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Journal of Virology, August 1999, p. 7093-7095, Vol. 73, No. 8
Laboratory of
Immunopathology,1 National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892, and Department of Immunology and Infectious
Diseases,
Received 26 February 1999/Accepted 4 May 1999
The unique Gag polyprotein of the replication-defective virus
responsible for murine AIDS (MAIDS) induces B-cell activation, proliferation, and differentiation, including immunoglobulin class switch-recombination to immunoglobulin E (IgE). Secretion of IgE normally requires the serial induction of interleukin 4 (IL-4), engagement of the IL-4 receptor, activation of signal transducer and
activator of transcription (STAT) 6, and induction of I A murine retrovirus-induced
immunodeficiency syndrome, designated murine AIDS (MAIDS), develops
following infection of susceptible mice with a replication-defective
virus that encodes only a variant Pr60gag
polyprotein (1, 9, 15). The syndrome is characterized by
progressive lymphoproliferation and severe immune defects associated with enhanced susceptibility to infection. The mechanisms by which the
defective virus Gag induces disease are not known but may involve
aberrant activation of extra- or intracellular signaling pathways.
Although type 2 cytokines (interleukin 4 [IL-4] and IL-10) were once
considered possible driving forces in this disorder (2), recent results have shown that MAIDS develops normally in mice deficient in expression of IL-4 (12) and/or IL-10
(14). Unexpectedly, IL-4-deficient mice with MAIDS had
levels of immunoglobulin E (IgE) in serum comparable to those in
wild-type mice (13). Because IL-4 was previously thought to
be an absolute requirement for induction of IgE, this suggested that B
cells from IL-4-deficient infected mice were responding to signals
mimicking those activated by engagement of the IL-4 receptor (IL-4R).
The IL-4R consists of the IL-4R To determine the importance of STAT6 to induction of MAIDS and
expression of IgE in association with this disease, we infected STAT6 At 6 and 10 weeks after infection, wild-type, +/ In the FACS profiles, blast populations Immunodeficiency of increasing severity is a second prominent feature
of MAIDS (9, 15). Studies of proliferative responses to
lipopolysaccharide (LPS), concanavalin A (ConA), and phorbol myristate
acetate (PMA) plus ionomycin at 8 weeks after infection showed
similarly pronounced defects in
0022-538X/99/$04.00+0
STAT6-Deficient Mice Exhibit Normal Induction of
Murine AIDS and Expression of Immunoglobulin E following Infection with
LP-BM5 Murine Leukemia Viruses
ABSTRACT
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germline transcripts as a prelude to switching. Remarkably, expression of IgE is
equivalent in normal and IL-4-deficient mice with MAIDS (Morawetz et
al., J. Exp. Med. 184:1651-1661, 1996). To understand this anomaly, we
studied mice with a null mutation of STAT6. Lymphoproliferation and
immunodeficiency, the hallmarks of MAIDS, developed with comparable kinetics and degree in normal and mutant mice. In addition, serum IgE
levels were indistinguishable in mice of either genotype. We conclude
that B cells from mice with MAIDS activate unique IL-4- and
STAT6-independent signaling pathways for B-cell activation and differentiation.
TEXT
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chain and either of two additional
molecules, the IL-2R
common chain or the IL-13R chain (5,
16). Like other members of the hematopoietin receptor family,
IL-4Rs do not encode either tyrosine or serine-threonine kinases;
however, binding of IL-4 to the receptor complexes results in
activation of the Janus family tyrosine kinases, Jak-1 and Jak-3,
activation of two distinct signaling pathways by phosphorylation of an
insulin receptor substrate (IRS) designated IRS-1 or 4PS (8), and activation of the signal transducer and activator of transcription, STAT6 (7). Phosphorylated IRS-1 interacts with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) and mediates proliferative and antiapoptotic responses to
stimulation with IL-4 (20). Phosphorylated STAT6
translocates to the nucleus, binds to -activated sequences, and
activates transcription of several genes, including CD23 and the IgE
germline sequence, I (17). Studies of mice deficient in
STAT6 due to gene targeting in embryonic cells showed that STAT6 is
critical for induction of T helper 2 responses and production of IgE
following immunization with anti-IgD or infection with a nematode
(10, 18). Proliferative responses to IL-4 were moderately to
severely depressed in these mice, depending on the assay system
(10, 18).
