By
From the * Immunobiologie Moléculaire, Unité Mixte de Recherche 49, Centre National de la Recherche
Scientifique-Ecole Normale Supérieure Lyon, 69364 Lyon Cedex 07, France; U396, Institut des
Cordeliers, 75006 Paris, France; § U404, Institut Pasteur de Lyon, 69365 Lyon Cedex 07, France;
and
U391, 35043 Rennes Cedex, France
Despite the development of an efficient specific immune response during measles virus (MV)
infection, an immunosuppression occurs contributing to secondary infections. To study the
role of nucleocapsid protein (NP) in MV-induced immunosuppression, we produced recombinant MV NP. Purified recombinant NP exhibited biochemical, antigenic, and tridimensional
structure similar to viral NP. By flow cytometry, we showed that viral or recombinant NP
bound to human and murine B lymphocytes, but not to T lymphocytes. This binding was specific, independent of MHC class II expression, and dependent of the B lymphocyte activation
state. The murine IIA1.6 B cell line, deficient in the Fc receptor for IgG (FcRII) expression,
did not bind NP efficiently. Transfected IIA1.6 cells expressing either murine Fc
RIIb1 or b2,
or human Fc
RIIa, b1*, or b2 isoforms efficiently bound NP. Furthermore, this binding was
inhibited up to 90% by monoclonal antibodies 2.4G2 or KB61 specific for murine and human
Fc
RII, respectively. Finally, the in vitro Ig synthesis of CD40- or Ig-activated human B lymphocytes in the presence of interleukin (IL)-2 and IL-10 was reduced by 50% in the presence
of recombinant NP. These data demonstrate that MV NP binds to human and murine Fc
RII
and inhibits in vitro antibody production, and therefore suggests a role for NP in MV-induced immunosuppression.
Measles virus (MV)1 is responsible for an acute childhood disease that is benign in industrialized countries, but is among the primary causes of infant death in developing countries. MV belongs to the Morbillivirus genus
of the Paramyxoviridae family and its genome is a nonsegmented negative strand RNA encoding six structural proteins: nucleocapsid protein (NP; 60 kD), phosphoprotein (70 kD), matrix protein (37 kD), fusion (F) protein (with
subunits F1, 40 kD, and F2, 20 kD), hemagglutinin (H)
protein (80 kD), and large protein (250 kD). The minimal
infectious unit is the ribonucleoprotein complex composed
of the RNA tightly associated with the NP, phosphoprotein, and large protein.
MV infection is initiated by interaction between a viral
protein, the glycoprotein H, and a cellular receptor, the
human CD46 molecule (1, 2). The release of ribonucleoprotein complex into the cytosol leads to genome transcription, viral protein synthesis, and MV replication. The
humoral immune response is detected at the onset of the
rash, and the most abundant and rapidly produced antibodies are specific for NP (3, 4). The cellular component of the
immune response against MV involves MHC class I-restricted
CD8+ T cells and MHC class II-restricted CD4+ T cells.
MV-specific MHC class II-restricted CD4+ T cells clones
have been isolated from PBL of healthy donors with a history of MV infection. Interestingly, the CD4+ T cell clones
were specific for the H, F, matrix, and NP proteins (5-7)
and most of them displayed cytotoxic activity. The anti-NP T cells constitute an important component of the cellular
response against MV as NP-specific CD4+ T lymphocytes
can protect rats against MV-induced encephalitic disease (8).
In spite of the fact that NP is synthesized as a cytosolic
protein, the dual humoral and cellular CD4+ responses
against NP indicate that NP is both accessible to the B cell
receptor (BCR) after its release in the extracellular compartment and to the peptide-loading compartment after its
uptake by the APC. In this context, NP could be internalized by APC either by fluid-phase endocytosis or by receptor-mediated endocytosis. Targeting a soluble exogenous
antigen to antigen-specific B cells via their BCR (9) or to
macrophages and dendritic cells via their FcR after its opsonization with specific antibodies (10, 11) results in an enhancement of MHC class II-restricted antigen presentation to CD4+ T cells. However, it remains unknown whether
receptor-mediated endocytosis via BCR, FcR, or an NP-specific cellular receptor could play a role in the induction
of the MHC class II-restricted NP presentation to CD4+ T
helper cells and subsequently in the high anti-NP antibody synthesis.
