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Division of Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, MD 21224; and
Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
complexes. Therefore, to determine whether infection with MV can also
induce IgE class switching, we infected the human Ramos B cell line
with the Edmonston strain of MV. Infecting Ramos cells with MV did not
result directly in either the activation of dsRNA-activated protein
kinase or IgE class switching. However, a synergistic effect on IgE
class switching was observed when Ramos cells were infected with MV
before IL-4 treatment. Ab cross-linking of the MV receptor, CD46,
mimicked the effects of MV infection in synergizing with IL-4 to induce
IgE class switching, suggesting that viral hemagglutinin is involved in
this synergistic effect. These data provide the first indication of a
potential mechanism for MV-induced IgE up-regulation and suggest a
model for a viral-induced exacerbation of IgE-mediated disorders such
as asthma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
which induces human B cells to switch to produce IgG subtypes (except
for IgG4), the presence of IL-4 and IL-13 induces the production of IgE
and IgG4 (4, 5).
An early step in IgE class switching is the transcription of an
immature RNA from the C
region of the Ig gene (6). This transcript,
termed germline (G),3
is induced by IL-4 and to a lesser extent by IL-13 and cannot be
translated into mature IgE protein (7). However, the presence of this
transcript is essential for final switch recombination events leading
to the production of the mature IgE transcript (8). Therefore, the
expression of the G transcript is generally accepted as the first
step of IgE class switching.
In addition to cytokines, the physical contact between T and B cells
provides a second signal (costimulatory signal) that is necessary for
Ig class switching. This second signal, which is provided by surface
molecules such as CD40 and CD40 ligand (CD40L) (glycoprotein
39), is required for the activation of B cells (9). Several
other surface molecules, such as CD28/B7 and ICAM-1/LFA-1 participate
in T and B cell interaction and Ig class switching (10, 11). Also,
transmembrane-bound TNF-
, CD23/CD23 receptor, and LFA-3/CD2
reportedly cooperate with IL-4 for the production of IgE (12, 13, 14).
Although the exact nature of the signals induced by the T and B cell
contact is not yet clear, it is known that this interaction is
essential for Ig class switching to any isotype.
Although it is commonly accepted that viral infections lead to
increased Ab responses that are characterized by the IgG isotype, there
is increasing evidence that infections with several viruses can lead to
an expression of the IgE isotype (15, 16, 17, 18). One of the viruses
associated with a polyclonal increase in IgE expression is measles
virus (MV) (18). We reported previously that viral activation of
dsRNA-activated antiviral protein kinase (dsRNA-activated protein
kinase (PKR), protein kinase dsRNA-activated) and subsequent
activation of the NF-B complex in human B cells leads to an
induction of IgE class switching (19). Therefore, we determined whether
MV infection can also induce IgE class switching through the activation
of PKR. Infecting Ramos cells with MV alone did not lead to the
activation of PKR or to the induction of G, suggesting that an
alternative MV-induced mechanism may be responsible for the IgE
up-regulation. We now report that MV infection acts synergistically
with IL-4 to increase IgE class switching, and that this effect can be
mimicked by cross-linking of the MV receptor CD46.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human Burkitt’s lymphoma B cell line Ramos 2G6.4C10 was purchased from American Type Culture Collection (Manassas, VA). Cells (1 x 105–106/ml) were grown in RPMI 1640 medium supplemented with 10% FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and gentamicin sulfate at 5 µg/ml at 37°C in a 5% CO2 humidified chamber. The synthetic dsRNA polyinosinic:polycytidilic (poly(I:C)) was obtained from Sigma (St. Louis, MO); all other reagents were the highest quality available.
IFN treatment and in vitro kinase reactions
Ramos cells were treated with 100 U/ml of human IFN- (Lee
Biomolecular, San Diego, CA). After 24 h, cells were washed twice
with isotonic buffer containing 20 mM HEPES (pH 7.5), 120 mM KCl, 5 mM
MgOAc, and 1 mM DTT. Cells were then lysed in buffer containing 20 mM
HEPES, 120 mM KCl, 5 mM MgOAc, 1 mM benzamidine, 1 mM DTT, and 1%
Nonidet P-40.
