(Journal of Leukocyte Biology. 2001;70:745-748.)
© 2001
by Society for Leukocyte Biology
Treatment of multiple sclerosis patients with interferon-ß primes monocyte-derived macrophages for apoptotic cell death
Johan Van Weyenbergh*,
Juana Wietzerbin*,
Dany Rouillard
,
Manoel Barral-Netto
and
Roland Liblau
* U365 INSERM,
Section de Recherche, Institut Curie
U546 INSERM, Hôpital La Pitié-Salpêtrière, Paris, France
Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Salvador-Bahia, Brazil
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ABSTRACT
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Although interferon (IFN)-ß has shown a significant clinical benefit
in multiple sclerosis (MS), its mechanism of action remains unclear. We
found that IFN-ß treatment of patients with MS resulted in a
significant increase in apoptotic cell death (measured by annexin V
staining and nuclear fragmentation) of monocyte-derived macrophages, as
compared with cells derived from patients before treatment. Stimulation
of the cells with IFN-ß in vitro resulted in an even further increase
of annexin V binding, as well as increased Fas (CD 95, APO-1)
expression. However, no increased Fas expression, apoptotic monocytes,
or monocytopenia were observed upon in vivo treatment. This indicates
that IFN-ß does not deliver a death signal to monocytes but
rather primes for subsequent macrophage apoptosis upon activation
or differentiation.
Key Words: in vivo • CD 14 • CD 64 • CD 95 (Fas)
Multiple sclerosis (MS) is a chronic inflammatory disorder of the
central nervous system (CNS), debilitating mostly young adults. The
immunomodulatory and antiviral cytokine interferon (IFN)-ß is the
first drug to show therapeutic effect in relapsing-remitting and
secondary-progressive MS [1
2
3
], but its mechanism of
action remains as enigmatic as the etiology of MS itself
[4
, 5
]. In an animal model of MS,
experimental autoimmune encephalomyelitis (EAE), myelin-specific
autoreactive T cells have been shown to initiate an autoimmune attack
leading to demyelination. It has been proposed that apoptosis of T
cells in EAE is a mechanism to control inflammation in the brain
[6
]. In MS patients, incubation with IFN-ß in vitro
has been shown to augment activation-induced T cell death
[7
]. However, in later stages of tissue pathology, T
cells are greatly outnumbered by macrophages, which in fact execute the
final step in demyelination, i.e., myelin phagocytosis
[8
, 9
]. Therefore, we investigated the
effect of IFN-ß treatment in vivo and in vitro upon
monocyte/macrophage apoptosis in patients with MS.
Seven patients (four women, all relapsing-remitting) with clinically
definite MS were selected for this study. Informed consent was
obtained, and blood was drawn before and at 1, 2, and 3 months of
treatment with IFN-ß1a, 72 h after injection (AvonexTM, 30 µg
intramuscularly once a week). All of the patients were free of
exacerbations and had not received corticosteroids or immunosuppressive
drugs during the 3 months preceding treatment. All patients had a
clinically satisfactory response to IFN-ß treatment, because none of
them manifested progression of handicap after 1 year of treatment.
Mononuclear cells were isolated by density-gradient centrifugation
(Ficoll-Paque, Pharmacia, Sweden) and cryopreserved at each time point.
The four samples from each patient were defrosted and processed
simultaneously after verifying cell viability, which was identical to
defrosted cells from healthy controls processed in parallel. Cells were
stained with annexin V-fluorescein isothiocyanate (FITC), anti-CD
14-FITC, anti-CD 64-FITC, anti-Fas (CD 95, APO-1)-phycoerythrin
(PE), or isotype-matched control antibodies (all from
Immunotech-Coulter, Marseilles, France) before and after in vitro
culture. Cells were cultured for 40 h in RPMI medium supplemented
with 2 mM L-glutamine, 50 µg/ml gentamycin, and 10%
fetal calf serum (all from Gibco-BRL, Cergy Pontoise, France), in the
absence or presence of IFN-ß (1000 U/ml). Cells were analyzed in a
cytofluorometer (FACScan, Becton-Dickinson, San Jose, CA) using Lysis
II software. Monocytes were identified as CD 14+, CD
64+ cells with characteristic forward- and side-scatter.