/ (referred to hereafter by genotype
only) mice with LP-BM5 murine leukemia virus (MuLV). The
STAT6-deficient mice used in this study derived from targeted D3 ES
cells injected into BALB/c blastocysts. Heterozygous knockout mice were
crossed to generate mice homozygous for the Stat6 mutation
(10). These mice were crossed to C57BL/6 (B6) mice and
intercrossed. Progeny homozygous for H-2b and
the Stat6 mutation were identified and crossed. Progeny
homozygous for the Fv1b allele of B6 were
selected and intercrossed for infection with stocks of the LP-BM5 virus
mixture prepared from the G6 clone of SC-1 cells (4).
, and / mice were
found to have equivalent levels of lymphadenopathy and splenomegaly
(Table 1). Fluorescence-activated cell
sorter (FACS) (Fig. 1) and
histopathologic studies (not shown) revealed that mice of all three
genotypes had comparably advanced disease at these time points.
TABLE 1.
Development of MAIDS in STAT6
knockout micea
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FIG. 1.
FACS analyses of spleen cells from mice infected with
LP-BM5 viruses for 8 weeks. Cells were stained with the indicated
antibodies to cell surface antigens, and 2 × 105
viable cells, as determined by size and exclusion of propidium iodide,
were evaluated. Similar results were obtained with mice infected for 8 to 12 weeks. FSC, forward angle scatter.
indicated by increased forward
angle scatterwere seen along with reduced expression of
immunoglobulin kappa light chain and CD45R (B220) as signs of B-cell
activation in infected +/+, +/, and / mice; profiles of spleen
cells from uninfected +/ and / mice were indistinguishable from
those of +/+ mice (11). Interestingly, expression of CD23, a
gene regulated by IL-4 and STAT6, was almost completely inhibited in
all infected mice. The unusual Thy-1 CD4+
T-cell population expanded in MAIDS (6) was readily
detected. Expression of the IgG FcR was greatly increased, and the size of the CD11b+ population was expanded. Finally, mice of
each genotype showed almost identical profiles for CD69, CD25, CD62L,
CD44, NK1.1, GR-1, CD43, CD24a, IgD, Ia, and TCR-/
(data not shown).
/ and +/ mice (Fig. 2). It has been suggested that altered
expression of cytokines may contribute to this phenotype (3,
19). Semiquantitative reverse transcription-PCR analyses of
BM5def, I, and cytokine transcripts demonstrated comparable levels
of p12def expression and germline induction (data not shown).
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FIG. 2.
Proliferative responses of spleen cells to stimulation
with mitogens or PMA plus ionomycin. Cells (2 × 105)
from spleens of uninfected mice or mice infected for 8 weeks were
cultured for 48 h in triplicate in 96-well plates with ConA (2 µg/ml), LPS (20 µg/ml), or PMA (20 ng/ml) plus ionomycin (250 ng/ml). Cultures were pulsed with [3H]thymidine for the
last 6 h of culture. Data are average values ± 1 standard
error of the mean for three mice per time point. Similar results were
obtained in a second experiment. CPM, counts per minute.
We used an enzyme-linked immunosorbent assay (ELISA) (13) to
measure serum IgE levels in sera from mice infected for 3 to 10 weeks
(Fig. 3). Sera of uninfected +/+ mice
contained 5 ng of IgE/ml, while sera of uninfected / mice contained
less than 1 ng/ml. At 10 weeks after infection, the levels of IgE in
serum were statistically similar among all three groups of mice (data not shown). Digestion circularization-PCR analyses of spleens from
infected / and +/+ mice demonstrated the presence of the predicted
550-bp PCR fragment that hybridized with a specific C
membrane region probe (not shown), indicating that expression of IgE
was a consequence of deletional switch recombination.
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These results provide unequivocal evidence that STAT6 is not required for induction of MAIDS or for IgE switch recombination and secretion as part of this disorder. These findings exclude any possibility that IL-4-independent induction of IgE in MAIDS can be ascribed to an IL-4-like activity of the BM5-defective virus and demonstrate the involvement of a STAT6-independent pathway. It will be important to determine whether alterations in other transcription factors binding to the cis-controlling elements of the IgE locus are responsible for driving IL-4- and STAT6-independent high-level IgE expression.
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ACKNOWLEDGMENTS |
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We thank Brenda Marshall for excellent editorial assistance.
M.J.G. is a Scholar of the Leukemia Society of America. This work was supported in part by contract N01-AI-45203 at MA Bioservices, Inc. (Rockville, Md.), by NIH grant AI40171, and by a gift from the Mathers Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: LIP, NIAID, NIH, Building 7, Room 304, 9000 Rockville Pike, Bethesda, MD 20892-0760. Phone: (301) 496-6379. Fax: (301) 402-0077. E-mail: hmorse{at}atlas.niaid.nih.gov.
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