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*
Department of Neurology, Neuroimmunology Branch and Clinical Research Group for Multiple Sclerosis, Julius-Maximilians Universität, Würzburg, Germany;
Department of Neurology, Heinrich-Heine-Universität, Düsseldorf, Germany;
Department of Neuropathology, University of Berlin, Charite Campus Virchow, Berlin, Germany; and
§
Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
, the
anti-inflammatory cytokine IL-10, and the cytokine-inducible NO
synthase (iNOS) during high-dose Ag therapy of adoptive transfer
experimental autoimmune encephalomyelitis (AT-EAE) in the Lewis rat.
Using semiquantitative and competitive RT-PCR, we found 5- to 6-fold
induction of TNF- mRNA and 3-fold induction of IFN- mRNA in the
spinal cord that occurred within 1 h after i.v. injection of Ag
and was accompanied by a 2-fold increase of iNOS mRNA. Both IFN- and
iNOS mRNA remained elevated for at least 6 h, whereas TNF- mRNA
was already down-regulated 6 h after Ag injection. A comparable
time course was found for circulating serum levels of TNF- and
IFN-. IL-10 mRNA levels did not change significantly following Ag
injection. Neutralization of TNF- by anti-TNF- antiserum in
vivo led to a significant decrease in the rate of T cell and
oligodendrocyte apoptosis induced by high-dose Ag administration, but
did not change the beneficial clinical effect of Ag therapy. Our data
suggest profound activation of proinflammatory but not of
anti-inflammatory cytokine gene expression by high-dose Ag
injection. Functionally, TNF- contributes to increased apoptosis of
both autoaggressive T cells and oligodendrocytes in the target organ
and may thereby play a dual role in this model of Ag-specific therapy
of CNS autoimmune diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-,
neutralization or genetic targeting of these cytokines in many cases
aggravates clinical disease (7, 8). For
anti-inflammatory cytokines such as IL-10 and TGF-ß, available
evidence also suggests a range of divergent and sometimes paradoxical
effects (9, 10, 11). NO generated by the inducible isoform of
NO synthase (iNOS) may act as a critical downstream mediator of both
harmful and protective cytokine effects in EAE
(12, 13, 14, 15, 16).
High doses of the specific Ag can suppress immune responses both in
vitro and in vivo (17, 18, 19). Intravenous Ag administration
induces apoptotic T cell death and results in prevention of
experimental autoimmune diseases of the central and peripheral nervous
system (Refs. 20 and 21 ; see review in Ref.
22). The molecular mechanisms of high-dose Ag therapy are
currently unknown. During Ag therapy, increased levels of TNF- were
reported (23). Recently, we have shown by inhibition
experiments in experimental autoimmune neuritis that TNF-
participates in T cell apoptosis in the inflammatory lesion in the
sciatic nerve and liver during high-dose Ag therapy (24).
To elucidate the contribution of proinflammatory vs
anti-inflammatory cytokines during high-dose Ag therapy in the CNS
model EAE, we used semiquantitative and competitive RT-PCR and ELISA
techniques to analyze the temporal pattern of TNF- as well as of
IFN-, iNOS, and IL-10 expression. The functional role of TNF- was
addressed using a neutralizing antiserum during Ag therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Lewis rats from Charles River Breeding Laboratories (Sulzfeld, Germany) were 6–8 wk old and had a body weight of 125–160 g. Animals were housed in plastic cages in a room with natural lighting and given commercial food pellets and water ad libitum. All experiments were conducted according to Bavarian state regulations for animal experimentation and approved by the responsible authorities.
Isolation of MBP
Guinea pig MBP (gpMBP) was purified according to established protocols (25). Briefly, homogenized brain was subjected sequentially to delipidation, neutralization, ammonium sulfate precipitation, and aceton precipitation. Purity of the gpMBP preparation was assessed by gel electrophoresis.