Nuclear fragmentation was demonstrated by fluorescence microscopy
following Hoechst 33258 staining. Statististical analysis was performed
using Prism-Graph Pad software; two-tailed paired Student’s
t-test was used to compare patients before and after
treatment; and two-way analysis of variance (ANOVA) was used to examine
interaction between in vivo and in vitro treatment. A
P-value < 0.05 was considered significant.
We found that treatment of patients with IFN-ß in vivo had no effect
on annexin V-binding or Fas surface expression in freshly isolated
monocytes (unpublished results); i.e., apoptotic features were absent
in peripheral blood monocytes. Accordingly, as shown in Table 1
, no monocytopenia was detected in any of the patients.
However, when comparing monocytes after 40 h of stimulation by
plastic adherence (i.e., after differentiation into macrophages), we
observed an increase in annexin V staining induced by IFN-ß treatment
in vivo, as compared with cells derived before treatment. The
percentage of annexin V-positive macrophages rose from 16.7 (±4.7)%
before treatment to 57.2 (± 18.4)% after 3 months of treatment
(Fig. 1A
, open bars, P=0.02, paired t-test). In
addition, nuclear fragmentation, the hallmark of apoptotic cell death,
was observed almost exclusively in annexin V-positive monocytes derived
from patients after IFN-ß treatment (Fig. 2A
), consistent with the annexin V-staining results. Also,
stimulation of the cells with IFN-ß in vitro resulted in an even
further increase of annexin V binding, up to 43.1 (±7.8)% in
untreated patients and 67.5 (±4.1)% at 3 months of treatment (Fig. 1A
, solid bars). Although a trend for time-dependent increase can be
observed for in vitro- and in vivo-induced apoptosis (Fig. 1A)
, a
statistically significant increase in apoptosis induced by IFN-ß in
vitro was observed only at 0 and 2 months, whereas in vivo treatment
led to significant apoptosis at 1 and 3 months only. Analysis by
two-way ANOVA revealed that the in vivo and in vitro effects of IFN-ß
on apoptosis occurred independently (P=0.84 for interaction
between in vitro and in vivo). This is somewhat unexpected, because in
vivo treatment by IFN-a2 has been shown to down-regulate the IFN-
/b
receptor [10
], thereby probably precluding in vitro
restimulation by IFN- or -ß. The apparent absence of in vitro
unresponsiveness induced by previous in vivo treatment might rely in
differences between IFN species, dose or route of administration.
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Figure 1. Samples from seven MS patients treated in vivo with IFN-ß were
obtained before (0 months) and at 1, 2, and 3 months of treatment.
Mononuclear cells were cultured for 40 h in the absence (open
bars) or presence of 1000 U/ml IFN-ß (solid bars) and gated on
monocytes/macrophages in a cytofluorometer. Data are expressed as: (A)
% annexin V-positive cells (SE); (B) mean fluorescence intensity (MFI,
SE) for anti-Fas antibody staining, all corrected for background
fluorescence. Statistical analysis (paired
t-test) was performed comparing unstimulated
cells at each time point with unstimulated cells before treatment
(**P<0.05) and comparing
IFN-ß-stimulated cells with unstimulated cells at the same time point
(*P<0.05).
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Figure 2. Mononuclear cells were cultured for 40 h,
stained with annexinV-FITC, fixed, stained with Hoechst 33258, and
analyzed by fluorescence microscopy (Zeiss). (A) Nuclear fragmentation
was evident almost exclusively in macrophages derived from patients
after treatment and correlated with annexin V staining (arrow). (B) In
cells derived from patients before treatment, annexin V staining and
nuclear fragmentation were virtually absent. (C) Anti-HLA-ABC-FITC
staining is shown as a positive control. Paired samples from a
representative patient are shown.
