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High-Dose Methylprednisolone Therapy in Multiple Sclerosis Induces Apoptosis in Peripheral Blood Leukocytes
Verena I. Leussink, MD;
Stefan Jung, MD;
Ursula Merschdorf, MD;
Klaus V. Toyka, MD;
Ralf Gold, MD
Arch Neurol. 2001;58:91-97.
ABSTRACT
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Background Apoptosis is supposed to contribute to the elimination of T cells from
the inflamed central nervous system in the natural disease course of multiple
sclerosis (MS). In the animal model experimental autoimmune encephalomyelitis,
T-cell apoptosis can be induced by high-dose glucocorticoid (GC) administration.
Objective To study the effects of intravenous high-dose GC therapy in MS on T-cell
apoptosis ex vivo.
Patients Sixty-six patients with MS (28 with relapsing-remitting MS, 22 with
secondary chronic progressive MS, and 16 with primary chronic progressive
MS) and 16 control patients receiving corticosteroids for other disorders
were included in the study.
Methods Blood samples were collected before and immediately after the first
infusion of 500 to 1000 mg of methylprednisolone given during 2 hours in the
early morning. Gradient-isolated peripheral blood leukocytes (PBLs) were cultured,
unstimulated, with corticosteroids (positive control), the mitogen phytohemagglutinin,
or anti–T-cell receptor monoclonal antibody. For investigation of apoptosis,
PBLs were cultured overnight and analyzed by immunoflow cytometry using TUNEL
(terminal transferase-mediated dUTP biotin nick end labeling) or annexin labeling
in combination with CD4, CD8, CD22, CD56, or bcl-2 staining. Proliferation
was measured by 3H-thymidine incorporation. For cytokine determination,
supernatants were collected after 48 hours of culture.
Results After in vivo corticosteroid treatment, apoptosis of unstimulated PBLs
was markedly and significantly augmented in all 3 MS subgroups. Fluorescence-activated
cell sorter analysis showed that apoptosis affected predominantly CD4 T cells.
Natural killer cells showed a relative increase after GC therapy without a
change in the rate of apoptotic cells. Expression of bcl-2 in T-cell subpopulations
was not significantly modified by high-dose GC therapy. Culture supernatants
of T-cell receptor–stimulated PBLs after GC therapy contained lower
concentrations of interleukin 2, interferon gamma, and tumor necrosis factor 
than those from PBLs taken before pulse therapy. Similar changes in the rate
of apoptosis and cytokine production were seen in controls.
Conclusions Corticosteroid pulse therapy is a strong inducer of leukocyte apoptosis.
Induction of apoptosis might contribute to the down-regulation of T-cell activity
and thereby terminate inflammation in the central nervous system.
INTRODUCTION
THERE IS EVIDENCE that multiple sclerosis (MS) is a disease in which,
among others, autoimmune processes play a central role.1, 2, 3
Inflammatory attack results from the interaction of T lymphocytes with other
components of the immune system. Intravenous (IV) treatment with high-dose
glucocorticoids (GCs) has now become the standard therapy in patients with
an acute relapse of disease or a rapidly progressive deterioration of chronic
progressive MS.4 Short-term high-dose corticosteroid
therapy does not affect the hypothalamic-pituitary-adrenal axis in these patients5 and does not reduce bone density.6, 7
Several studies4, 8, 9, 10, 11, 12
on GC treatment yielded controversial results as to the most effective dose
and form of application. Results of the North American Optic Neuritis Treatment
Trial9 indicated that high-dose IV methylprednisolone
treatment slightly accelerates recovery from relapses and might even reduce
the risk of subsequent attacks,13 whereas low-dose
oral prednisolone therapy is ineffective and might even be associated with
an increased risk of subsequent development of MS. In a small, double-blind,
controlled trial,8 the effect of 500 mg of
methylprednisolone administered orally for 5 days was not different from giving
the same dosage intravenously.