T cell culture
Encephalitogenic MBP-specific T cell line MBP13 was established from Lewis rats as described previously (26, 27). T cell specificity was tested in vitro in 96-well microtiter plates with 1.5 x 104 responder T cells, 7.5 x 105 irradiated (3000 rad) thymocytes, and graded doses of gpMBP using RPMI 1640 supplemented with 1% normal rat serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine (Life Technologies, Eggenstein, Germany). For animal experimentation, all T cells were taken from the same stage of activation.
Induction of adoptive transfer (AT)-EAE and therapy protocols
AT-EAE was induced by tail vein injection of freshly activated
MBP-specific CD4+ T cells. For in vivo
neutralization of TNF-, 300 µl polyclonal sheep
anti-recombinant murine TNF--antiserum cross-reactive to rat
TNF- (28, 29), or 300 µl control serum, was given 15
min before Ag injection or in the absence of Ag. For the kinetic
analysis of cytokine induction, cellular infiltration, and apoptosis,
respectively, spinal cord tissue as well as serum samples were obtained
1, 6, and 20 h after a single i.v. injection of 500 µg gpMBP
(Table I
). To assess the clinical consequences of Ag injection and
TNF- neutralization, we followed a prolonged treatment schedule with
repeated Ag injections as described previously (21).
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Animals were weighed and inspected for clinical signs of disease on a daily basis. Disease severity of EAE was assessed according to the following scale: 0, normal; 1, limp tail; 2, mild paraparesis of the hind limbs, unsteady gait; 3, moderate paraparesis, voluntary movements still possible; 4, paraplegia or tetraparesis; and 5, moribund.
Semiquantitative RT-PCR
After sacrifice of the animals with CO2
inhalation, the lumbal and lower thoracic spinal cord (
300 mg wet
weight/animal) was rapidly prepared by air insufflation into the spinal
canal (30) and immediately homogenized in 5 ml Trizol
reagent (Life Technologies). Total RNA was isolated according to the
manufacturer’s protocol and reverse transcribed using
oligo(dT)20 primers and SuperscriptII-Reverse
Transcriptase (Life Technologies) as detailed elsewhere
(30). cDNA according to 20 ng of total RNA was subjected
to subsequent PCR analysis in total volume of 30 µl containing
containing 25 pmol of each primer (see below): 10 mM Tris-HCl, pH 8.3
(at 25°C), 50 mM KCl, 10% DMSO, 1.25 mM MgCl2,
250 µM each dATP, dCTP, dGTP, and dTTP, and 1.5 U AmpliTaq
DNA-polymerase (Perkin-Elmer, Oak Brook, IL). Rat IFN- and TNF-
primers were purchased from Clontech (Palo Alto, CA) and iNOS primers
from Biosource International (Camarillo, CA). The sequences of the
IL-10 and GAPDH primers have been described previously
(30). PCR was performed in a TRIO-Block thermocycler
(Biometra, Göttingen, Germany) at the following conditions: 1) 2
min at 93°C; 2) 30 s at 93°C, 30 s at 60°C (IFN-,
TNF-, iNOS), or 58°C (GAPDH), 45 s at 72°C for 27 cycles
(IFN-), 25 cycles (TNF-, iNOS), 30 cycles (IL-10), or 18 cycles
(GAPDH); and 3) 10 min at 72°C. For each gene, preliminary
experiments were conducted to ascertain that amplification of cDNA was
in the linear range under the respective cycling conditions
(31). PCR products were analyzed on ethidium
bromide-stained agarose gels and quantified densitometrically as
described elsewhere (31).
Competitive PCR
Competitive PCR analysis of TNF- mRNA was performed using
CytoXpress quantitative PCR kits from Biosource, essentially
according to the manufacturer’s protocol. In initial titration assays,
serial dilutions of cDNA corresponding to 50, 5, and 0.5 ng starting
RNA were coamplified with 1000 copies of an internal calibration
standard (ICS) for 30 cycles and analyzed on 1.5% agarose gels.
