ARTICLE
INTRODUCTION
Systemic lupus erythematosus (SLE) is characterized by the presence
of auto-antibodies reactive with various intracellular macromolecules,
such as nucleosomes, dsDNA, and small nuclear ribonucleoproteins (snRNP)
[1]. The cause of SLE is still unknown, although several etiological factors
have been suggested [1]. Previous studies have shown the presence of IFN-alpha
in the serum of 70 to 80% of patients with active SLE [2-4], and in the
cerebro-spinal fluid (CSF) in case of neurological complications [5].
IFN-alpha is also associated with tubulo-reticular inclusions observed
in endothelial and circulating cells of SLE patients [6]. Moreover, an
indirect argument for a role of IFN-alpha in the pathogenesis of SLE is
the appearance of a SLE-like syndrome in some patients receiving long
term IFN-alpha therapy [7-9]. IFN-gamma represents a group of homologous
proteins with antiviral and immunomodulating properties. In addition to
the antiviral activity, IFN-alpha increases the cytotoxic activity of
NK cells and of CD8+ T lymphocytes. Moreover, IFN-alpha, like
IFN-gamma, induces a shift of the T cell cytokine profile towards a Th1
pattern [10]. IFN-alpha genes are normally repressed but will be induced
by double stranded (ds) viral RNA or by viral glycoproteins [11-17]. Recently,
it has been reported that IFN-alpha inducing activity in the serum of
patient with SLE was associated with a reduced number of IFN-alpha producing
cells [18]. It has been suggested that the cells responsible for IFN-alpha
production could be immature dendritic cells [19], however, the mechanism
by which those cells would produce IFN-alpha remains unknown. Therefore,
our aim was to confirm the presence of both IFN-alpha and IFN-gamma-inducing
activity in the sera from patients with SLE. Since we had previously shown
that some virus complexed with antibodies can induce IFN-alpha via
the Fc gammaRII [20], we wished to establish if the same mechanism was
involved in SLE.
METHODS
Patients and serum
Fifteen serum samples from patients with active SLE, 2 samples from
patients with influenza virus infection, 18 samples from patients with
other autoimmune disorders (rheumatoid arthritis n = 6; systemic sclerosis
n = 5; primary Sjögren syndrome n = 6; pemphigus foliaceus n = 1),
and 16 serum samples from healthy control subjects were studied. All of
the patients with SLE or rheumatoid arthritis met the ACR criteria for
the respective diseases. SLE activity was assessed according the SLEDAI
[21]. This study was performed in accordance with the bioethical guidelines
of our institution.
Endogenous IFN-alpha assay
Endogenous IFN-alpha was assayed by determining the antiviral effect
on MDBK cells as previously described [22]. Results were expressed as
IU/ml relative to an international standard (NIH Ga 023-902-530).
Herpes simplex virus (HSV1)
Stocks of HSV1 were prepared from supernatant of infected Vero cells
cultured in RPMI-1% fetal bovine serum, 48 hours after infection, at a
multiplicity of infection (m.o.i.) of 0.1 and had a titer of 107
PFU/ml. HSV1 was used as the positive control in the IFN induction tests.
IFN-alpha induction experiments
Human mononuclear cells were isolated from the venous blood of healthy
subjects, on a Ficoll gradient. The cells were washed in RPMI without
serum and the suspension adjusted to 2 x 106 cells/ml in RPMI
1640, supplemented with 10% fetal calf serum. One hundred microliters
of serially diluted test serum or 50 µl of optimal dilution of HSV
were added to 400 µl of the mononuclear cell suspension. After 18
hours of incubation at 37° C, the supernatants were collected and
IFN-alpha assayed on MDBK cells. Results are expressed as the reciprocal
of the serum dilution which induce IFN-alpha activity.
Serum protein separation by chromatography
Gel filtration of SLE or normal serum was performed using a Sephacryl
S200 column at 0.3 ml/mn. Affinity chromatography was performed on a protein
G Sepharose column at 1 ml/mn. Individual fractions were immediately tested
for IFN inducing activity at the dilution of 1/2 in culture medium.
IFN-alpha characterization
The antiviral activity induced in PBMC by SLE serum was further characterized
as IFN-alpha in neutralization tests with polyclonal anti-IFN-alpha, anti-IFN-gamma
(INSERM U. 43, anti-IFN-ß, anti-IFN-omega antibodies [23] (a gift
from M Tovey, IRSC Villejuif), and anti-IFN-gamma monoclonal antibody
(Boeringer-Mannheim, Mannheim Germany).
Resistance to acid pH, a characteristic of IFN-alpha was tested in serum
fractions obtained after gel filtration or affinity chromatography. After
incubation of PBMC with aliquots of such fractions at 37° C for 18
hours, supernatants were exposed to glycine buffer at pH 2 or pH 7 overnight
at 4° C. Then, remaining antiviral activity was tested by biological
assay.