Glucocorticoids exert their anti-inflammatory effect at different levels:
modulation of cell activation, cytokine expression, secretion of inflammatory
mediators, leukocyte migration, reduction of tissue edema,14, 15, 16, 17
and, at high doses, T-cell apoptosis in the nervous system18, 19
and other tissues.20 Use of GCs probably leads
to short-term, transient functional changes in leukocytes, including the induction
of apoptosis. It is still unknown whether certain leukocyte subpopulations
are particularly susceptible. The present study focused on the effects of
IV high-dose GC therapy on T-cell apoptosis ex vivo.
PATIENTS, MATERIALS, AND METHODS
PATIENTS
A total of 66 patients (49 women and 17 men) with MS were included in
this study: 28 (21 women and 7 men) with relapsing-remitting (RR) MS, 22 (19
women and 3 men) with secondary chronic progressive (SP) MS, and 16 (9 women
and 7 men) with primary chronic progressive (PP) MS. Sixteen patients (10
women and 6 men) who received GC pulse therapy for other disorders, such as
inflammatory neuropathy,4 plexus neuritis,5 vasculitis,1 lumbar
disc herniation,1 myositis,1
myelitis,3 and papillitis,1
served as control subjects.
The mean ± SEM ages of the groups were 35 ± 2 years for
the RRMS patients, 44 ± 2 years for the SPMS patients, 51 ±
2 years for the PPMS patients, and 51 ± 4 years for the control group.
Of the 66 MS patients, 9 (5 with SPMS and 4 with RRMS) were pretreated
with azathioprine (100-200 mg/d), 2 (both with SPMS) with cyclophosphamide,
2 (both with RRMS) with ß-interferon, and 1 (with SPMS) with methotrexate
(7.5 mg/wk) in a long-term immunosuppressive therapy regimen for more than
12 months, ie, in a steady state situation.
Except for one patient receiving 8 mg of methylprednisolone orally daily,
the patients had not undergone GC pulse therapy for a minimum of 2 months.
METHODS
GC Pulse Therapy
Typically, 1000 mg of methylprednisolone (Urbason; Hoechst, Frankfurt,
Germany) IV over 3 days (500 mg IV over 5 days in 6 MS patients and 6 controls
and 250 mg IV over 5 days in 5 controls) was given in the morning with an
infusion time of 2 hours. Blood samples (20 mL of EDTA) were taken by venipuncture
from each patient before and immediately after the first GC infusion, taking
into account the plasma half-time for methylprednisolone of 1.5 to 3 hours.
The indication for GC therapy was made by physicians not involved in this
study. The study was approved by the local ethics committee, and consistent
informed consent was obtained.
Cell Culture
All culture media and supplements were purchased from Gibco BRL (Eggenstein,
Germany). Immediately after blood samples had been taken, peripheral blood
leukocytes (PBLs) were separated by gradient centrifugation using Ficoll (Nycomed
AS, Oslo, Norway), washed twice, and seeded at 4 x 105 cells
per well in 96-well round bottom microtiter plates (Nunc, Wiesbaden, Germany)
in 100 µL of standardized cell culture medium (RPMI-1640 supplemented
with glutamine, 2 mmol/L; penicillin G sodium, 100 U/mL; streptomycin sulfate,
100 µg/mL; and 5% fetal calf serum). Cultures were kept unstimulated
or methylprednisolone, 0.1 µg/mL—which equaled 0.4 µg/mL
in serum achieved after methylprednisolone pulse therapy19—was
added as a positive control for apoptosis; phytohemagglutinin, 5 µg/mL
(Sigma, Deisenhofen, Germany), was added as mitogenic lectin; or anti–CD3
monoclonal antibody X35, 0.1 µg/mL (Coulter, Krefeld, Germany), was
added for T-cell receptor activation.
Flow Cytometry and Proliferation Assays
After overnight culture for detection of DNA fragmentation as an indicator
for apoptosis, PBLs were fixed with 4% freshly prepared paraformaldehyde and
0.025% Nonidet P 40 as described previously.21
The same fixation was applied for labeling of bcl-2. Labeling of cells in
vitro was conducted by incubation in tubes with 50 µL of 5x tailing
buffer (Promega, Heidelberg, Germany); 0.15 nmol of fluorescein-12-deoxyuridine
triphosphate (dUTP) (Boehringer, Mannheim, Germany); 0.85 nmol of deoxythymidine
triphosphate; and 1 nmol of deoxyadenosine triphosphate, deoxycytidine triphosphate,
and deoxyguanosine triphosphate (all from Pharmacia Biotech, Freiburg, Germany).
