Jeroen Bastiaans

Kiki van Bilsen

Marion Kolijn

Martin van Hagen

Nicoline M Korthagen

Robert W Kuijpers

Willem A Dik

Journal of Translational Medicine

PubMed

Springer - Publisher Connector

FcRn mediated IgG transport by retinal pigment epithelium cells

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POSTER PRESENTATION Open AccessFcRn mediated IgG transport by retinal pigmentepithelium cellsNicoline M Korthagen1*, Kiki van Bilsen1,2, Willem A Dik1, Jeroen Bastiaans1,3, Marion Kolijn1, Robert W Kuijpers4,Martin van Hagen1,2From 7th European Workshop on Immune-Mediated Inflammatory DiseasesNoordwijk aan Zee, the Netherlands. 28-30 November 2012BackgroundThe neonatal Fc receptor (FcRn) binds immunoglobulinG (IgG), protects it from degradation and can transportIgG from apical to basolateral side of epithelial cells [1].Previously, we showed that human retinal pigmentepithelial (RPE) cells express FcRn and that, in contrastto non-ocular cells, TNF-a downregulates FcRn expres-sion in RPE cells [2]. We speculated that FcRn in RPEplays a pivotal role in ocular IgG metabolism. The aim ofthis study was to develop a method to investigatewhether RPE cells transport IgG molecules.Materials and methodsMonolayers of RPE cells, ARPE-19 (immortalized cell line)and human primary RPE cells (both fetal and adult), wereestablished on Transwell culture inserts using fibronectincoated membranes. Trans-epithelial resistance (TER) mea-surements were used to evaluate monolayer formation.Plates with an average TER >35Ω*cm2 were used for sub-sequent experiments. Monoclonal IgG was added eitherapically or basolaterally and after incubation IgG was mea-sured in the culture supernatant using an ELISA that wasdeveloped inhouse. To distinguish leakage of IgG throughthe cell layer from active transport by the cells we usedNH4Cl or Chloroquine to block cytoplasmic vesicles.ResultsThe highest TERs were observed with ARPE-19 cells,while TERs often remained low with the primary cellcultures. The IgG level that crossed the membrane cor-related positively incubation time, but this associationwas not linear. The observed effects of NH4Cl andChloroquine were limited.ConclusionsFurther experiments are needed to determine whetherthere is active transport by the RPEs. Paracellular diffusionwill be investigated using dextran and specific blockingantibodies for the FcRn. In addition, experiments will beperformed with RPE cells where FcRn is overexpressed orknocked-out.AcknowledgementsFetal human RPE cells were kindly provided by Sheldon Miller (National EyeInstitute, Bethesda, MD, USA).This research project was funded by grants from UitZicht and theInternational Retinal Research Foundation.Author details1Dept. of Immunology, Erasmus University Medical Center, Rotterdam, theNetherlands. 2Dept. of Internal Medicine, Erasmus University Medical Center,Rotterdam, the Netherlands. 3Rotterdam Eye Hospital, Rotterdam, theNetherlands. 4Dept. of Ophthalmology, Erasmus University Medical Center,Rotterdam, the Netherlands.Published: 28 November 2012References1. Roopenian DC, Akilesh S: FcRn: the neonatal Fc receptor comes of age.Nat Rev Immunol 2007, 7(9):715-25.2. van Bilsen K, van Hagen PM, Bastiaans J: The neonatal Fc receptor isexpressed by human retinal pigment epithelial cells and isdownregulated by tumour necrosis factor-alpha. Br J Ophthalmol 2011,95(6):864-868.doi:10.1186/1479-5876-10-S3-P2Cite this article as: Korthagen et al.: FcRn mediated IgG transport byretinal pigment epithelium cells. Journal of Translational Medicine 201210(Suppl 3):P2.1Dept. of Immunology, Erasmus University Medical Center, Rotterdam, theNetherlandsFull list of author information is available at the end of the articleKorthagen et al. Journal of Translational Medicine 2012, 10(Suppl 3):P2http://www.translational-medicine.com/content/10/S3/P2© 2012 Korthagen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