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Because the importance of apoptotic cell death in MS and EAE pathology
has been become clear in recent years [5
6
7
], a central
role of the Fas/FasL pathway has been proposed [11
12
13
14
],
although clear data about participating cell types are lacking. To our
surprise, we observed no significant increase in Fas expression on
macrophages after in vivo treatment with IFN-ß (Fig. 1B
, open bars,
P>0.1 at all time points). However, Fas expression was
strongly increased by IFN-ß stimulation in vitro (Fig. 1B
, solid
bars), reaching statistical significance at 0 and 2 months of
treatment. It is interesting that IFN-ß stimulation in vitro resulted
in a significant increase in macrophage apoptosis at these same time
points, as described above. Again, no interaction was observed between
in vivo and in vitro treatment, as determined by two-way ANOVA
(P=0.63). Addition and cross-linking of an agonist anti-Fas
monoclonal antibody had no effect on spontaneous or IFN-ß-induced
macrophage apoptosis in three patients tested (unpublished
results). A similar strong resistance to Fas-induced cell death has
been described recently in T cell lines derived from MS patients [15
]. Taken together, our results demonstrate
that IFN-ß can prime macrophage apoptosis in vivo as well as in
vitro, possibly through a Fas-independent mechanism. A recent study
describes the ability of IFN-ß to induce apoptosis in melanoma
cell lines through a TRAIL-dependent mechanism [16
].
Clearly, further studies are warranted to investigate the molecular
mechanism(s) of IFN-ß-induced apoptosis in MS.
Finally, our findings extend those of Kaser et al. [7
]
to cells of the monocytic lineage and to the in vivo level; i.e.,
monocytes primed by IFN-ß treatment of MS patients undergo apoptotic
cell death upon subsequent activation and differentiation, which is
mimicked by adherence to plastic in vitro. This phenomenon is likely to
occur in the CNS of MS patients, because monocytes inevitably become
activated and differentiate into macrophages when crossing the
blood-brain barrier. It should be stated, however, that no apoptotic
features were observed in freshly isolated peripheral blood monocytes
before or after treatment. IFN-ß-induced apoptosis in circulating
monocytes, leading to monocytopenia and eventually immunosuppression,
would indeed represent a highly undesirable side effect, which was not
observed in our patients (see Table 1
) nor in any of the large clinical
trials [1
2
3
]. Rather, IFN-ß-induced apoptosis seems
to require prior activation of T lymphocytes [7
] or
macrophages (this study) and hence to selectively affect cells with
potentially demyelinating capacities. Similarly, elimination by
apoptosis of T lymphocytes and macrophages has been shown to correlate
with clinical recovery in the EAE model [14
]. Therefore,
triggering programmed cell death in effector cells in situ might be one
possible mechanism through which IFN-ß reduces active lesions, as
determined by magnetic resonance imaging, and exerts its
beneficial effect in MS. We have shown previously that the
high-affinity immunoglobulin G receptor (CD 64), preferentially
expressed on activated macrophages, is a target for antagonistic
regulation by IFN-ß and IFN-
in MS patients [18
],
which might incriminate CD 64 as an effector molecule in demyelination.
Based on the results of this study, we are led to believe that
selective triggering of apoptosis in activated macrophages, which can
be achieved by a CD 64-directed immunotoxin—recently demonstrated in a
transgenic mouse model [19
], might be considered as a
future adjunctive or alternative to IFN-ß therapy in MS.
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ACKNOWLEDGEMENTS
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This study was supported in part by the Association de Recherche
contre le Cancer (to J. W.) and the French Ministry of
Health AOM 95083 (to R. L.). M. B-N. is a CNPq senior
investigator. The authors thank Silvia Cardoso for skillful practical
assistance and Drs. Jean-Pierre Kolb and Albert Ko for helpful
discussions.
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FOOTNOTES
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Correspondence at current address: Dr. J. Van Weyenbergh,
Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (FIOCRUZ)
Rua Waldemar Falcao 121—Brotas, 40295-001 Salvador-BA, Brazil. E-mail:
ejomaflo{at}svn.com.br or u365{at}curie.fr
Received August 21, 2001;
accepted August 27, 2001.
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