MV infection gives rise to a paradoxical situation: despite
the development of an efficient immune response establishing long-term immunity and virus elimination, an immunosuppression occurs that contributes to secondary infections
and mortality. This immunosuppression was first described
by Von Pirquet (12) who observed a suppression of tuberculin skin test reactivity during the acute phase of MV infection and for several weeks thereafter. During the acute
phase of measles, lymphocytes from infected individuals respond poorly to mitogens like PHA or PWM (13). Moreover, the production of cytokines from both lymphocytes
and monocytes is dysregulated (14) and antibody production to the antigens of Salmonella typhi vaccine is impaired
(15, 16). Finally, a suppression of IgG synthesis was recently reported in MV-infected human SCID mice (17).
The respective role of viral proteins in this immunosuppression remains unclear.
After vaccination, both immune response and immune
suppression are observed. In the majority of cases, children
immunized with live MV vaccine develop antibodies against
NP (18). Recent data reported that mitogen-induced lymphoproliferation was decreased at 3 mo after vaccination
with live attenuated MV, and was more pronounced in
children with the highest antibody response and immune
activation (19). MV vaccination may alter the T cell repertoire. Vaccination of infants with live attenuated MV vaccine induce changes in the circulating TCR V Cells and Antibodies.
Human cells were cultured in RPMI
1640 (GIBCO BRL, Life Technologies SARL, Cergy Pontoise,
France) supplemented with 10% FCS (TechGen, International,
Les Ulis, France), 10 mM Hepes, 2 mM glutamine, and 50 µg/
ml gentamicin (GIBCO BRL). Human B cells were purified
from tonsils by two successive rounds of T cells depletion by rosetting with sheep red blood cells (Biomérieux, Lyon, France).
Tonsils were finely minced, the cell suspension (107 cells/ml) was
mixed with 0.02 vol of packed sheep red blood cells, and 30 ml
were layered over 15 ml of Ficoll (Eurobio, Les Ulis, France).
Cells were centrifuged at 50 g for 15 min and at 800 g for 30 min.
B cells were recovered from the interface between medium and
Ficoll and were washed before another round of depletion. T
cells were purified from tonsils by two successive passages on nylon wool columns. B and T cell preparations were routinely >98% and >90% pure, respectively, as revealed by their pattern of expression of CD20 and CD3 (Dako, Trappes, France). Rabbit anti-human Igs coupled to polyacrylamide beads were purchased from Bio-Rad S.A. (Ivry Sur Seine, France) and used at a final dilution of 1:600 to stimulate B cells during 2 d. PHA (Sigma-Aldrich Chimie SARL, Saint Quentin Fallavier, France) was used
at 5 µg/ml to stimulate T cells during 3 d.
repertoire
since the V
4 subset was decreased and the V
2 subset was
increased (20). Such an alteration in the T cell repertoire
could be due to the presence of a viral superantigen as reported for the rabies virus nucleocapsid that binds to V
8
and HLA class II
chain (21). In the present study, we
show that measles virus NP binds to human and murine Fc
receptor for IgG (Fc
RII) and that NP inhibits the in vitro
antibody synthesis from activated human B lymphocytes.
Production of Recombinant NP. AcNPV (Autographa californica nuclear polyedrosis virus) and recombinant virus stocks were grown and assayed in confluent monolayers of Sf (Sporodoptera frugiperda)-9 cells in TGV 3 (TC 100 modified medium) containing 10% FCS. Sf 9 cells and AcNPV virus were obtained from G. Devauchelle (Institut National de la Recherche Agronomique-Centre National de Recherche Scientifique, St. Christol lez Alès, France).
The cDNA for the NP of measles virus (Hallé strain) was described by R. Buckland et al. (29) and was inserted in the transfer vector pGmAc 34 (G. Devauchelle) at the SmaI site. The recombinant transfer vector pGmacNP contained the cDNA of NP under the control of the polyhedrin promoter. Sf9 cells were transfected with a mixture of plasmid pGmacNP and wild-type AcNPV DNA. Recombinant baculovirus AcNPVNP were plaque purified.Purification of Recombinant and Viral NP.