Five units of terminal transferase and bidistilled water to 50 µL were
added. After incubation for 1 hour at 37°C on a rotating shaker, immunofluorescence
was measured with a fluorescence-activated cell sorter (FACScan; Becton Dickinson,
Heidelberg) using the CellQuest software (Becton Dickinson).
For bcl-2 double staining, PBLs were fixed (see the previous paragraph)
and then incubated with a hamster anti–human bcl-2 IgG antibody (Pharmingen,
Hamburg, Germany) and fluorescein isothiocyanate–labeled goat anti–hamster
IgG antibody (Dianova, Hamburg) at a dilution of 1:100, followed by addition
of a mouse anti–human CD4 antibody (IgG1; Becton Dickinson, San Jose,
Calif) detected with goat anti–mouse IgG1-Tricolor (Medac, Hamburg)
or addition of mouse anti–human CD8 antibody (IgG2a; Becton Dickinson)
labeled with goat anti–mouse IgG2a-Tricolor (Medac) to achieve differential
detection on a FACScan. The antibodies used for fluorescence-activated cell
sorter analyses are listed in Table 1.
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Table 1. Cell Specificity, Concentration and Dilution, and Supplier
of Antibodies Used for Fluorescence-Activated Cell Sorter Analysis
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After overnight culture, apoptotic cells were detected by surface labeling
with annexin–fluorescein isothiocyanate (Boehringer). A total of 2 x
105cells were incubated in tubes with 5-mmol/L Ca2+
buffer. After addition of annexin according to the instructions of the supplier
in a final concentration of 2.5 µg/mL, incubation at 4°C for 10
minutes, and washing, cells were light protected and diluted in 80 µL
of phosphate-buffered saline solution, 1% bovine serum albumin, and 0.1% sodiumazide
for further processing. Peripheral blood leukocytes were then incubated with
either anti–human CD4 Tricolor/anti–human CD22 phycoerythrein
or anti–human CD8 Tricolor/anti–human CD56 phycoerythrein at 4°C
for 10 minutes, washed, and measured by fluorescence-activated cell sorter
analysis.
Proliferative responses were determined by 3H-thymidine uptake.
After 48 hours, cultures were pulsed with 3H-thymidine, 0.007 MBq/well
(Amersham-Buchler, Braunschweig, Germany), for 16 hours. The cells were then
collected on fiberglass filter paper in a Betaplate 96-well harvester (Pharmacia
Biotec), and the incorporated radioactivity was quantified in a Betaplate
96-well liquid scintillation counter (Pharmacia Biotec). Values were expressed
as counts per minute (mean of triplicate cultures).
Cytokine Analysis by Enzyme-Linked Immunosorbent Assay
For determination of cytokine production by cultured PBLs (interleukin
[IL]-2, interferon gamma [IFN- ], and tumor necrosis factor
[TNF-] as TH1 cytokines and IL-4 and transforming growth
factor ß [TGF-ß] as TH2 cytokines), supernatants of unstimulated
and anti-CD3–stimulated cells were collected after 48 hours of culture
and stored at –80°C.
Transforming growth factor ß was measured in acid-activated supernatants
in duplicates using a diagnostic enzyme-linked immunosorbent assay (ELISA)
kit (Genzyme, Karlsruhe, Germany).
For measurement of IL-2 and IL-4, Duosets (Genzyme) were used; for measurement
of IFN- and TNF-, complementary antibody pairs and standards
(R&D Systems, Abingdon, England) were adapted for optimal sensitivity
of the ELISA following the instructions given by the suppliers. Concentrations
of these cytokines in the supernatants were determined in triplicate, and
probes of 1 donor (before and after GC infusion) were always measured in the
same assay to exclude interassay variation.
