Quantifizierung der IFN-γ-mRNA in Lymphknotenzellen von Lewis-Ratten mit Experimenteller Autoimmuner Enzphalomyelitis (EAE) und ihre Beeinflussung durch Kaliumkanalmodulatoren
Kirsten Wissel
ISBN 978-3-89722-429-2
230 pages, year of publication: 2000
price: 40.50 €
Abstract: Alkoxypsoralens, known as DNA photomodifying agents, have
been shown to block voltage-dependent K+ channels (Kv) as well as to
alleviate functional deficits in certain multiple sclerosis (MS)
patients in a manner similar to 4-aminopyridine. Since Kv channel
blockers are known to inhibit T cell-mediated immune responses both
in vitro and in vivo, we investigated the effects of
5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), Margatoxin
(MgTX), known as specific blocker of the Kv channel subtype Kv1.3,
and Quinidine, blocking agent of Ca2+- and voltage-dependent K+
channels, by (1) proliferation of lymph node cells (LNC) from Lewis
rats challenged for experimental autoimmune encephalomyelitis (EAE)
by immunisation with spinal cord homogenate as measured by
3H-thymidine incorporation and (2) IFN-y gene expression of LNC as
measured by quantitative RT-PCR. The effects of the K+ channel
blockers were determined in unstimulated (control), guinea pig (GP)
myelin- and Concanavalin A (ConA)-stimulated LNC at different phases
of the disease. For accurate quantitation of small differences in
IFN-g mRNA levels we established a non-competitive RT-PCR that based
on the internal standardization by using a synthetic homologous
internal RNA standard which was added to a certain number of LNC
prior to the RNA extraction. The co-preparation of both internal RNA
standard and wildtype total RNA was followed by co-amplification in
the same PCR and co-quantification by differential liquid
phase-hybridization/ELISA in microtiter plates. A linear measuring
range was achieved comprising up to 40fold changes of wildtype mRNA
copy numbers in cell cultures, and the accuracy of quantitation
method was confirmed by a variance of about 23 %. The maximum IFN-g
wildtype mRNA copies per lymph node cell in the control and GP-myelin
cultures were found at the height of disease. Contrastly the highest
number of IFN-g mRNA copies in ConA-cultures was attained in the
early phase. We found suppressive effects of 5-MOP and 8-MOP on cell
proliferation as well as on gene expression of IFN-g in control,
GP-myelin and ConA-stimulated cultures. Thereby a significant
stronger inhibition of 5-MOP compared to 8-MOP was monitored.
Quinidine exhibited high blocking effects on cell proliferation and
IFN-g gene expression in all cell cultures. Contrastly MgTX did not
suppress significantly the cell proliferation nor IFN-g gene
expression in control as well as in in vitro stimulated cell
cultures. Due to our findings we conclude that 5-MOP, 8-MOP and
Quinidine interfere with voltage-controlled signal transduction in
T-lymphocytes and might therefore suppress immune responses in
autoimmune diseases of the central nervous system. In particular the
alkoxypsoralens and their derivates without phototoxic effects are
new candidates for further studies on K+ channel blocking
immunosuppressive drugs. The agents may exert a dual beneficial
effect on demyelinating diseases like MS due to their ability to
attenuate the inflammatoryprocess and to improve axonal conductivity.