Recombinant NP was
purified from confluent monolayers of Sf9 cells infected with virus AcNPVNP at a multiplicity of 1 PFU/cell. 2 × 108 infected
cells were harvested 3 d after infection by centrifugation at 200 g
and washed 3 times in PBS. Purified NP were prepared from
these cells using modifications of the previously described method
for preparation of intracellular measles virus nucleocapsids (30).
Cell pellets were resuspended in hypotonic buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl) at 2 × 107 cells/ml in the presence
of proteinase inhibitors (0.1 mM 3,4-dichloroisocoumarin, 0.02 mM E-64, 0.1 mM 1,10-Phenantroline; Sigma-Aldrich Chimie SARL). Cells were lysed by the addition of the nonionic detergent Nonidet P-40 to a final concentration of 1% (vol/vol) and
left on ice for 30 min. Nuclei were removed by centrifugation at
1,000 g at 4°C for 5 min, and the supernatant was adjusted to
contain 10 mM EDTA. This fraction was then clarified of particulate material by centrifugation at 10,000 g at 4°C for 10 min.
This cytoplasmic extract was centrifuged through a discontinuous
density gradient in SW 41 centrifuge tubes (Beckman Instrs.,
France SA, Gagny, France). The step gradient was comprised of 2 ml of 40% (wt/vol) CsCl, 2 ml of 30% CsCl, 2 ml of 25% CsCl,
and 2 ml of 5% (wt/vol) saccharose each containing 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, and 0.2% (wt/vol)
sodium lauroyl sarcosinate. Centrifugation was performed at
12°C for 2 h at 36,000 rpm. The dense visible band observed in
the 30% CsCl step was removed by needle aspiration, diluted in
PBS, and centrifuged in a rotor (SW 41; Beckman Instrs. France
SA) at 12°C for 2 h at 36,000 rpm. The NP pellet was solubilized in
1 ml of PBS, aliquoted, and stored at 80°C.
Protein Analyses.
Cytoplasmic extract of Sf9-infected cells or
purified NP were subjected to 10% SDS-PAGE. Gels were stained
with Coomassie brilliant blue R-250 or electroblotted onto a nitrocellulose membrane using a semidry blotting system (Biometra,
Göttingen, Germany). The NP was identified with mAb 25NP
and alkaline phosphatase-conjugated anti-mouse Ig (Bio-Rad S.A.).
The NBT/BCIP system (Bio-Rad S.A.) was used for colorimetric detection. The protein concentration was determined using
the copper-dependent BCA assay (Pierce Chem. Co., Rockford,
IL) in microtiter plate with bovine serum albumin as a standard.
Flow Cytometry.
Cells (5 × 105) were incubated for 1 h at
4°C with purified NP (5 µg) in 100 µl of PBS containing 1%
BSA and 0.1% NaN3. Cells were then washed three times in this
solution and incubated with mAb 25NP or biotinylated 25
NP
for 30 min at 4°C. After three washes, cells were incubated with
FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Labs., West Grove, PA) or with streptavidin conjugated to
FITC (Jackson Laboratory) for 30 min at 4°C, and then washed.
Flow cytometric analyses were performed on a FACScan® (Becton
Dickinson, Immunocytometry Systems, Rungis, France). The
median fluorescence intensity value was chosen to quantify NP
binding. For antibody inhibition experiments, the value obtained for control without NP was subtracted for each point and the
value obtained without antibody was considered as 100%.
NP Biotinylation and Competition. NP was biotinylated with succinimidyl-6-(biotinamido)hexanoate (Pierce Chem. Co.) for 2 h at 4°C and then dialyzed in PBS. Biotinylated NP binding on Cl.13 cell line was detected with FITC-conjugated streptavidin by FACS® analysis. For competition experiments, 2 µg of biotinylated NP were incubated with 0-85 µg of unlabeled NP. FITC-conjugated streptavidin was used to detect the remaining biotinylated NP binding and values of median fluorescence intensity measured. The value obtained for the control without NP was subtracted for each point to determine the percentage of NP binding. The number obtained for 2 µg of biotinylated NP was considered as 100%.
In Vitro Human Antibody Production.
