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,
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*
Division of Developmental and Clinical Immunology, Departments of
Medicine,
Pediatrics, and
§
Microbiology, University of Alabama, and the
¶
Howard Hughes Medical Institute, Birmingham, AL 35294; and
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Washington University Center for Immunology and Department of Pathology, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ß, and -
, are cytokine family members
with multiple immune response roles, including the promotion of cell
growth and differentiation. Conversely, the type I IFNs are potent
inhibitors of IL-7-dependent growth of early B lineage progenitors,
effectively aborting further B lineage differentiation at the pro-B
cell stage. Type I IFNs and ß function via receptor-mediated
activation of a Jak/Stat signaling pathway in which Stat-1 is
functionally important, because many IFN-induced responses are
abrogated in Stat-1-deficient mice. To the contrary, we show here that
the inhibition of IL-7-dependent B lymphopoiesis by IFN-ß is
unaffected in Stat-1-deficient mice. The present data indicate that the
type I IFNs can activate an alternative signaling pathway in which
neither Stat-1 nor phosphatidylinositol 3'-kinase are essential
components. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5
(5). Productive light chain gene rearrangement occurs in
postmitotic pre-B cells (6), and the subsequent expression
of an IgM receptor characterizes the immature B cell
(B220+CD43-BP-1+IgM+).
Finally, coexpression of IgD begins when the newly formed B cells
migrate to the periphery (7).
IL-7, an essential growth factor for B and T lymphopoiesis in mice
(8), may also influence the rearrangement of B and TCR
genes (9, 10). The IL-7 receptor is composed by the
IL-7R-chain and the common 
-chain, which is shared by the IL-2,
IL-4, IL-9, and IL-15 receptors (11). In keeping with the
onset of IL-7 receptor expression in pro-B cells (9), B
cell development is blocked at this differentiation stage in mice
deficient in either IL-7 or its receptor (12, 13).
The type I IFNs, IFN-, -ß, and -, are members of a family of
pleiotropic cytokines that participate in antiviral responses as well
as in other physiological processes, such as cell growth and
differentiation (14). In earlier studies, we found that
the type I IFNs selectively inhibit IL-7 promoted growth of early B
lineage and T lineage cells, whereas having no effect on cell growth
induced by other cytokines (15, 16). The IFN-ß
inhibition of IL-7-dependent cells or cell lines is featured by the
induction of apoptosis. Because type I IFNs have been shown to be
constitutively produced by resident BM macrophages (15),
they could play a regulatory role in normal B cell development.
IFNs bind to their cell surface receptors to activate the Jak-Stat
signaling system (17, 18, 19). Janus family kinase 1 (Jak1)
and tyrosine kinase 2 (Tyk2) are closely related cytoplasmic tyrosine
kinases that constitutively associate with the - and ß-chains of
the type I IFN receptor (IFNAR). Receptor interaction with IFN- or
-ß promotes Jak1 and Tyk2 phosphorylation of tyrosine residues and
activates the signal activating and transcription factors Stat-1,
Stat-2, and Stat-3. The Stat proteins thereby acquire the capacity to
oligomerize whereupon they migrate into the nucleus and bind to
regulatory motifs in the promoter regions of many genes to modulate
their transcription (20). IL-7 signaling activates a
partially overlapping Jak-Stat signaling pathway. Both the Jak1 and
Jak3 kinases are activated after IL-7 receptor ligation, and these
kinases phosphorylate multiple substrates, including the Stat-1 and the
Stat-5 transcription factors (21). However, among the
multiple components of the type I IFN activation pathway, Stat-1
appeared essential in that all IFN-induced responses originally
examined were defective in Stat-1-deficient mice (22, 23). Nevertheless, this assumption can be questioned because
alternative IFN signaling pathways have been identified (14, 20)
The inhibition of B and T lymphopoiesis by type I IFNs could reflect cross-talk between the type I IFN and IL-7 signaling pathways (15, 16). As a first step in examining this interaction, we examined the effects of IFN on IL-7-mediated B lymphopoiesis in Stat-1-deficient mice. Contrary to our expectation, Stat-1 does not appear to be essential for the IFN inhibition of IL-7-promoted B lymphopoiesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FITC-labeled mAbs to S7/CD43, Sca-1 and IgD, PE-labeled Abs to
B220 and BP-1, CyChrome-labeled Abs to CD19 and µ heavy chains, and
biotin-labeled Abs to the IL-7R-chain were obtained from PharMingen
(San Diego, CA); streptavidin (SA)-CyChrome and SA-PE were obtained
from Southern Biotechnology Associates (Birmingham, AL). Cell
suspensions were incubated on ice for 25 min with the FITC-, PE-,
CyChrome-, or biotin-conjugated mAbs, washed with PBS containing 3%
FCS and 0.02% NaN3, and counterstained with
SA-CyChrome or SA-PE to reveal biotin conjugates. Before analysis with
a Becton Dickinson (Mountain View, CA) FACScalibur flow cytometer, 1
µg/ml propidium iodide in PBS was added to allow identification of
dead cells for exclusion from the analysis. The data was analyzed with
the WinMDI 2.8 (Trotter@scripps.edu) software program.