STATISTICAL ANALYSIS
Statistical analysis was performed using a software program (GraphPad
Prism TM, Version 2.0; GraphPad Software Inc, San Diego, Calif). After checking
for a symmetrical distribution of data, the t test
for grouped data was used, with P<.05 and P<.01 considered statistically significant. Data are
given as mean ± SEM. After classification of cytokine modulation by
GC infusion (increase of >20%, decrease of >20%, and unchanged [increase or
decrease  20%]), results were tested by the Dixon and Mood test for statistical
significance.
RESULTS
METHYLPREDNISOLONE PULSE THERAPY IN VIVO AUGMENTS PBL APOPTOSIS IN
VITRO
We found a significant increase of apoptotic PBLs in all subgroups of
MS after IV high-dose GC treatment. In unstimulated cultures, the percentage
of apoptotic T cells increased from 2.3% ± 0.2% to 4.5% ± 0.4%
in RRMS (P<.01), from 2.1% ± 0.2% to 3.5%
± 0.3% in SPMS (P<.01), and from 2.6% ±
0.4% to 3.6% ± 0.5% in PPMS (P<.05) as
measured by TUNEL (terminal transferase-mediated dUTP biotin nick end labeling)
(Figure 1). The 2 methods of TUNEL
staining and annexin labeling yielded comparable results. When GCs were added
directly to the culture, similar rates of apoptosis were observed from cells
taken before and after pulse therapy (data not shown), speaking for a similar
corticosteroid susceptibility of PBLs taken before pulse therapy.
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Figure 1. Percentage of apoptotic peripheral
blood leukocytes before and after glucocorticoid (GC) pulse therapy in patients
with 3 types of multiple sclerosis—relapsing remitting (A), secondary
chronic progressive (B), and primary chronic progressive (C)—as measured
by TUNEL and flow cytometry after in vitro culture. Data represent mean ±
SEM.
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METHYLPREDNISOLONE PULSE THERAPY PREDOMINANTLY AFFECTS CD4 T CELLS
After GC pulse therapy, immunofluorometric analysis showed a marked
and significant increase of apoptosis of CD4 T cells in RRMS (3.5% ±
0.6% before vs 6.4% ± 1.0% after GC therapy; P<.01),
in SPMS (3.8% ± 0.8% vs 6.6% ± 1.2%; P<.01),
and to a lesser degree in PPMS (4.3% ± 1.1% vs 6.2% ± 1.2%; P = .02). Concerning the number of CD4 T cells, IV GC therapy
caused a marked and significant reduction of CD4 T cells in PBLs of all MS
patients (after an in vitro culture even in the absence of GC therapy), which
did not differ between the various MS subgroups (RRMS, 47.1% ± 2.8%
before vs 27.9% ± 3.0% after GC therapy; SPMS, 54.4% ± 4.6%
before vs 36.5% ± 4.9% after GC therapy; and PPMS, 49.9% ± 2.3%
before vs 31.2% ± 4.4% after GC therapy; P<.001
for each group).
Because of the marked reduction of CD4 T cells, the proportion but not
the absolute number of CD8 T cells in MS patients tended to increase after
GC infusion in RRMS (35.6% ± 2.2% before vs 39.0% ± 2.9% after
GC therapy; P = .2) (Figure 2), (more clearly) in PPMS (31.7% ± 3.1% before vs
41.0% ± 4.1% after GC therapy; P = .03), and
(significantly) in SPMS (32.9% ± 3.4% before vs 40.7% ± 3.6%
after GC therapy; P<.01). Flow cytometric double
labeling of apoptotic CD8 T cells also revealed a significant increase in
RRMS (3.9% ± 0.3% before vs 5.7% ± 0.7% after GC therapy; P = .03) and in SPMS (4.5% ± 0.7% before vs 6.9%
± 0.9% after GC therapy; P = .01) but not
in PPMS (6.3% ± 1.8% before vs 6.8% ± 1.3% after GC therapy; P = .64).