Acid stability of IFN-alpha-inducing activity
Serum or fractions from sephacryl filtration were incubated under various
conditions: either pH 2 at 4° C overnight, or pH 4.5 at 37°
C overnight, or at pH 7 at 56° C for one hour. Then, the residual
IFN-inducing activity was measured as described above.
Effects of monoclonal antibodies for Fc-gamma receptors
or Fcgamma fragments on IFN-alpha induction
Inhibition of IFN-alpha induction was attempted by preincubating PBMC
for 30 min at 37° C with either 2 to 10 µg/ml of anti-CD16,
anti-CD23 (Immunotech, Marseille Lumigny, France) or anti-CD64 monoclonal
antibodies (Cymbus, Hants, UK) or 1 to 10 µg/ml anti-CD32 monoclonal
antibodies clone IV.3 [24] and clone AT 10 [25], purified by affinity
chromatography on protein G sepharose [26]. In other experiments, the
cells were preincubated with 10 mg/ml of purified Fcgamma fragments (gift
from M.F. Makula Pasteur-Mérieux Serum et Vaccin, Lyon, France)
or the F(ab)'2 fragment of anti-CD32 monoclonal antibodies IV.3 and AT10
prepared as previously described [26] and used at various dilutions as
described in the results section. IV.3 F(ab')2 fragments were previously
shown to recognize FcgammaRIIA/C, but not FcgammaRIIB while AT10 antibody
recognized both [26, 27]. Herpes simplex virus type 1 was used as IFN-alpha
inducer control in all inhibition tests with antibodies. The IFN-alpha
assay was then performed as described above.
RESULTS
Endogenous IFN-alpha assay
Endogenous IFN-alpha was detected (from 2 to 400 IU/ml) in the serum
of 12 out of 15 SLE patients. No endogenous IFN-alpha was detected (<
2 IU/ml) in the serum of all 18 patients with other autoimmune disorders
and in all 16 serum from healthy subjects. The IFN-alpha titers were up
to 200 IU/ml in the two serum samples from patients with influenza virus
infection (Table 1).
Assay of IFN-alpha induced by serum
Eleven out of fifteen SLE serum samples induced IFN-alpha production
by PBMC at dilutions ranging from 1/400 to 1/1600. Ten of the SLE serum
had both endogenous IFN-alpha and IFN-alpha-inducing activity (Table
1). Two of the six patients with Sjögren syndrome and one
of the five with scleroderma induced only a low IFN-alpha production (5
to 10 I.U./ml) by PBMC with a serum dilution of 1/400. None of the serum
samples from healthy subjects or from patients with influenza viral infection
induced an antiviral activity (data not shown).
IFN-alpha characterization
The IFN-antiviral activity induced by SLE serum in PBMC was abolished
by polyclonal or monoclonal anti-IFN-alpha antibodies but not by anti-IFN-ß,
-gamma or -omega antibodies. In addition, the IFN-alpha induced under
those experimental conditions was resistant to acid pH and protected the
bovine cells line MDBK, which is sensitive to human type 1 IFN only.
Acid stability of
IFN-alpha inducing activity
Serum or fractions from sephacryl filtration were sensitive to acidic
pH since treatment of those samples at pH 2 overnight at 4° C, inhibited
the IFN-alpha-inducing potential by 60 to 90% (n = 3). Moreover, treatment
at pH 4.5 overnight at 37° C completely suppressed their IFN-alpha-inducing
activity (n = 3).
Separation of serum proteins by chromatography
After fractionation by gel-filtration, most of the IFN-alpha-inducing
activity was detected in the IgG fraction (Figure
1). No IFN-alpha inducing activity was found in the corresponding
fractions of normal serum.
IgG from two SLE serum samples and one control serum sample were purified
using a Protein G Sepharose column; IFN-alpha-inducing activity was detected
in the eluted fraction containing IgG molecules of SLE serum but not of
the control serum.
Inhibition of IFN-alpha production by monoclonal
antibodies to Fc-gamma receptors
When PBMC were preincubated with anti-Fc-gamma receptor monoclonal antibodies,
IFN-alpha-inducing activity was neutralized by anti-CD32 antibody (Table
2), but not by anti-CD16, anti-CD23 or anti-CD64. The monoclonal
anti-CD32 antibody IV.3, which recognizes FcgammaRIIA/C, and AT10, which
recognizes, both FcgammaRIIA/C and FcgammaRIIB [25], completely abolished
the IFN-alpha production by PBMC. F(ab)'2 fragments of the same two monoclonal
antibodies also abolished the IFN-alpha induction, although IV.3 F(ab')2
fragments were less inhibitory than AT10 F(ab')2 fragments (Table
2). The decrease in IFN-alpha production was related to the concentration
of Fc gammaRII mAb (Figure 2).