Reactions were performed as described previously (20). Briefly,
mixtures for in vitro phosphorylation of cellular extracts contained 20
mM HEPES (pH 7.5), 90 mM KCl, 5 mM MgOAc, 1 mM DTT, 100 µM
[-32P]ATP (specific activity 1 Ci/mM) (Amersham,
Arlington Heights, IL), 100 µM ATP (Sigma), and equal amounts
of detergent extract prepared from 1 x 106 cells in a
final volume of 25 µl. dsRNA (poly(I:C)) was added to the reaction
mixtures at 1 µg/ml followed by incubation at 30°C. After 10 min,
the reactions were quenched by adding an equal volume of 2x SDS sample
buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS,
0.0125% bromophenol blue, and 5% 2-ME. After boiling for 2
min, the reduced, denatured proteins were then subjected to
electrophoresis through 10% SDS-PAGE. The labeled proteins were
visualized by autoradiography of the dried gels.
IL-4 treatment, viral infections, and Ab treatment
Ramos cells were treated with 5 ng/ml of human rIL-4 (Sigma). The Edmonston wild-type strain of MV at multiplicity of infection (MOI) of 2.5 plaque-forming units (PFU)/cell was used for infections of Ramos cells. Mouse anti-CD46 mAb (GB24) was kindly provided by Dr. Christopher L. Karp (Johns Hopkins University) and was originally obtained from Dr. John P. Atkinson (Washington University, St. Louis, MO); the mAb was used at the indicated concentrations. At 48 h after infection or Ab treatment, cells were harvested and total cellular RNA was extracted.
RNA extraction, detection of G, and quantitative analysis
RNA was isolated using the TRIzol total RNA isolation reagent
(Life Technologies, Gaithersburg, MD). After reverse transcription, the
cDNA was amplified in the presence of 2 µg/ml of primers, 100 µM
dNTPs, 0.25 U of Taq polymerase (Perkin-Elmer, Foster City,
CA), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM
MgCl2 and 0.001% gelatin in a final volume of 25 µl.
Primers for the C exon-derived sequence
(5'-AGAGGTCGGGCATTGGAGGGAATGT-3') and the G exon-derived
sequence (5'-AGGCTCCACTGCCCGGCACAGAAAT-3') described by Gauchat et al.
(21) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward
primer (5'-CACAGTCCATGCCATCACTG-3') and reverse primer
(5'-TACTCCTTGGAGGCCATGTG-3') were used in the PCR reactions. PCR was
performed in a DNA Thermocycler (Perkin-Elmer) for 42 cycles for G
and for 25 cycles for GAPDH. For restriction endonuclease mapping, the
210-bp PCR product corresponding to G cDNA was purified using the
QIAquick gel extraction kit (Qiagen, Chatsworth, CA). The purified
fragment was digested with BglI enzyme (Life Technologies)
for 2 h at 37°C, and the products were resolved on a 2% agarose
gel. For the detection of MV infection primers,
5'-TCAGTAATGATCTCAGCAACTG-3' and 5'-TTCAATGGTGCCCCACTCGGGA-3'
corresponding to viral hemagglutinin (HA) were synthesized and used in
PCR reactions. A 100-bp ladder (Life Technologies) was used to
provide m.w. markers.
To determine the relative increase of the G, the agarose gels were
visualized using an Ultraviolet Products digitizing camera (San
Gabriel, CA). Quantitation was then performed using National Institutes
of Health Image analysis software. The increase in G expression was
normalized against expression of the housekeeping gene GAPDH.