POSTER PRESENTATION Open Access
FcRn mediated IgG transport by retinal pigment
epithelium cells
Nicoline M Korthagen1*, Kiki van Bilsen1,2, Willem A Dik1, Jeroen Bastiaans1,3, Marion Kolijn1, Robert W Kuijpers4,
Martin van Hagen1,2
From 7th European Workshop on Immune-Mediated Inflammatory Diseases
Noordwijk aan Zee, the Netherlands. 28-30 November 2012
Background
The neonatal Fc receptor (FcRn) binds immunoglobulin
G (IgG), protects it from degradation and can transport
IgG from apical to basolateral side of epithelial cells [1].
Previously, we showed that human retinal pigment
epithelial (RPE) cells express FcRn and that, in contrast
to non-ocular cells, TNF-a downregulates FcRn expres-
sion in RPE cells [2]. We speculated that FcRn in RPE
plays a pivotal role in ocular IgG metabolism. The aim of
this study was to develop a method to investigate
whether RPE cells transport IgG molecules.
Materials and methods
Monolayers of RPE cells, ARPE-19 (immortalized cell line)
and human primary RPE cells (both fetal and adult), were
established on Transwell culture inserts using fibronectin
coated membranes. Trans-epithelial resistance (TER) mea-
surements were used to evaluate monolayer formation.
Plates with an average TER >35Ω*cm2 were used for sub-
sequent experiments. Monoclonal IgG was added either
apically or basolaterally and after incubation IgG was mea-
sured in the culture supernatant using an ELISA that was
developed inhouse. To distinguish leakage of IgG through
the cell layer from active transport by the cells we used
NH4Cl or Chloroquine to block cytoplasmic vesicles.
Results
The highest TERs were observed with ARPE-19 cells,
while TERs often remained low with the primary cell
cultures. The IgG level that crossed the membrane cor-
related positively incubation time, but this association
was not linear. The observed effects of NH4Cl and
Chloroquine were limited.
Conclusions
Further experiments are needed to determine whether
there is active transport by the RPEs. Paracellular diffusion
will be investigated using dextran and specific blocking
antibodies for the FcRn. In addition, experiments will be
performed with RPE cells where FcRn is overexpressed or
knocked-out.
Acknowledgements
Fetal human RPE cells were kindly provided by Sheldon Miller (National Eye
Institute, Bethesda, MD, USA).
This research project was funded by grants from UitZicht and the
International Retinal Research Foundation.
Author details
1Dept. of Immunology, Erasmus University Medical Center, Rotterdam, the
Netherlands. 2Dept. of Internal Medicine, Erasmus University Medical Center,
Rotterdam, the Netherlands. 3Rotterdam Eye Hospital, Rotterdam, the
Netherlands. 4Dept. of Ophthalmology, Erasmus University Medical Center,
Rotterdam, the Netherlands.
Published: 28 November 2012
References
1. Roopenian DC, Akilesh S: FcRn: the neonatal Fc receptor comes of age.
Nat Rev Immunol 2007, 7(9):715-25.
2. van Bilsen K, van Hagen PM, Bastiaans J: The neonatal Fc receptor is
expressed by human retinal pigment epithelial cells and is
downregulated by tumour necrosis factor-alpha. Br J Ophthalmol 2011,
95(6):864-868.
doi:10.1186/1479-5876-10-S3-P2
Cite this article as: Korthagen et al.: FcRn mediated IgG transport by
retinal pigment epithelium cells. Journal of Translational Medicine 2012
10(Suppl 3):P2.
1Dept. of Immunology, Erasmus University Medical Center, Rotterdam, the
Netherlands
Full list of author information is available at the end of the article
Korthagen et al. Journal of Translational Medicine 2012, 10(Suppl 3):P2
http://www.translational-medicine.com/content/10/S3/P2
© 2012 Korthagen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.


Akilesh S: FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol

Rotterdam, the Netherlands Full list of author information is available at the end of the article Korthagen et al.

The neonatal Fc receptor is expressed by human retinal pigment epithelial cells and is downregulated by tumour necrosis factor-alpha.

https://translational-medicine.biomedcentral.com/track/pdf/10.1186/1479-5876-10-S3-P2?site=translational-medicine.biomedcentral.com

FcRn mediated IgG transport by retinal pigment epithelium cells

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