Dilutions with visibly equivalent intensities of the cDNA and ICS bands
were used for subsequent quantification in a microplate hybridization
assay employing colorimetric detection of biotinylated primer
incorporated into both cytokine and ICS amplicons during the
PCR.
Cytokine ELISA
For the measurement of TNF- and IFN- serum levels, we used
commercially available sandwich ELISA systems from PharMingen (San
Diego, CA) following the instructions given by the supplier. The
sensitivity of the TNF- assay for serum samples was 2 pg/ml, and the
sensitivity of the IFN- assay was 30 pg/ml.
Histological analysis and immunocytochemistry
For histological analysis, animals were sacrificed by intracardiac perfusion with Ringer’s solution containing 2 x 104 U/L heparin followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 (32). Tissue samples were embedded in paraffin according to standard procedures. Five-micrometer paraffin sections were mounted on poly-L-lysine coated slides and processed as described previously (32). Tissue sections were deparaffinized in xylene and 96% ethanol, treated with chloroform, washed in 0.05 M Tris buffer, and routinely stained with hematoxylin/eosin. For the detection of pan-T cells, serial sections were stained with mAb B 115-1 (dilution 1:500; Holland Biotechnology, Leiden, The Netherlands). For oligodendrocyte staining, the monoclonal anti-2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) Ab (dilution 1:1000; Chemicon, Hofheim, Germany) or the monoclonal anti-myelin/oligodendrocyte glycoprotein Ab 8-18C5 were used (33). For detection, we then used the avidin-biotin complex system (Dako, Hamburg, Germany) with 3,3'-diaminobenzidine as peroxidase substrate, or New fuchsin as alkaline phosphatase substrate. Finally, sections were counterstained with hemalaun, dehydrated, and mounted in Eukitt (Kindler, Freiburg, Germany). Coded sections from the spinal cord were examined by masked observers. The number of B 115-1-stained cells was evaluated quantitatively in transverse spinal cord sections in five fields of 0.8 mm2 from five sections obtained at different levels of the tissues. Each field was rated at x200 primary magnification per marker and animal by a masked observer.
Detection of apoptosis by in situ tailing (IST)
IST was performed on paraffin-embedded tissue. Tissue sections were incubated for 1 h with 50 µl of a reaction mixture containing 1 µl of digoxigenin-labeled nucleotides (Dig DNA labeling mixture; Roche, Mannheim, Germany) and 12 U of terminal transferase (Promega, Heidelberg, Germany) as described previously (34). The reaction was stopped by adding 0.5 M EDTA. Sections were then treated for 1 h with an alkaline phosphatase-labeled anti-digoxigenin Ab (Roche) at a dilution of 1:600. Color reaction was visualized by alkaline phosphatase histochemistry using 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate (Roche) as a chromogen. After IST, the same sections were stained for T cells using the B 115-1 Ab and the avidin-biotin complex detection system with alkaline phosphatase. Fast red salts (Roche) served as a chromogen.
Statistical evaluation
Statistical analysis was performed using ANOVA and Mann-Whitney U rank sum tests using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize the expression of TNF-, IFN-, IL-10, and
iNOS mRNA after Ag therapy, we performed RT-PCR analysis of total
spinal cord RNA isolated 1, 6, and 20 h after i.v. injection of
MBP. The treatment schedule is given in Table I
. DNA sequencing of representative PCR
products confirmed their identity as cytokine or iNOS cDNA,
respectively. For semiquantitative evaluation, cytokine expression
levels were normalized against those of the housekeeping gene GAPDH
(Figs. 1 and 2).
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(mean 5-fold), IFN- (3-fold), and iNOS (2-fold) mRNA
levels (Fig. 1). Six hours after Ag administration, mRNA levels of
TNF- had already decreased to levels of untreated EAE animals. In
contrast, the increase of IFN- and iNOS mRNA levels persisted at
6 h and had declined by 20 h. Throughout, IL-10 mRNA levels
in MBP-injected rats were not significantly different from those in
untreated EAE animals (Fig. 1).