Mice, cell lines, and IFN
Stat-1- (22) and
Ifnar1-deficient mice (B&K Universal Group, North
Humberside, U.K.) were of 129Sv/Ev background. Although the type I IFN
receptor (IFNAR) is formed by two chains, IFNAR1 and IFNR2,
IFN-ß-induced responses were undetectable in
Ifnar1-/- mice (16).
Stat-1 mice were bred as -/- male x +/- female to
derive +/- and -/- littermates. BALB/c mice were bred at our animal
facility at the University of Alabama (Birmingham, AL). Four- to
6-wk-old animals were used as BM donors, and fetal liver cells were
obtained 15 days postcoitus. Stat-1 genotyping was conducted
on DNA samples from tails or whole embryos, employing a PCR-based
analysis with previously described primers (22).
The Scid7 IL-7-dependent cell line, a gift from Dr. S. I.
Nishikawa (Kyoto University, Kyoto, Japan), has a pro-B phenotype
(CD19+CD43+BP-1+µHC-)
and is sensitive to IL-7 deprivation and the inhibitory effect of
IFN-ß (15).
An IFN- and -ß (IFN-ß) combination isolated from
viral-infected cultures and control supernatants from mock-infected
cultures were purchased from Access Biomedical (San Diego, CA) and used
at an equivalent concentration of 103
U/ml.
Ex vivo B lymphopoiesis assay
BM mononuclear cells from 6- to 8-wk-old mice or fetal liver
mononuclear cells from 15-day-old embryos were cultured with
IL-7-transfected NIH3T3 fibroblasts (24) at a
concentration of 106 cells/ml in RPMI 1640 medium
with 5% FCS, L-glutamine, penicillin/streptomycin, and 50
µM 2-ME in the presence or absence of IFN-ß. Ly294002 and
wortmannin inhibitors were obtained from Calbiochem (La Jolla, CA).
Cultured cells were harvested by treatment with 0.02% EDTA, and viable
lymphoid cells were enumerated by phase microscopy on the basis of
trypan blue exclusion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abnormalities of B lymphopoiesis have not been noted in Stat-1-/- mice (22, 23), but a detailed analysis of B lineage development in these mice has not been reported. Therefore, in our initial studies, we examined B lineage cells for the expression of B220, CD19, CD43, BP-1, IgM, and IgD in BM samples from Stat-1-/- mice and their +/- littermate controls at 4–6 mo of age. This comparative analysis confirmed the expectation that B cell development occurs normally in Stat-1-deficient mice (data not shown).
Inhibition of B lymphopoiesis by type I IFNs in Stat-1-deficient mice
The possible role of Stat-1 in the IFN-ß-mediated inhibition
of B cell development was tested initially in an ex vivo system wherein
progenitor cells from adult BM or fetal liver are cultivated with
IL-7-transfected fibroblasts. In this ex vivo model,
B220- progenitor cells undergo proliferation and
progressive differentiation into IgM+ B cells,
whereas untransfected fibroblasts do not support B cell development
(data not shown). Contrary to our expectation, in these experiments,
the growth of B220+ B lineage cells was inhibited
by IFN-ß treatment regardless of the Stat-1 genotype.
Comparable reductions in the numbers of viable
B220+ cells were observed in IFN-treated cultures
of cells from the Stat-1+/- and
Stat-1-/- mice (Fig. 1
). Phenotypic analyses over the 7-day
culture interval further indicated comparable cellular composition of
the surviving cell populations (Fig. 2).
