Bollati-Fogolín, Mariela

Bürgi, Milagros

Etcheverrigaray, Marina

Kratje, Ricardo

Oggero, Marcos

Prieto, Claudio

English

PubMed

Milagros Bürgi

Claudio Prieto

Marcos Oggero

Mariela Bollati-Fogolín

Marina Etcheverrigaray

Ricardo Kratje

Springer - Publisher Connector

MEETING ABSTRACT Open Access
New reporter cell clones to determine the
biological activity of human type I interferons
Milagros Bürgi1, Claudio Prieto1, Marcos Oggero1, Mariela Bollati-Fogolín2, Marina Etcheverrigaray1, Ricardo Kratje1*
From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies
Vienna, Austria. 15-18 May 2011
Background
Interferons (IFNs) are potent biologically active proteins
synthesized and secreted by somatic cells of all mamma-
lian species. They play an important role in the immune
response and defence against viruses because they have
antiproliferative, antiviral and immunomodulatory activ-
ities [1]. They are widely used as biopharmaceuticals, so
their potency must be correctly identified. Usually, the
biological activity is quantified by a bioassay based on
its capacity to induce an antiviral state in target cells.
Antiviral assays may be subject to high inter- and intra-
assay variations having the drawbacks of using viruses
[2]. Therefore, a set a new human cell lines derived
from different tissues were developed using the
enhanced green fluorescent protein (eGFP) gene under
the control of type I IFN-inducible Mx2 promoter. In
addition, having reporter gene assays derived from cells
of different tissue origins would allow us to design stu-
dies aiming to evaluate how IFNs induce their actions
through the human body.
Results
Three human tissue-derived cell lines: A549 (lung can-
cer cells), HEp-2 (epidermoid larynx carcinoma cells)
and HeLa (cervical adenocarcinoma cells) were used to
develop new reporter gene assays based on the expres-
sion of the eGFP gene under the control of type I IFN-
inducible Mx2 promoter.
Six stably transfected Mx2/eGFP lines (two of each
wild type cell line) were obtained and selected during 10
days with the antibiotic neomycin.
The Mx promoter sequence, cloned upstream to the
eGFP, responded specifically and quantitatively to type I
IFNs (IFN-a and IFN-b) using the new reporter cell
lines (Figure 1). Therefore, the three cell lines showed
that eGFP percentage (measured by flow cytometry)
rose as IFN increased, reaching maximum values of 25-
85% activated cells, depending on the cell lines and the
type of IFNs. No eGFP expression was observed for
negative controls, indicating that there was no basal
expression of the reporter protein.
Two subtypes of IFN-a and IFN-b were assayed: the
E. coli-derived IFNs-a having the amino acid position
23 occupied by the residue Lys (IFN-a2a) or by the resi-
due Arg (IFN-a2b) and the E.coli-derived IFN-b with
the residue Cys 17 replaced by Ser (IFN-b1b) or the gly-
cosylated CHO cell-derived IFN-b (IFN-b1a). The dose-
response relationships corresponding to both IFN-b sub-
types showed higher slopes (43 ± 5 IU/ml) than those of
IFN-a subtypes (23 ± 12 IU/ml). Considering that type-I
IFNs act through a common receptor complex (ifnar)
and that mutational analysis revealed distinct binding
sites for these IFNs on ifnar [3], the above mentioned
results give more evidences that differences in IFN a/b
recognition may be associated with cytoplasmic
signaling.
Analyzing in more detail the linear dose-response rela-
tionships for each cell line (Table 1), A549 cells showed
the lowest detection limit for all IFN subtypes (0.24 –
0.48 IU/ml). Comparing IFN-a subtypes, no differences
in detection limit were observed using any of the cell
lines. Contrarily, differences in responses were observed
when comparing the activity of IFN-b subtypes in HEp