Purified B cells were seeded
at 2 × 105 cells/well in round-bottomed 96-well microtiter trays
in a final volume of 0.25 ml and were stimulated with rabbit anti-
human Igs coupled to polyacrylamide beads (1/600) or 2 × 104
mitomycin C-treated Ltk cells stably transfected with the CD40
ligand (CD40L; gift of M. Bakkins, Vrije Universiteit, Brussels,
Belgium). Purified recombinant IL-2 and IL-10, provided by A. Minty (Sanofi, Labège, France) and J. Banchereau (Schering-Plough, Dardilly, France), respectively, were used at 10 U/ml
and 50 ng/ml. Purified NP was added at the onset of the culture
at the optimal concentration of 30 ng/ml. Ig secretion (IgG, IgM,
and IgA) was determined in 12-d culture supernatants by standard
ELISA techniques, as described elsewhere (31). The percentage
of inhibition in the presence of NP was calculated on the basis of
maximal stimulation without NP.
As the measles nucleocapsids are difficult to purify and are often degraded in mammalian cells (32), we used the recombinant baculovirus technology to produce high level of measles NP in insect cells. We observed that the recombinant NP reached the maximal level of expression in Sf9 cells by 3 d after infection with recombinant AcNPVNP.
As estimated from SDS-PAGE analysis, NP represented
~10% of total soluble proteins 3 d after infection (Fig. 1 A,
lanes 1 and 2). We took advantage of the nucleocapsid formation in insect cells to purify them using centrifugation
on CsCl gradients as previously described for viral nucleocapsids isolation (30). The recombinant NP formed a dense
visible band in the 30% CsCl step gradient. This single-step
gradient purification was sufficient to obtain homogenously
purified NP that could be pelleted. Purified NP analyzed
by SDS-PAGE (Fig. 1 A, lane 3) migrated as two molecular species of about 60 kD; the larger one was predominant
and co-migrated with the viral NP (data not shown). Both
bands were recognized by mAb 25NP (Fig. 1 B, lane 3)
that recognized no polypeptide in insect cells infected with
wild-type AcNPV (Fig. 1 B, lane 1). The minor band
probably represents a cleavage product of the major one as
it became more abundant in cells harvested later. Under
these conditions, the purification yield was ~50% and resulted in the isolation of 1 mg of pure recombinant NP
from 108 infected insect cells.
Electron microscopy analysis (data not shown) confirmed the assembly of recombinant NP in nucleocapsids similar to measles nucleocapsids (33). These observations show that the insect cell expression system permits the production of large quantities of purified NP that preserve its native structure.
NP Binds to Human and Murine B Cells.Tonsillar human
B and T lymphocytes were incubated with variable amounts
of recombinant or viral NP and assayed for NP binding by
flow cytometry. Only B lymphocytes bound both recombinant and viral NP (Fig. 2, A and C). T lymphocytes
failed to do so (Fig. 2, B and D).
In the course of this study, we observed that the proportion of B cells susceptible to bind NP was variable and
ranged between 65 and 90%. Since different tonsil specimens were used for each experiment, this observation suggested that NP binding could be modulated after B cell activation. The data shown in Fig. 3 indicate that the level of
NP binding to anti-Ig-activated B lymphocytes (Fig. 3 B;
median 107.5) was significantly higher than for unstimulated B lymphocytes (Fig. 3 A; median 62.6). As summarized in Table 1, a similar increase in NP binding was also
observed in the human B lymphoblastoid cell line JY after
anti-Ig stimulation. In contrast, PHA-activated T lymphocytes or HUT78 lymphoma cells did not bind NP (Table
1). Moreover, Burkitt lymphoma or lymphoblastoid cell
lines were both able to bind NP (Table 1).
|
Binding studies were performed with the Cl.13 cell line,
a mutant cell line derived from JiJoye, that bound NP very
efficiently (Table 1) and biotinylated recombinant NP. NP
binding was saturable and the maximal binding capacity
derived from these experiments was 8 µg/5 × 105 cells
(133 pmol/5 × 105 cells; Fig. 4 A). Competition experiments indicated that a 25-fold excess of unlabeled NP was
required to inhibit, by 93%, biotinylated NP binding (Fig.
4 B). Altogether, these results indicate that the Cl.13 cell
line express a specific membrane receptor for NP.