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Figure 2. Percentage of CD4+
T cells (A), CD8+ T cells (B), and natural killer (NK) cells (C)
within the whole population of peripheral blood leukocytes before and after
glucocorticoid (GC) pulse therapy in patients with relapsing-remitting multiple
sclerosis. Data represent mean ± SEM.
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Natural killer (NK) cells exhibited a significant relative increase
after GC pulse therapy (RRMS, 28.2% ± 3.4% before vs 47.5% ±
4.02% after GC therapy [Figure 2];
SPMS, 24.2% ± 3.9% before vs 40.1% ± 5.5% after GC therapy;
and PPMS, 27.5% ± 4.2% before vs 48.4% ± 6.6% after GC therapy; P<.01 for all groups). However, the rate of apoptotic
NK cells was not changed by precedent GC infusion. We did not find significant
changes in the relative number of B-cell lymphocytes in any of the groups
after methylprednisolone pulse therapy. The percentage of apoptotic B cells
revealed a significant increase only in patients with SPMS (16.2% ±
11.1% before vs 22.7% ± 11.9% after methylprednisolone therapy; P = .03).
Because NK cells are partly positive for CD4, the total number of cells
staining for CD4, CD8, NK, and B cells in some patients exceeded 100%.
EXPRESSION OF bcl-2 IN T-CELL SUBPOPULATIONS WAS NOT MODIFIED BY GC
THERAPY
In any group of patients, analysis of bcl-2 expression in subpopulations
of lymphocytes by double staining in combination with CD4 or CD8 did not show
a significant effect of GC pulse therapy (data not shown). Note that these
analyses were performed after 16 hours of cell culture only, and thus early
changes in bcl-2 expression were not studied.
METHYLPREDNISOLONE PULSE THERAPY REDUCES CELLULAR PROLIFERATION
On stimulation with the mitogen phytohemagglutinin, PBLs isolated after
GC pulse therapy exhibited significantly reduced cellular proliferation (RRMS,
14 708 ± 1773 cpm before vs 5118 ± 1355 cpm after GC therapy;
SPMS, 12 555 ± 1621 cpm before vs 5429 ± 1695 cpm after
GC therapy; P<.01 for both). In PPMS, the reduction
of cellular proliferation after GC treatment was not significant (8097 ±
1470 cpm before vs 6450 ± 1589 cpm after GC therapy; P = .25). Stimulation with the T-cell receptor–activating monoclonal
antibody X35 revealed comparable results in RRMS and SPMS (P<.01) but also displayed significant effects in PPMS (P = .02). An example of the proliferation data from X35-stimulated
PBL cultures is illustrated in Figure 3.
Similar results were seen in unstimulated PBL cultures and after addition
of methylprednisolone to cultures, yet overall incorporated radioactivity
was less than 1000 cpm.
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Figure 3. Proliferation of X35 (anti–T-cell
receptor)–stimulated peripheral blood leukocytes (PBLs) before and after
glucocorticoid (GC) pulse therapy in patients with 3 types of multiple sclerosis—relapsing-remitting
(A), secondary chronic progressive (B), and primary chronic progressive (C).
Error bars represent mean ± SEM.
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HIGH-DOSE METHYLPREDNISOLONE INHIBITS THE PRODUCTION OF TH1
BUT NOT TH2 CYTOKINES BY T-CELL RECEPTOR–STIMULATED PBLs
Analysis of cytokine secretion of T-cell receptor–stimulated PBLs
from 41 MS patients from any subgroup showed a decrease of the TH1
cytokines IL-2, IFN-, and TNF- after GC pulse therapy by more
than 20% in most of the cultures, which all reached statistical significance
(P<.05-.01). In contrast, the TH2 cytokines
IL-4 and TGF-ß were not remarkably affected by GC therapy (Table 2).
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Table 2. Changes in X35-Stimulated Cytokine Production After Intravenous
High-Dose Methylprednisolone Therapy in Patients With Multiple Sclerosis*
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Cytokine levels in the various subgroups of MS before and after GC therapy
in unstimulated and X35-stimulated cultures are illustrated in Figure 4.