To corroborate the semiquantitative PCR findings, we performed
competitive PCR analysis of TNF- mRNA levels in Ag-injected and
control EAE animals. Representative findings from titration experiments
are shown in Fig. 2. Increasing dilutions of cDNA were coamplified with
a fixed amount (1000 copies) of an internal calibration standard. In
RNA isolated from control EAE spinal cord, a weak band corresponding to
TNF- cDNA was seen only at 5 ng total starting cDNA, whereas in EAE
rats 1 h after Ag injection, PCR products were obtained at 0.5 ng
starting cDNA. Accordingly, the subsequent microplate-based
quantification revealed a significant increase in the copy number of
TNF- mRNA from 326 ± 125 per ng starting total RNA (mean
± SEM, n = 4) in control EAE animals up to 1957
± 159 per ng RNA 1 h after Ag injection (mean ± SEM,
n = 4), corresponding to an average 6-fold induction in
the latter group (p < 0.001).
Administration of neutralizing TNF- antiserum led to an 50%
decrease in the induction of iNOS mRNA at 1 h after Ag injection.
At 6 h after MBP injection, iNOS mRNA levels in animals treated
with TNF--specific antiserum were similar to those in animals
receiving control serum (data not shown). All cytokine mRNA levels were
essentially unaffected by neutralization of TNF-.
Cytokine serum levels during Ag therapy
To corroborate our mRNA data at the protein level, we determined
TNF- and IFN- serum levels in samples from EAE rats 1 h,
6 h, and 20 h after i.v. injection of gpMBP. One hour after
injection of gpMBP/control serum, we observed a strong increase in
TNF- and IFN- serum levels (Fig. 3). Six hours after administration of
gpMBP protein, levels of TNF- slightly decreased, and, after 20
h, TNF- was no longer detectable. Similar to the RT-PCR data,
IFN- exhibited a more sustained increase until 6 h, and was
still elevated at 20 h after Ag injection (Fig. 3). In animals
treated with neutralizing TNF- antiserum, the rise in TNF- serum
level was not detectable. Moreover, TNF- blockade down-regulated
IFN- protein secretion, which could only be detected 6 h after
treatment (Fig. 3).
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Ab on cellular
infiltration and apoptosis in spinal cord
After a single injection of gpMBP/control serum (Fig. 4A), histological analysis of
the spinal cord showed an immediate decrease of T cell inflammation and
an increase in the percentage of apoptotic T cells that reached its
maximum 20 h after injection (Table II). When a combination of gpMBP and
anti-TNF- antiserum (Fig. 4B) was administered, no
significant increase of T cell apoptosis compared with untreated EAE
animals was observed (Table II). Because our subsequent clinical
studies required more extended treatment protocols (see below), we
comparatively studied the effects of repeated Ag injections on T cell
infiltration and apoptosis in the spinal cord. When gpMBP was
administered twice within 12 h and rats were perfused 6 h
later (21), we observed further augmentation of T cell
apoptosis (gpMBP/control serum recipients, 36.9 ± 20.3%
apoptotic T cells; control serum recipients, 18.1 ± 8.6%;
p < 0.05). Thus, repeated Ag injections induced
qualitatively identical, but quantitatively more pronounced
changes.
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, C and D) that was most pronounced at 6 h
and 20 h after Ag injection (Table III). With concomitant neutralization of
TNF-, we observed a significant decrease of apoptotic
oligodendrocytes compared with the control serum group (Table III).
When Ag therapy was given twice (21), oligodendrocyte
apoptosis was even more pronounced (gpMBP/control serum recipients,
18.0 ± 7.8% apoptotic oligodendrocytes; control serum
recipients, 9.8 ± 3.1%; p < 0.05). Under these
conditions, we additionally found elevated serum levels of liver
enzymes, indicating TNF--induced liver injury as a side effect of
high-dose Ag therapy similar as shown previously in P2-specific Ag
therapy of experimental autoimmune neuritis (24).