Electrophoretic mobility shift assays (EMSAs)
Cell extracts for EMSAs were prepared according to Schreiber et
al. (22). EMSAs were performed using a
[-32P]-end-labeled NF-B (from light chain)
consensus oligonucleotide (Promega, Madison, WI) and a custom
synthesized oligonucleotide corresponding to CAAAT/enhancer-binding
protein (C/EBP) and the STAT-6 response element of IgE germline
promoter (5'-CGCTGTTGCTCAATCGACTTCCCAAGA ACA-3') as described
previously (23). The reactions (20 µL) consist of 2 µL of nuclear
extract in buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 0.2 mM
EDTA, 10% glycerol, 40 µg/ml poly dI.C:dI.C, 0.05% Nonidet
P-40 (which was omitted for C/EBP-STAT-6 assays), and 0.5 µl of
labeled probe. After 30 min of incubation at 37°C, the protein/DNA
complexes were resolved on 4.5% nondenaturing polyacrylamide gel and
visualized by autoradiography of the dried gels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether MV-induced IgE up-regulation was due to the
activation of PKR, Ramos cells were infected with MV at an MOI of 2.5
PFU/cell. After 24 h, cells were harvested and detergent extracts
were prepared. In vitro kinase reactions were performed in the presence
or absence of 1 µg/ml of poly(I:C), which is a concentration of dsRNA
previously determined to be optimal for PKR activation in Ramos cells
(19). The data revealed that infecting Ramos cells with MV did not
induce or activate PKR. However, treating cells with IFN- did result
in an induction of PKR that was activated in the presence of dsRNA
(Fig. 1
A).
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B). This finding is in agreement
with previous studies showing that human B cells can support MV
replication (24). MV synergizes with IL-4 in IgE class switching
Our data showed that PKR was not activated by MV infection. To
determine whether MV infection could, nevertheless, induce IgE class
switching in human B cells by an alternative mechanism, Ramos cells
were infected with the Edmonston strain of MV at an MOI of 2.5
PFU/cell. At 48 h postinfection, cells were harvested and total
cellular RNA was extracted. RT-PCR was performed using primers specific
to G. Consistent with our data showing that MV infection does not
activate PKR, infecting the B cells with MV alone also did not result
in IgE class switching. Treating the cells with IL-4, a potent inducer
of IgE class switching, resulted in an induction of IgE class
switching, as evidenced by the expression of G (Fig. 2A). The identity of G was
confirmed by BglI digestion of the PCR product (data not
shown).
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was
determined by RT-PCR. Treating MV-infected cells with IL-4
resulted in a significant increase (
11-fold) in G expression over
IL-4 treatment alone. However, as noted above, MV infection alone did
not result in an induction of G (Fig. 2A).
Because MV infection did not directly induce or activate PKR, we
determined whether an alternative mechanism was involved in this
synergistic effect. It is known that CD46 is the receptor for MV and
that viral HA mediates the initial virus/cell interaction (25). It is
also known that Ab-mediated ligation of CD46 results in some biological
events that are similar to those induced by MV infection (26).
Therefore, we determined whether ligation of CD46 would provide a
costimulatory signal for IgE class switching. Ramos cells were treated
with 5 ng/ml of IL-4 and subsequently treated with anti-CD46 Ab.
After 48 h, the expression of G was determined by RT-PCR. The
data revealed that the addition anti-CD46 Ab to IL-4-treated Ramos
cells induced a significant increase (5.5-fold) in the expression of
G (Fig. 2C). Therefore, the effect of anti-CD46 Ab
treatment was similar to those observed with MV infection. It is
interesting to note that the anti-CD46-induced IgE class switching
costimulatory effect was less evident at 2 µg/ml than at 1 µg/ml.
This may be due to an increase in the monovalent association of Ig
molecules at higher concentrations.
These data suggest that the synergistic effect induced by MV infection may be mediated by ligation of CD46 by viral HA.