APOPTOSIS OF PBLs, DECREASE OF CD4 T CELLS, AND REDUCED PROLIFERATION
AND TH1CYTOKINE PRODUCTION RATE AFTER METHYLPREDNISOLONE
PULSE THERAPY ARE NOT SPECIFIC FOR MS PATIENTS
In controls with inflammatory and noninflammatory disorders, GC pulse
therapy also led to a marked and significant increase of apoptotic PBLs (2.4%
± 0.4% before vs 3.2% ± 0.6% after GC therapy; P = .02) and to a significant decrease of CD4 T cells (52.5% ±
2.6% before vs 34.6% ± 3.0% after GC therapy; P<.01).
The increase of NK cells after high-dose GC therapy (24.8% ± 3.0% before
vs 46.3% ± 3.4% after GC therapy) and of CD8 T cells (31.9% ±
1.7% before vs 42.4% ± 2.3% after GC therapy; P<.01
for both) was comparable to the results in MS patients. Similar to MS patients,
only the rate of apoptotic CD8 T cells was not affected by GC therapy (P = .9), whereas the rate of apoptotic CD4 T cells increased
from 4.8% ± 0.75% before to 8.3% ± 0.75% after GC therapy (P<.01).
Proliferation assays of unstimulated (737 ± 168 cpm before vs
405 ± 76 cpm after GC therapy; P<.01),
phytohemagglutinin-stimulated (9296 ± 1863 cpm before vs 3764 ±
1196 cpm after GC therapy; P<.01), and X35-stimulated
(8589 ± 2198 cpm before vs 3828 ± 1011 cpm after GC therapy; P = .02) PBL cultures displayed a significant decrease
of cellular proliferation after GC pulse therapy.
Similarly, levels of the TH1 cytokines IL-2, IFN-,
and TNF-ß in controls showed decreases after GC treatment comparable
to those in MS patients, whereas IL-4 and TGF-ß levels were not affected.
COMMENT
Glucocorticoids are potent anti-inflammatory drugs commonly used in
the treatment of autoimmune disorders. In this study, we investigated the
effects of high-dose IV GC therapy on subsequent PBL apoptosis in vitro and
on leukocytic production of cytokines in vitro in MS patients and in non-MS
patients receiving GC for other indications. Using flow cytometry and proliferation
assays, we demonstrated that GC pulse therapy rapidly triggers PBLs in vivo
for subsequent apoptosis in vitro. CD4 T cells showed the highest rate of
apoptosis in vitro. Besides T-cell migration in vivo, the observed susceptibility
of CD4 T cells to corticosteroid treatment and apoptosis of CD4 T cells might
partly be the reason for the preferential decrease of circulating CD4 T cells
after GC infusion.22, 23, 24, 25
Previous investigators revealed a transient decrease of CD4 T cells
and concomitant down-regulation of IL-2 and IFN- after high-dose IV
methylprednisolone therapy in MS patients26, 27
and a decrease of proliferation of CD45RA+ cells after high-dose
methylprednisolone therapy measured by 3H-thymidine uptake in an
ex vivo study.25 In experimental autoimmune
encephalomyelitis, the animal model for MS, it has been reported19
that the elimination of T-cell inflammation after IV methylprednisolone treatment
was based on an increased induction of apoptosis in situ. For obvious reasons,
this survey is not transferable to human conditions. The dosage of 0.1 µg/mL
used in cell culture was roughly equivalent to the serum level measured after
IV pulse therapy in experimental autoimmune encephalomyelitis.19
In the present study, at least in blood, similar changes were now observed
after pulse therapy. In experimental autoimmune encephalomyelitis also, a
dose-dependent increase of apoptosis was reported.19
This question was not addressed in our study.
Anti-inflammatory effects of GC therapy on immune cells may result in
cell death or anergy. Both conditions are associated with reduced proliferative
capacity of PBLs. This was shown by 3H-thymidine incorporation.