Notably, the development of µHC+ pre-B and B
cells was profoundly inhibited by IFN-ß regardless of whether the
progenitor cells were obtained from
Stat-1+/- or from
Stat-1-/- mice. The number of
B220- cells was also reduced by the IFN-ß
treatment, although the limited growth of non-B lineage cells in this
ex vivo system made it difficult to discern whether this was a primary
or secondary effect. Titration of the IFN dosage resulted in a
graduated decrease in the inhibition with lower concentrations, but
differential levels of inhibition were not evident for the
Stat-1+/- and
Stat-1-/- cells at any IFN concentration
(not shown). In keeping with previous results obtained for
IL-7-dependent cell lines (15), the levels of IL-7R
expression were not reduced after IFN-ß treatment, indicating that
the inhibition cannot be explained simply by deprivation of the IL-7
signal (not shown).
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ß inhibitory effect, we
examined progenitor cells from mice deficient in the type I IFN
receptor (Ifnar1-/-). In these
experiments, fetal liver cells from
Stat-1+/- and
Ifnar1-/- mice were cultured with the
IL-7-producing fibroblasts either in the presence or absence of
IFN-ß. Whereas IFN-ß inhibited the development of
Stat-1+/- progenitors, the development of
B lineage cells from the Ifnar1-/-
progenitors was unaffected by IFN-ß treatment (Fig. 3). These results indicate that the
IFN-ß inhibitory effect is mediated specifically via the type I
IFN receptor and cannot be attributed to a nonspecific toxin in the
IFN-ß preparation.
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ß-mediated up-regulation of
Sca-1 in early B lineage cells
The foregoing evidence indicating that Stat-1 is nonessential for
the type I IFN-induced inhibition of IL-7-induced B lymphopoiesis was
surprising, given that Stat-1 is essential for all of the IFN-ß
responses assessed in mature B cells (23). In view of this
paradox, we examined whether Stat-1 was essential for another type of
IFN response in the fetal liver progenitors, namely the up-regulation
of transcription and expression of the Sca-1 Ag (Ly-6A/E), a cell
surface accessory molecule expressed on BM and fetal liver cells
(25, 26). When we examined Sca-1 expression by early B
lineage cells, an IFN-induced up-regulation of this Ag could be easily
detected for cells from the Stat-1+/-
mice, but not for cells from the
Stat-1-/- animals. IFN-ß also failed
to enhance the expression of this Ag on cells from
Ifnar1-/- mice, thereby confirming the
IFN-ß specificity of this response (Fig. 4). Therefore, this type I IFN response
is Stat-1-dependent in B cell progenitors as is the case for
B cells.
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ß-mediated inhibition of B lymphopoiesis
PI 3'-kinase, which is involved in many signaling cascades that
influence cell growth, is also activated following type I IFN
stimulation (27, 28, 29). To examine its possible influence in
the growth inhibition induced by IFN-ß, we examined the role of PI
3'-kinase in the IFN-mediated inhibition of B lymphopoiesis. The
initial assessment was conducted by adding Ly294002, an inhibitor of PI
3'-kinase (30), to cultures of fetal liver cells from
BALB/c mice. Although relatively high concentrations of Ly294002 could
inhibit B lymphopoiesis, this PI 3'-kinase inhibitor had no
demonstrable counter effect on the IFN-mediated inhibition of cell
growth at any of the concentrations employed (Fig. 5A). The effect of this PI
3'-kinase inhibitor was also evaluated in experiments in which an
IL-7-dependent pro-B cell line, Scid7, was cultured with IL-7. In these
experiments, the Scid7 cells were inhibited by IFN-ß at all
inhibitor concentrations tested. Ly294002 alone was again inhibitory at
the highest concentrations employed, but had no demonstrable counter
effect on the IFN-ß-mediated inhibition (Fig. 5B).
Wortmannin, another inhibitor of PI 3'-kinase, was also tested in these
experiments with the same outcome (data not shown). The inhibitory
effect of Ly294002 alone, most evident at the highest concentration
employed (1 µM), can be attributed to the compromise of IL-7
signaling that involves PI 3'-kinase activity (10). If the
IFN-ß-mediated inhibition of cell growth also required PI
3'-kinase activity, an increase would be expected in the numbers of
cells treated with both IFN-ß and the inhibitor Ly290042. This
outcome was not observed at any Ly290042 concentration, thereby
suggesting that integrity of the PI 3'-kinase signaling pathway is not
essential for the type I IFN-mediated inhibition of B
lymphopoiesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß-mediated inhibition of B lymphopoiesis, in contrast with
previously described biological models wherein type I IFN-induced
growth inhibition was abolished in Stat-1-deficient mice and cell lines
(22, 23, 31).