Activation of NF by IL-4 treatment, MV infection, and anti-CD46 treatment
Genetic knockout studies as well as C promoter studies have
shown that NF-B complexes and STAT-6 are involved in IgE class
switching (27, 28). Furthermore, the cooperation between NF-B and
STAT-6 binding sites in IgE up-regulation has been documented (29). To
determine whether IL-4 could activate STAT-6 in Ramos cells, we treated
the cells with 5 ng/ml of IL-4; at different times posttreatment, the
cells were harvested and extracts were prepared. Data from EMSAs showed
that a STAT-6-reactive complex was induced within 3 min after IL-4
treatment (Fig. 3A). The
identity of the polypeptide in the complex was determined by
competition assays using STAT-6- and C/EBP-specific probes (Fig. 3B).
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B activation in Ramos
cells, Ramos cells were infected with MV at an MOI of 2.5; whole cell
extracts were prepared after indicated times posttreatment. Data from
EMSAs showed that NF-B was activated upon MV infection (Fig. 3C). The maximal level of NF-B activation was observed at
24 h posttreatment. To determine whether anti-CD46 treatment
could also activate NF-B, we treated the cells with 1 µg/ml of
anti-CD46 Ab. The data revealed that, similar to MV infection,
treating Ramos cells with anti-CD46 resulted in a time-dependent
activation of the NF-B complex (Fig. 3D). However,
infection with MV or treatment with anti-CD46 did not result in
STAT-6 activation in Ramos cells (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction of several cell surface molecules such as CD40/CD40L, LFA-1/ICAM-1, and CD23 may be necessary for efficient IgE class switching (9, 10, 11). However, using flow cytometry experiments, our data revealed that MV infection of Ramos cells did not result in an increase in the surface expression of LFA-1, ICAM-1, or CD23 (data not shown). It is known that adhesion molecules may be activated without any detectable increase in surface expression; therefore, our data suggest but do not prove that up-regulation of these adhesion molecules is not involved in the MV-induced synergistic effect. It is also known that the costimulatory signal exerted by CD40/CD40L interaction coincides with homotypic aggregation of cells (30). Because our initial experiments showed that infecting Ramos cells with MV induced homotypic aggregation, we hypothesized that MV infections may provide a costimulatory signal for IgE class switching through other surface molecules.
MV gains entry into cells by attaching viral HA to its putative receptor CD46 (membrane cofactor protein) (25). This cell surface molecule is a member of a family of regulators of complement activation, and Ab cross-linking of CD46 has been shown to result in biological effects similar to MV infection (25, 31). Therefore, we determined whether MV-induced CD46 cross-linking was the mechanism of the observed synergistic effect. Cross-linking experiments revealed that Abs to CD46 mimicked the effects of MV infection, suggesting a molecular pathway for MV-induced IgE up-regulation. It is important to note that the synergistic effect observed by IL-4 and MV infection was consistently greater than that observed by IL-4 and anti-CD46. The exact reason for this difference is not clear; however, it is possible that in contrast to anti-CD46 Ab, which provides dimeric cross-links, the presence of MV HA protein on the surface of infected cells provides multimeric cross-linking of CD46 molecules. It is important to note that previous in vivo studies have shown that monocytes are the major MV-infected cell population (32). Therefore, during in vivo MV infections, the IgE class switching costimulatory effect may be provided by the interaction of infected monocytes with B cells. Furthermore, it has recently been reported that, in addition to CD46, another cell surface molecule may be used by MV for cell entry (33), suggesting that the cross-linking of other surface molecules may contribute to this synergistic effect.
It is also known that MV infection leads to an elaboration of IL-4 (33). IL-4 is a potent inducer of IgE class switching; however, the increase in IgE that is seen during MV infection is probably not due to an elaboration of IL-4, because IL-4 was induced in vivo as a secondary response to MV infection (34). Furthermore, in our in vitro assays, MV infection alone did not result in IgE class switching, nor did it result in expression of IL-4 mRNA (data not shown).
Promoter studies on the transcriptional events leading to IgE
expression have shown that activation of both NF-B and STAT-6
molecules is necessary for the efficient induction of IgE class
switching, as detected by expression of G (27, 28). The data from
our EMSA experiments showed that STAT-6 was activated within 3 min
after IL-4 treatment of Ramos cells, and that infecting Ramos cells
with MV induced activation of the NF-B complex. Therefore, we
believe that the observed synergistic effect is mediated by activation
of the two necessary IgE regulatory NF, namely STAT-6 and NF-B (29).