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Ab on disease course
To obtain significant clinical effects during treatment of severe
EAE, a single injection of Ag was generally not sufficient. To assess
the clinical consequences of TNF- neutralization during Ag therapy,
we therefore followed a prolonged treatment schedule with repeated Ag
injections. Starting at the onset of overt disease on day 2, EAE
animals received 100 µg gpMBP twice daily for 4 days in combination
with either anti-TNF- serum or control serum. Additional groups
of EAE animals received only anti-TNF- serum or control serum
without concomitant Ag therapy.
EAE rats treated with control serum alone developed severe signs of
disease (Fig. 5). Two of four rats even
died. Administration of gpMBP had a significant beneficial effect that
was not changed by concomitant neutralization of TNF-.
Administration of anti-TNF--Ab without Ag injection only
slightly ameliorated the disease course (p <
0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-. This was shown at the level of spinal
cord mRNA expression and at the protein level in serum samples.
Therefore, it appears that both infiltrating cells in the CNS
compartment and circulating immune cells in the blood contribute to
proinflammatory gene induction following Ag-specific therapy of EAE.
The concomitant up-regulation of iNOS mRNA in the spinal cord of
Ag-treated animals suggests profound activation of proinflammatory
effector pathways that correlates to the increased apoptosis of both T
cells and oligodendrocytes found in quantitative histological analysis.
In contrast, IL-10 mRNA levels did not change following Ag
administration, arguing against a major role of this
anti-inflammatory cytokine in high-dose Ag therapy of EAE.
In view of the established Th1-mediated autoimmune pathogenesis of EAE
(5, 6), the induction of TNF- and IFN- mRNA and
secretion of these cytokines after therapeutic Ag injection is
unexpected. Both cytokines exert potent proinflammatory effects in
numerous in vivo and in vitro models, and thus should be expected to
exacerbate the local inflammatory reaction in EAE, thereby causing an
aggravation of clinical disease (5, 6). Moreover,
experiments in transgenic animals suggest a direct toxic effect of
TNF- on oligodendrocytes and myelin (35). The
cytokine-dependent up-regulation of iNOS could equally play a
detrimental role by promoting the release of large amounts of toxic NO
that has been implicated as a mediator of oligodendrocyte and myelin
damage in EAE and multiple sclerosis (36). However, this
traditional view has been challenged recently by a number of studies
describing unexpected disease-ameliorating effects of proinflammatory
cytokines and iNOS in EAE (7, 12, 14, 37). In
TNF--deficient mice, Liu et al. (8) showed an
aggravation of clinical and histological signs of EAE. TNF- is a
potent inducer of T cell apoptosis (38), a mechanism that
has been implicated as a major cause of spontaneous recovery from EAE
(39, 40). Accordingly, TNF receptor-deficient mice exhibit
a significant decrease of T cell apoptosis in this model
(41). Moreover, recent studies in both EAE and
experimental autoimmune neuritis indicate that the therapeutic effect
of high-dose Ag administration is also mainly mediated by an increase
in the rate of T cell apoptosis (20, 21).
For the first time, we describe here increased apoptosis of
oligodendrocytes as a potentially harmful side effect of high-dose Ag
therapy. The neutralization of TNF- led to a decrease in the rate of
both T cell and oligodendrocyte apoptosis, thereby inhibiting pathways
with opposite functional impact. This may explain why, in our study,
the beneficial clinical effect of Ag administration was overall not
affected by concomitant neutralization of TNF-. An alternative
explanation would be that the effect of Ag therapy on the small
subpopulation of Ag-specific cells (42) in the infiltrate
is not mediated by TNF- and therefore sustained.
