* 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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Key Words: in vivo • CD 14 • CD 64 • CD 95 (Fas)
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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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View this table: [in a new window] |
Table 1. Clinical and Hematological Features of the MS Patients
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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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 View larger version (47K): [in a new window] |
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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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.
Received August 21, 2001; accepted August 27, 2001.
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on high affinity Fc receptor expression in healthy controls and multiple sclerosis patients J. Immunol. 161,1568-1574This article has been cited by other articles:
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