However, at the present time, we cannot exclude the involvement of
other NF that are involved in IgE class switching, such as B
cell-specific activating protein and C/EBP (35, 36).
In addition to infection with MV, IgE levels are elevated in infections with respiratory syncytial virus, influenza virus, and members of picornaviruses and flaviviruses (15, 16, 17, 18, 37, 38). Based on our previous data, we believe that viral infections that lead to the activation of PKR can directly induce IgE class switching and consequently increase the risk of primary induction of IgE-mediated disorders such as asthma and allergy. However, infections with viral strains that can cross-link relevant B cell surface molecules and synergize with IL-4 may also lead to exacerbation of IgE-mediated disorders. If so, viral infections that can activate PKR and cross-link costimulatory surface receptors will provide the strongest signal for the induction and exacerbation of IgE-mediated disorders. Further studies are needed to test this concept.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Farhad Imani, Division of Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address:
3 Abbreviations used in this paper: G, germline ; CD40L, CD40 ligand; MV, measles virus; PKR, dsRNA-activated protein kinase; poly(I:C), polyinosinic:polycytidilic acid; MOI, multiplicity of infection; PFU, plaque-forming units; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; EMSA, electrophoretic mobility shift assay; CEBP, CAAAT/enhancer-binding protein.
Received for publication July 30, 1998. Accepted for publication October 21, 1998.
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transcripts in B lymphoid cells. J. Exp. Med. 168:2385. RNA synthesis in human B cells by tumor necrosis factor-, anti-CD40 monoclonal antibodies, or transforming growth factor-ß correlates with levels of IgE production. Int. Immunol. 4:397.
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promoter mediated by STAT/6 and C/EBPß. J. Immunol. 161:1822.This article has been cited by other articles:
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  S. R. Permar, D. E. Griffin, and N. L. Letvin Immune Containment and Consequences of Measles Virus Infection in Healthy and Immunocompromised Individuals Clin. Vaccine Immunol., April 1, 2006; 13(4): 437 - 443. [Full Text] [PDF]
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M. Kurita-Taniguchi, A. Fukui, K. Hazeki, A. Hirano, S. Tsuji, M. Matsumoto, M. Watanabe, S. Ueda, and T. Seya Functional Modulation of Human Macrophages Through CD46 (Measles Virus Receptor): Production of IL-12 p40 and Nitric Oxide in Association with Recruitment of Protein-Tyrosine Phosphatase SHP-1 to CD46 J. Immunol., November 1, 2000; 165(9): 5143 - 5152. [Abstract] [Full Text] [PDF]
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H. Matsuse, A. K. Behera, M. Kumar, H. Rabb, R. F. Lockey, and S. S. Mohapatra Recurrent Respiratory Syncytial Virus Infections in Allergen-Sensitized Mice Lead to Persistent Airway Inflammation and Hyperresponsiveness J. Immunol., June 15, 2000; 164(12): 6583 - 6592. [Abstract] [Full Text] [PDF]
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 A. Hirano, Z. Yang, Y. Katayama, J. Korte-Sarfaty, and T. C. Wong Human CD46 Enhances Nitric Oxide Production in Mouse Macrophages in Response to Measles Virus Infection in the Presence of Gamma Interferon: Dependence on the CD46 Cytoplasmic Domains J. Virol., June 1, 1999; 73(6): 4776 - 4785. [Abstract] [Full Text]
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 M. J. Ludford-Menting, S. J. Thomas, B. Crimeen, L. J. Harris, B. E. Loveland, M. Bills, S. Ellis, and S. M. Russell A Functional Interaction between CD46 and DLG4. A ROLE FOR DLG4 IN EPITHELIAL POLARIZATION J. Biol. Chem., February 1, 2002; 277(6): 4477 - 4484. [Abstract] [Full Text] [PDF]
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