To determine whether NP could be a superantigen, we tested if the NP binding was dependent on MHC class II molecule expression. As mentioned before, PHA-activated T cells or HUT78 cells, which both express MHC class II molecules, did not bind NP. Moreover, equivalent levels of NP binding were obtained using RJ2.2.5, RM2, and RM3 MHC class II-deficient mutants of the Raji cell lines as well as the wild-type MHC class II-expressing Raji (Table 1). These results clearly indicate that NP binding is independent of MHC class II expression.
Murine B lymphocytes and various murine B cell lines were then tested for their ability to bind NP. NP binding was observed to murine splenic B cells and to the B lymphoma cell line M12 (Table 1). In contrast, no significant NP binding was detected to splenic T cells or the T cell hybridoma 408 (Table 1).
Taken together, these findings indicate that the binding of NP to lymphocytes is: (a) specific and restricted to the B cell lineage, (b) increased after B cell activation, and (c) independent of MHC class II expression. The only exception was the murine IIA1.6 cell line derived from the B cell lymphoma A20 (Table 1).
FcAs IIA1.6 cells
are deficient for FcRII expression, we investigated whether
Fc
RII could be involved in NP binding. To test this hypothesis, murine splenocytes were incubated with recombinant NP in the presence or absence of mAb 2.4G2 recognizing murine Fc
RII. As shown in Fig. 5 B, the NP
binding was abolished by mAb 2.4G2. To further explore
the nature of the NP receptor expressed by B cells, two
variants of the IIA1.6 cell line expressing the murine
Fc
RIIb1 or b2 isoforms were used. As illustrated in Fig. 5,
expression of Fc
RIIb1 or b2 allowed IIA1.6 cells to efficiently bind NP. Moreover, the addition of mAb 2.4G2
inhibited NP binding to both transfectants by 86 and 88%
for the Fc
RIIb1 and b2, respectively. These results support
the notion that murine Fc
RII are specific NP receptors.
We next tested the ability of NP to bind to human FcRII
using IIA1.6 cell line expressing the human Fc
RIIa, b1*,
or b2 isoforms. As shown in Fig. 6, all Fc
RII transfectants
bind NP efficiently. Furthermore, the NP binding was inhibited by the CD32-specific mAb KB61 to IIA1.6 cells
expressing Fc
RIIa, b1*, or b2 (92, 89, and 81% inhibition
of NP binding, respectively). Similar binding experiments
performed on tonsillar B lymphocytes showed that mAb KB61 also inhibits the NP binding on normal B cells by up
to 76% (data not shown). Taken together, these observations demonstrate that both murine and human Fc
RII are
receptors for measles virus NP.
NP Inhibits the In Vitro Ig Synthesis.
It has long been known
that immune complexes can exert a negative feedback on B
cell responses via FcRII (34). The three-dimensional structure of the NP could induce cross-linking of the Fc
RII
expressed on B cells and mimics the antagonistic signal evoked
by immune complexes. To test this possibility, purified tonsillar B cells were costimulated with IL-2, IL-10, and either immobilized anti-Ig antibodies or a CD40L transfectant, in the
presence or absence of an optimal dose of recombinant NP.
The combination of IL-2 and IL-10 was chosen with regard
to its capacity to efficiently stimulate Ig synthesis (35). Determination of the polyclonal IgM, IgG, and IgA secretion
after a 12-d culture period was chosen as the read-out system.
As shown in Table 2, IL-2 and IL-10 elicited a strong
polyclonal antibody response both in the anti-Ig and in the
CD40L systems. Addition of NP in the cultures resulted in
a significant inhibition of the Ig response (ranging between
49 and 62%), irrespective of the isotype considered. This
finding suggests that the binding of NP to the FcRII
downmodulates the capacity of B cells to differentiate into
Ig-secreting cells.
In the present study, we have shown that viral or recombinant NP can bind to the surface of B cells. This NP binding is specific, restricted to the B cell lineage both in humans
and mice, increased after B cell activation, and does not require MHC class II expression. We also demonstrated that
the murine and human FcRII are receptors for measles virus NP and that the binding of NP inhibits the Ig synthesis
by activated B cells.