Two major transcription factor complexes are formed in response to
ligation of the type I IFN receptor: IFN- activation factor (GAF)
and IFN-stimulated gene factor 3 (ISGF3) (17, 19, 20). GAF
is a Stat-1 homodimer, whereas ISGF3 is formed by activated Stat-1,
Stat-2, and p48 (32). Although Stat-1-deficient
mice cannot form GAF, the possibility of a hypothetical ISGF3 complex
lacking Stat-1 but still functional cannot be excluded for at least
some responses. In this regard, we found that the expression of
Sca-1, a gene containing targets for both GAF and ISGF3
factors in its promoter (26), was not up-regulated in B
cell progenitors following IFN treatment of
Stat-1-deficient mice.
The role of Stat-3 in type I IFN signaling is not as well documented as
that for Stat-1 in part because the Stat-3 knockout is early
embryonically lethal (33). Stat-3 may also be involved in
the antiviral and antiproliferative activities of type I IFNs through
up-regulation of NF-
B binding activity (34) and by
functioning as an adapter to couple PI 3'-kinase to the IFN receptor,
thereby activating a new signaling pathway (28, 29). PI
3'-kinase is also associated with IL-7 receptor signaling, and its
activity is required for the proliferative response of B cell
progenitors (10). The essential role of this signaling
component is evidenced by the block in B lymphopoiesis at the pro-B
cell stage in PI 3'-kinase-deficient mice (35, 36). Using
inhibitors of PI-3'-kinase, Ly294002 and wortmannin, we were unable to
alter the inhibitory effect of IFN-ß. Nevertheless, the
possibility of competition between IL-7 and IFN signaling pathways for
the available pool of PI 3'-kinase was not tested in these
experiments.
The indication that Stat-1 is not essential for the inhibition of B
lymphopoiesis by type I IFNs suggests that another, as yet
uncharacterized, transcription complex (lacking Stat-1) or a different
IFN signaling pathway is responsible for this inhibition of B cell
development. Interferons can inhibit the growth of embryonic
fibroblasts in a Stat-1-dependent fashion involving regulation of
c-myc expression. In this model system IFN- was recently
shown to suppress c-myc expression in cells from wild-type
mice, but not in those from Stat-1-/- mice
(37). Indeed, in the Stat-1-deficient cells both
c-myc and c-jun expression were rapidly
up-regulated in response to IFN-, thereby providing evidence for
Stat-1-independent signaling in cells of another differentiation
pathway.
As components of the innate immune system, IFNs are produced in response to external aggression (14). They also may be constitutively produced in a normal organism. IFN-ß is one of many lymphopoietic regulatory factors (reviewed in Ref. 38) produced locally in normal BM (15), where it may play a role in selection of the B cell repertoire (39). Dissociation between the IFN-induced antiviral and growth signaling pathways has also been described (40). The complexity of the IFN signaling cascades, together with the results described above, suggest an interesting versatility in the signaling components responsible for the rich diversity of IFN responses. Elucidation of the Stat-1-independent pathway that is activated in early B lineage cells via the type I IFN receptor may also reveal the mechanism whereby this signaling pathway counteracts with that of the IL-7 receptor.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Max D. Cooper, University of Alabama, WTI 378, 1824 6th Avenue South, Birmingham, AL 35294-3300.
3 Abbreviations used in this paper: BM, bone marrow; Jak, Janus-activated kinase; IFNAR, type I IFN receptor; SA, streptavidin; PI, phosphatidylinositol; GAF, IFN- activation factor; ISGF3, IFN-stimulated gene factor 3.
Received for publication February 4, 2000. Accepted for publication June 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß (IFN-/ß) and interferon--mediated induction of Ly-6E gene in B cells. Blood 92:2399.. J. Immunol. 158:2390.[Abstract]
and interferon . Proc. Natl. Acad. Sci. USA 93:7637.
. Nature 283:393.
subunit of phosphoinositide 3-kinase. Nature 283:390.
trough Stat-1 dependent and -independent
pathways. EMBO J. 19:263.
induced anti-viral and growth signaling pathways. J. Immunol. 162:3289.This article has been cited by other articles:
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