cell lines. Therefore, the standardization of several
assays to measure the potency of IFNs might be carried
out using the cell lines herein documented.* Correspondence: rkratje@fbcb.unl.edu.ar1Cell Culture Laboratory, School of Biochemistry and Biological Sciences,
Universidad Nacional del Litoral. Ciudad Universitaria – C.C. 242 –
(S3000ZAA) Santa Fe, Provincia de Santa Fe, Argentina
Full list of author information is available at the end of the article
Bürgi et al. BMC Proceedings 2011, 5(Suppl 8):P4
http://www.biomedcentral.com/1753-6561/5/S8/P4
© 2011 Bürgi 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.
Conclusions
These systems have several advantages when compared
to antiviral activity assays and other reporter gene sys-
tems: they can determine the potency of type I IFNs
using only one cell line; they are very fast having the
specificity of the Mx promoter. Also, they are sensitive
and safe, showing reproducible responses in a dose-
dependent manner. Outstandingly, the main contribu-
tion of this work was the development of alternative
reporter systems as suitable candidates to evaluate the
way that IFNs induce their activity in different human
tissues.
Author details
1Cell Culture Laboratory, School of Biochemistry and Biological Sciences,
Universidad Nacional del Litoral. Ciudad Universitaria – C.C. 242 –
(S3000ZAA) Santa Fe, Provincia de Santa Fe, Argentina. 2Cell Biology Unit,
Institut Pasteur de Montevideo. Mataojo 2020 (11400) Montevideo, Uruguay.
Published: 22 November 2011
References
1. Billiau A: Interferon: the pathways of discovery I. Molecular and cellular
aspects. Cytokine Growth Factor Rev 2006, 17(5):381-409.
2. Girad DJ, Fleischaker RJ: A study showing a high degree of
interlaboratory variation in the assay of human interferon. J Biol Stand
1984, 12(3):265-269.
3. Piehler J, Schreiber G: Mutational and structural analysis of the binding
interface between type I interferons and their receptor ifnar2. J Mol Biol
1999, 294(1):223-237.
doi:10.1186/1753-6561-5-S8-P4
Cite this article as: Bürgi et al.: New reporter cell clones to determine
the biological activity of human type I interferons. BMC Proceedings 2011
5(Suppl 8):P4.
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Figure 1 Reporter gene assays based on the eGFP gene expression after the induction of Mx2 promoter by type I IFNs.
Table 1 Linear dose-response relationships from reporter gene assays employing different human cell lines.
IFN HEp L1D7 HEp L1G5 HeLa C5G6 HeLa C6C3 A549L2A6 A549L2G9
IFN-a2a 1.52 – 390 12.20 – 25,000 7.8 – 1,000 1.95 – 500 0.24 – 16 0.24 – 125
IFN-a2b 0.76 – 390 12.20 – 50,000 7.8 – 1,000 1.95 – 500 0.24 – 4 0.24 – 8
IFN-b1a 0.05 – 200 3.05 – 6,250 3.9 – 250 1.95 – 31.25 0.24 – 32 0.48 – 62
IFN-b1b 3.05 – 200 12.20 – 50,000 3.9 – 1,000 3.90 – 500 0.24 – 32 0.48 – 62
Bürgi et al. BMC Proceedings 2011, 5(Suppl 8):P4
http://www.biomedcentral.com/1753-6561/5/S8/P4
Page 2 of 2


New reporter cell clones to determine the biological activity of human type I interferons

Bürgi Milagros

Prieto Claudio

Oggero Marcos

Bollati-Fogolín Mariela

Etcheverrigaray Marina

Kratje Ricardo

Directory of Open Access Journals

BMC Proceedings

A: Interferon: the pathways of discovery I. Molecular and cellular aspects. Cytokine Growth Factor Rev

Fleischaker RJ: A study showing a high degree of interlaboratory variation in the assay of human interferon.

Mutational and structural analysis of the binding interface between type I interferons and their receptor ifnar2. J Mol Biol

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New reporter cell clones to determine the biological activity of human type I interferons

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