This reduction most likely exceeded the extent that may be explained by apoptosis
and favors other potent antiproliferative mechanisms of GC. To elucidate these
mechanisms, we investigated the effects of corticosteroids on cytokine production
using ELISA and revealed, in accord with other authors, inhibitory effects
on the secretion of IL-2,28, 29, 30, 31, 32
IFN-,33, 34 and TNF-.35, 36 The TH2 cytokines IL-4
and TGF-ß were not significantly affected by GC. In a recent study with
18 MS patients, Wandinger et al37 focused on
the production of IL-1, IL-2, IFN-, IFN-, and TNF- after
GC pulse therapy and measured a decrease of all mentioned cytokines. The selective
effects of methylprednisolone on the expression of TH1 cytokines
may relate to different numbers of GC receptors or different GC receptor affinities
in TH1 and TH2 cells. Besides this possibility, GC sensitivity
may change in relation to the activation stage of the respective T-cell subsets.29
The effects of GC pulse therapy on the rate of apoptotic CD4 and CD8
T cells and on the reduction of cellular proliferation were comparable in
RRMS and SPMS patients but less obvious in PPMS patients. This might be an
explanation for the limited efficacy of GC therapy on the course of the disease
in the latter patients. In principle, concomitant immunosuppressive treatment
could have impact on the results of our study. This reflects the daily situation
in the clinic. Because patients were all being treated for more than 12 months,
we assumed that they had achieved a stable condition.
Bcl-2 protein enhances cell survival in several experimental systems
through the inhibition of apoptosis38, 39, 40
and might explain the differential susceptibility of T-cell subsets to corticosteroid-induced
apoptosis. Expression of bcl-2 was not different between the CD4 and CD8 T
cells, in contrast to the findings of Migita et al,24
who detected higher levels of bcl-2 in CD8 T cells. The fact that the percentage
of bcl-2–expressing T-cell subpopulations did not change after high-dose
methylprednisolone therapy in our study suggests that increased apoptosis
after GC infusion is not due to the down-regulation of bcl-2 by GC. Recently,
Schmitz et al41 presented evidence for 2 different
cell types (CD95 type I and type II cells), of which only type II cells showed
a reduction of apoptosis due to overexpression of bcl-2. This implies that
bcl-2 might rescue cells from apoptosis only in certain PBL subpopulations.
Alternatively, one has to consider that our bcl-2 analyses were not performed
directly after GC therapy but in cells cultured for 16 hours. Thus, early
changes might have been missed.
In MS patients, elimination of inflammatory T cells by apoptosis occurs
as a physiologic defense mechanism of the central nervous system.42, 43 From our results and experiments
in the animal model experimental autoimmune encephalomyelitis,19
it can be anticipated that methylprednisolone pulse therapy not only augments
apoptosis of PBLs, but also of T cells in the inflamed nervous system. This
effect was not specific for MS, but could also be observed in PBL cultures
from controls. Yet, in patients with immune dysregulation, GC pulse therapy
might accelerate termination of inflammation locally and systemically. In
addition, our data suggest that the suppressive effect of methylprednisolone
on production of TH1 cytokines might be another reason for the
clinical benefit of GC therapy in MS. Future studies should aim at characterizing
the underlying molecular mechanisms of the anti-inflammatory effects of methylprednisolone.
AUTHOR INFORMATION
Accepted for publication April 10, 2000.
Drs Leussink and Jung contributed equally to this work.
This work was supported by a grant from the German MS Society (Drs Jung
and Gold) and by funds from the state of Bavaria, Hannover.
We thank Gabriele Köllner and Alexandra Bunz for excellent technical
assistance and Gregor Rothe, MD, University of Regensburg, Regensburg, Germany,
for helpful advice with the use of CD-specific antibodies on fixed human peripheral
blood leukocytes.
From the Department of Neurology, Julius-Maximilians Universität
Würzburg, Würzburg, Germany. Dr Merschdorf is now with the Department
of Psychiatry, Universität Würzburg.
Reprints: Ralf Gold, MD, Department of Neurology, Julius-Maximilians
Universität Würzburg, Josef-Schneider-Strasse 11, D-97080 Würzburg,
Germany (e-mail: r.gold{at}mail.uni-wuerzburg.de).
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