The IFN-alpha-inducing activity was also abolished after preincubation
with Fc-fragment preparations (Figure
2). The various monoclonal antibodies used in the experiments
and Fc-fragment preparations had no effect on the induction of IFN-alpha
by the Herpes simplex virus used as control with the same PBMC.
DISCUSSION
This study confirms the previously reported presence of IFN-alpha in
the serum of patients with SLE [2-4]. The presence of IFN-alpha seems
to be typical of SLE, since it was not detected in the serum from patients
with other autoimmune diseases. Furthermore, an IFN-alpha-inducing activity
was observed in the serum from patients with SLE but not in normal subjects
or in patients with influenza virus infection, although they had high
levels of endogenous IFN-alpha. In other connective tissue diseases, the
IFN-alpha-inducing activity is rarely found or is less than in SLE. Such
IFN-alpha-inducing activity in SLE has again been recently reported [18,
19]. In our study, IFN-alpha-inducing activity was associated with endogenous
IFN-alpha in ten out of twelve serum samples. However, IFN induction was
not due to a priming effect by endogenous IFN-alpha since it was observed
at serum dilutions with no detectable levels of IFN-alpha, and conversely
it was not observed in the serum of influenza-infected patients who had
high levels of endogenous IFN-alpha. The antiviral activity induced in
PBMC by SLE serum was characterized as IFN-alpha, as its antiviral activity
was neutralized by anti-IFN-alpha, but not by anti-IFN-ß, anti-IFN-alpha
or anti-IFN-gamma antibodies. This IFN-alpha was stable at pH 2, which
is a characteristic of type I IFN and contrasts with the partial acid
sensitivity of IFN-alpha originally described in SLE serum [2]. However,
this acid lability is not an intrinsic property of IFN itself, but is
due to a serum factor that can be separated from the antiviral activity
[28].
We undertook to further characterize the IFN-alpha-inducing activity.
This activity, detected in the same fractions as IgG and retained on sepharose
protein G column, is very likely an immunoglobulin or small sized immune-complexes.
This finding led us to investigate the involvement of Fcgamma receptors in
IFN-alpha induction. To this aim, blocking experiments were performed
using Fcgamma fragments and antibodies to various Fc receptors (CD16, CD23,
CD32 and CD64). Only Fcgamma fragments or anti-CD32 antibodies inhibited IFN-alpha
induction. IFN-alpha induction therefore appears to be dependent on an
interaction between IgG and FcgammaRII receptors. It is interesting to
note that a particular genotype of the FcgammaRII has been reported in
lupus patients [29-30].
The interaction between IgG and FcgammaRII receptors may involve one
or more of the three isoforms of these receptors. Fcgamma RIIB can mediate
endocytosis via intracytoplasmic dileucine motifs, whereas FcgammaRIIA/C
trigger both endocytosis and cell activation via immunoreceptor
tyrosine-based activation motifs [26-27]. We found that blocking all three
FcgammaRII abrogated IFN-alpha induction. Both FcgammaRIIA/C and FcgammaRIIB
may contribute to the induction of IFN-alpha since blocking FcgammaRIIA
with IV.3 F(ab')2 fragments only partially inhibited IFN-alpha production
whereas blocking FcgammaRIIA/C and FcgammaRIIB with AT10 F(ab')2 fragments
abolished IFN-alpha production.
Since FcgammaRIIA/C and Fcgamma RIIB can both mediate endocytosis, the
IFN-alpha-inducing activity could be related to engulfement of either
immune complexes or aggregated antibodies. In the case of immune complex
endocytosis, the internalization of nucleic acids or proteins complexed
with specific auto-antibodies could trigger IFN-alpha production. This
hypothesis is in agreement with the previous finding that IgG-virus complexes
can induce IFN-alpha via FcgammaRII receptors [20]. Moreover, previous
studies have shown that the IFN-alpha-inducing activity may be suppressed
after treatment with DNase I [19]. However, cell penetration by antibodies
does or does not require Fc binding according to which antibody or which
target cell is considered [31-32]. An alternative explanation for IFN-alpha
induction by SLE serum would be the presence of autoantibodies against
Fcgamma receptors which could possibly activate PBMC. However, in our hands,
none of the serum samples with IFN-alpha-inducer activity contained anti-CD32
auto-antibodies when tested on CD32-transfected L929 cells (data not shown).
CONCLUSION
SLE serum contains soluble factors resembling IgG or immune complexes
that can interact with FcgammaRII receptors to produce IFN-alpha. Our results
may allow better understand the origin of endogenous IFN-alpha, which has
a deleterious effect on the course of autoimmune diseases [33-36]. The
inhibition of this function by CD32 antibody could lead to new therapeutic
approach in SLE.
Acknowledgements.
We gratefully acknowledge Michael Tovey and Marie France Makula for supplying
us with materials for this study. We thank Helmut Ankel for critical comments
and assistance in editing the manuscript.
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