To produce large quantities of recombinant NP, we developed a baculovirus expression system. The recombinant NP exhibits similar biochemical and antigenic characteristics of viral NP. In agreement with Fooks et al. (36), we observed that in the absence of other viral proteins, NP could self-assemble into nucleocapsid-like structures very similar to those observed for measles nucleocapsids (data not shown and reference 33). Such particles were also observed in mammalian cells infected with recombinant vaccinia virus expressing NP (37). The recombinant NP thus presents a native structure suitable for binding and functional studies.
Binding studies using biotinylated NP showed a saturation of NP binding to Cl.13 cell line and a competition
with unlabeled NP indicating that NP binding on B cells is
specific. Rabies N protein was previously described as a
human viral superantigen that binds to the HLA class II chain (21). However, our results show that MV NP binding is independent of MHC class II expression, suggesting
that it is unlikely to behave as a superantigen. Consequently, NP is probably not directly involved in the alterations of the T cell repertoire observed after MV vaccination (20).
Among the various human or mouse B cell lines tested,
only one, the IIA1.6 mutant, did not bind NP efficiently. The
fact that this mutant does not express FcRII prompted us
to examine whether the low-affinity IgG receptor (Fc
RII;
CD32) could be a cellular receptor for NP. Human B cells
preferentially express Fc
RIIb and phagocytic cells Fc
RIIa
molecules. Two different Fc
RIIb isoforms are generated
by alternative splicing, IIb1 and IIb2, which are identical
except for a 19- or a 47-amino acid insertion in the cytoplasmic tail of the mouse or human IIb1 isoform, respectively. On mouse B cells, only the IIb1 form is expressed,
the IIb2 molecules being mainly expressed on myeloid
cells. Human and mouse IIb2 isoforms mediate endocytosis
of their ligands, whereas human IIa isoform is favoring
phagocytosis (10, 25). Fc
RIIa bears a 26-amino acid immunoreceptor tyrosine-based activation motif in its intracytoplasmic domain involved in stimulatory functions (38).
The immunoreceptor tyrosine-based activation motif is replaced by a 13-amino acid immunoreceptor tyrosine-based
inhibitory motif in the intracytoplasmic domain of Fc
RIIb
isoforms. This motif has been previously shown to be required for the inhibition of the antiimmunoglobulin-induced
B cell activation by immune complexes (10, 25). When
IIA1.6 cells transfected with expression vectors encoding
either for murine Fc
RIIb1 or b2 isoforms or human
Fc
RIIa, b1*, or b2 isoforms, all transfectants efficiently bound recombinant NP. This binding was specific since it
was inhibited by mAb directed against human or murine
Fc
RII. These results demonstrate that murine and human
Fc
RII are specific NP receptors. However, since the inhibition of NP binding with specific anti-Fc
RII antibody
was never complete and a very low NP binding could be detected to the IIA1.6 cells, we can not definitively exclude the presence of another cell-surface receptor for NP.
The mAb 2.4G2 blocks the interaction between murine
FcRII and immune complexes, but also inhibits the NP
binding to Fc
RII, thus suggesting that immune complexes and NP share the same binding site on Fc
RII. The
central core (amino acids 189-373) of NP is essential for
NP-NP interaction (39) and is probably not accessible for
Fc
RII interaction. By contrast, the NH2- or COOH-terminal regions seem to be accessible to antibodies directed
against NP (40), and thus could be involved in the interaction with Fc
RII. Several genotypes have been described
for MV, but variations between strains of a same genotype
is small. Moreover, for group A, comprising all the vaccine
strains like Hallé, no more than one nucleotide difference
was found in the nucleotide sequence encoding NP (41).
During the acute phase of measles infection, high amounts
of NP-specific antibodies are produced suggesting an interaction between the BCR and NP released from MV-infected cells. In this context, all APCs expressing FcRII
could efficiently internalize NP before any production of
specific anti-NP antibodies. This receptor-mediated endocytosis without opsonization could target NP to an endocytic compartment where formation of MHC class II-peptide occurs, and could explain the efficient CD4+ T helper cell
response against NP observed during MV infection.
Cross-linking of the BCR with either human FcRIIb1,
IIb2, or murine Fc
RIIb1 molecules results in inhibition of
antibody production. This inhibition involves the immunoreceptor tyrosine-based inhibitory motif of Fc
RIIb that
recruits phosphatases (42). It was originally thought that the
BCR and FcR have to be co-ligated for this mechanism of
inhibition. In the present study, we show that engagement
of the BCR is not required for the development of the
NP-mediated inhibition of Ig synthesis inasmuch as NP
downregulates the Ig response by CD40-activated B cells.
