and autocrine effects alike. Thus, neutralization assays are not suitable to irrevocably demonstrate that the examined factors exert their effect via a paracrine mechanism. We were interested in investigating the impact of immunosuppressive factors present in ovarian carcinoma-associated ascites by dissecting paracrine versus autocrine effects of interleukin 10 (IL-10) and prostaglandin E2 (PGE(2)) on the activation of monocyte-derived dendritic cells (DC). We explored several methods of depletion based on introduction of the neutralizing antibodies bound to beads. Here we describe the pitfalls of the investigated depletion approaches and show the importance of monitoring the presence of residual neutralizing antibodies in the sample upon depletion, which impacts on the suitability of the approach to distinguish paracrine from autocrine effects. Only one of three investigated approaches showed no dislocation of neutralizing antibody from the beads into the sample. This method, which is based on covalently linking antibody to magnetic beads harbouring a reactive group allowed for the complete removal of the investigated factors from ascites and represents an elegant tool to elucidate immunoregulatory or -stimulatory cytokine networks in considerably more depth than the use of neutralizing antibodies in cell cultures alone can contribute.
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spelling pubmed-53337922017-04-01 Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects Brencicova, E. Diebold, S.S. J Immunol Methods Research Paper Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble factors such as cytokines by the cultured cells is common and can have an impact via autocrine mechanisms. The addition of cytokine-specific neutralizing antibodies leads to neutralization of the targeted factors irrespective of their source and affects paracrine and autocrine effects alike. Thus, neutralization assays are not suitable to irrevocably demonstrate that the examined factors exert their effect via a paracrine mechanism. We were interested in investigating the impact of immunosuppressive factors present in ovarian carcinoma-associated ascites by dissecting paracrine versus autocrine effects of interleukin 10 (IL-10) and prostaglandin E2 (PGE(2)) on the activation of monocyte-derived dendritic cells (DC). We explored several methods of depletion based on introduction of the neutralizing antibodies bound to beads. Here we describe the pitfalls of the investigated depletion approaches and show the importance of monitoring the presence of residual neutralizing antibodies in the sample upon depletion, which impacts on the suitability of the approach to distinguish paracrine from autocrine effects. Only one of three investigated approaches showed no dislocation of neutralizing antibody from the beads into the sample. This method, which is based on covalently linking antibody to magnetic beads harbouring a reactive group allowed for the complete removal of the investigated factors from ascites and represents an elegant tool to elucidate immunoregulatory or -stimulatory cytokine networks in considerably more depth than the use of neutralizing antibodies in cell cultures alone can contribute. Elsevier 2017-04 /pmc/articles/PMC5333792/ /pubmed/28159551 http://dx.doi.org/10.1016/j.jim.2017.01.008 Text en © 2017 The Authors
spellingShingle Research Paper
Brencicova, E.
Diebold, S.S.
Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
title Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
title_full Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
title_fullStr Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
title_full_unstemmed Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
title_short Antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
title_sort antibody-mediated depletion of immunosuppressive factors from ovarian carcinoma-associated ascites for investigation of paracrine versus autocrine effects
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333792/
https://www.ncbi.nlm.nih.gov/pubmed/28159551
http://dx.doi.org/10.1016/j.jim.2017.01.008
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