In our study, the induction of TNF- was accompanied by considerable
induction of IFN- and iNOS expression. In IFN- receptor-deficient
mice Willenborg et al. (43) found an exacerbation of
clinical disease that may in part be attributable to the decreased
generation of NO. Accordingly, inhibition of iNOS in wild-type animals
similarly enhanced disease induction and worsened the clinical course
of EAE (14, 44). NO inhibits the production of IL-2 and
IFN-, has an antiproliferative effect on T cells, and inhibits Ag
presentation by down-regulating MHC class II molecules in macrophages.
Moreover, NO may induce apoptosis of myelin-reactive T cells in vitro
(45). Taken together, these findings suggest a potential
role of proinflammatory cytokines and NO in the limitation of T
cell-mediated CNS inflammation (16) and challenge more
simplistic concepts derived from earlier studies.
In conclusion, our study demonstrates that Ag therapy results in a
profound modulation of the cytokine network and supports the concept
that TNF- is one of the central molecular mediators of apoptosis.
Functionally, TNF- promotes apoptotic death of both autoaggressive T
cells and oligodendrocytes and therefore appears to play a dual role in
high-dose Ag therapy of EAE.
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Acknowledgments |
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Footnotes |
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2 A.W. and S.J. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Ralf Gold, Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Julius-Maximilians Universität, Josef-Schneider-Straße 11, D-97080 Würzburg, Germany.
4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; iNOS, inducible NO synthase; gp, guinea pig; AT, adoptive transfer; ICS, internal calibration standard; CNPase, 2',3'-cyclic nucleotide 3'-phosphodiesterase; IST, in situ tailing.
Received for publication May 23, 2000. Accepted for publication September 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
in high-dose antigen therapy in experimental autoimmune neuritis: inhibition of TNF- by neutralizing antibodies reduces T-cell apoptosis and prevents liver necrosis. J. Neuropathol. Exp. Neurol. 59:368.[Medline]
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confers resistance to experimental allergic encephalomyelitis. Eur. J. Immunol. 26:1641.[Medline]
is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide. J. Immunol. 163:5278.This article has been cited by other articles:
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  F. Odoardi, N. Kawakami, W. E. F. Klinkert, H. Wekerle, and A. Flugel Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease PNAS, November 20, 2007; 104(47): 18625 - 18630. [Abstract] [Full Text] [PDF]
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 T. A. Dietlin, F. M. Hofman, B. T. Lund, W. Gilmore, S. A. Stohlman, and R. C. van der Veen Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion J. Leukoc. Biol., May 1, 2007; 81(5): 1205 - 1212. [Abstract] [Full Text] [PDF]
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 R. Gold, C. Linington, and H. Lassmann Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research Brain, August 1, 2006; 129(8): 1953 - 1971. [Abstract] [Full Text] [PDF]
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 S. P. Zehntner, C. Brickman, L. Bourbonniere, L. Remington, M. Caruso, and T. Owens Neutrophils That Infiltrate the Central Nervous System Regulate T Cell Responses J. Immunol., April 15, 2005; 174(8): 5124 - 5131. [Abstract] [Full Text] [PDF]
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B. Marchetti, M. C. Morale, J. Brouwer, C. Tirolo, N. Testa, S. Caniglia, N. Barden, S. Amor, P. A. Smith, and C. D. Dijkstra Exposure to a Dysfunctional Glucocorticoid Receptor from Early Embryonic Life Programs the Resistance to Experimental Autoimmune Encephalomyelitis Via Nitric Oxide-Induced Immunosuppression J. Immunol., June 1, 2002; 168(11): 5848 - 5859. [Abstract] [Full Text] [PDF]
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M. Stienekemeier, K. Falk, O. Rotzschke, A. Weishaupt, C. Schneider, K. V. Toyka, R. Gold, and J. L. Strominger Vaccination, prevention, and treatment of experimental autoimmune neuritis (EAN) by an oligomerized T cell epitope PNAS, November 20, 2001; 98(24): 13872 - 13877. [Abstract] [Full Text] [PDF]
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