One plausible explanation is that the sole cross-linking of
FcR is sufficient to transduce a negative regulatory signal.
This hypothesis is compatible with the fact that the recombinant NP has an oligomeric structure thus susceptible to
efficiently cross-link the Fc
RII on B cells. It is noteworthy that no inhibition of B cell proliferation was observed in the presence of NP (data not shown). These results extend previous studies showing that antigen-antibody complexes bound to murine B lymphocytes via Fc
R inhibit
their antibody response, but not their proliferation in response to F(ab
)2 anti-µ and lymphokines (43, 44). Our data
thus support the notion that NP would rather interfere
with the plasma cell differentiation pathway. Determination of the exact nature of inhibitory signal(s) generated by
interaction between NP and Fc
RII will need further investigations. It was previously demonstrated that in vitro,
MV infection inhibits Ig secretion from PWM-stimulated
PBL cultures (45) and impairs the proliferative response of
B cells to mitogens (46). This inhibition affects the early
phase of B lymphocyte differentiation and could be due to
a blockade of the late G1 phase of the cell cycle (47). It is
thus probable that, besides NP, additional mechanisms operate during the MV-induced immunosuppression. In line
with such a hypothesis, recent data suggest that the MV H and F proteins could mediate inhibition of lymphocyte
proliferation in uninfected cells (48). In vivo, the downregulation of antibody production during measles infection
could involve two different mechanisms, one acting on B
cell proliferation, and the other one mediated by NP acting
on B cell differentiation in plasma cell.
During measles infection, defects in the responses of both
lymphocytes and monocytes have been reported (49). Measles virus infection of primary human monocytes leads to a
marked suppression of IL-12, some downregulation of IL-10,
and an unaltered TNF- and IL-6 production. The interaction between MV and its cellular receptor, the human
CD46, could inhibit IL-12 production (50). However, the
possibility that cross-linking of Fc
R by NP is involved in
modulating monokine production needs to be investigated.
It is very puzzling to note that viruses use molecules of the immune system for their entry, but also for the subversion of regulatory pathways. This subversion could not only implicate viral proteins of the envelope like H or F, but also internal viral protein such as MV NP that decreases in vitro Ig secretion. Other viral NP could perturb immune response as rabies and influenza NP. Rabies NP could act as a superantigen leading to T cell activation (21) and either clonal deletion or anergy. NP from influenza A virus inhibits polymorphonuclear neutrophils functions such as chemotaxis and superoxide production suggesting that NP could play a role in secondary bacterial infections observed in patients infected with influenza virus (51).
In conclusion, our present findings suggest that during
acute phase of measles virus infection, NP specifically targets to the FcRII-expressing cells and may play a role in
the MV-induced immunosuppression.
Address correspondence to Chantal Rabourdin-Combe, Immunobiologie Moléculaire, UMR 49, CNRS-ENS Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France. Phone: 33-4-72-72-80-16; FAX: 33-4-72-72-86-86.
Received for publication 11 April 1997 and in revised form 16 May 1997.
1 Abbreviations used in this paper: AcNPV, Autographa californica nuclear polyedrosis virus; BCR, B cell receptor; CD40L, CD40 ligand; F, fusion; FcWe thank Marie-Claude Biémont for technical help in construction of NP recombinant virus and Rob Ruigrock for electron microscopy analysis. We also thank Christian Bonnerot and Sebastian Amigorena for providing IIA1.6 cells transfected with murine FcRII, Jan G.J. Van de Winkel for providing IIA1.6 cells tranfected with human Fc
RII, and David Y. Mason for the KB61 antibody. We are grateful to Patrick
Bertolino, Patrice Dubois, and Jacqueline Marvel for helpful comments on the manuscript.
This work was supported by institutional grants from the Centre National de la Recherche Scientifique and Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, and by additional supports from Association pour la Recherche sur le Cancer (CRC 6108) and Ligue Nationale contre le Cancer (CRC). K. Ravanel is a recipient of a grant from the Fondation pour la Recherche Médicale.
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