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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1370-1377
IMMUNOBIOLOGY
Both Stat5a and Stat5b are required for antigen-induced
eosinophil and T-cell recruitment into the tissue
Shin-ichiro Kagami,
Hiroshi Nakajima,
Kotaro Kumano,
Kotaro Suzuki,
Akira Suto,
Kazunori Imada,
Helen W. Davey,
Yasushi Saito,
Kiyoshi Takatsu,
Warren J. Leonard, and
Itsuo Iwamoto
From the Department of Internal Medicine II, Chiba University School
of Medicine, Chiba, Japan; Laboratory of Molecular Immunology, National
Heart, Lung, and Blood Institute, Bethesda, MD; AgResearch, Ruakura,
Hamilton, New Zealand; and Department of Immunology, Institute of
Medical Science, University of Tokyo, Japan.
 |
Abstract |
Antigen-induced eosinophil recruitment into the airways of
sensitized mice is mediated by CD4+ T cells and their
cytokines, especially IL-5. In this study, we found that the
antigen-induced airway eosinophilia was diminished in Stat5a-deficient
(Stat5a /) mice and Stat5b-deficient
(Stat5b/) mice. We also found that antigen-induced
CD4+ T-cell infiltration and IL-5 production in the
airways were diminished in Stat5a/ mice and
Stat5b/ mice. Moreover, antigen-induced proliferation
of splenocytes was diminished in Stat5a/ mice and
Stat5b/ mice, suggesting that the generation of
antigen-primed T cells may be compromised in Stat5a/
mice and Stat5b/ mice and this defect may account for
the diminished antigen-induced T-cell infiltration into the airways.
Interestingly, IL-4 and IL-5 production from anti-CD3-stimulated
splenocytes was diminished in Stat5a/ mice and
Stat5b/ mice. However, antigen-specific IgE and IgG1
production was diminished in Stat5a/ mice but not in
Stat5b/ mice, whereas antigen-specific IgG2a
production was increased in Stat5a/ mice, suggesting
the enhanced Th1 responses in Stat5a/ mice. Finally,
we found that eosinophilopoiesis induced by the administration of
recombinant IL-5 was also diminished in Stat5a/ mice
and Stat5b/ mice. Together, these results indicate
that both Stat5a and Stat5b are essential for induction of
antigen-induced eosinophil recruitment into the airways and that the
defects in antigen-induced eosinophil recruitment in
Stat5a/ mice and Stat5b/ mice
result from both impaired IL-5 production in the airways and diminished
IL-5 responsiveness of eosinophils.
(Blood. 2000;95:1370-1377)
© 2000 by The American Society of Hematology.
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Introduction |
Allergic late-phase reactions provoked by specific
antigens are associated with intense eosinophil infiltration into the
site of antigen administration.1-3 In addition to the
infiltration of eosinophils, there is an increase in CD4+ T
cells and interleukin-5 (IL-5)-producing cells at the sites of
late-phase reactions,4,5 suggesting that CD4+ T
cells and IL-5 might be involved in antigen-induced eosinophil recruitment into the tissue. In a murine model of airway late-phase reaction, we and others have provided direct evidence that
CD4+ T cells and IL-5 mediate antigen-induced eosinophil
recruitment into the tissue of sensitized mice.6,7
Conversely, endogenous IFN- production down-regulates
antigen-induced eosinophil recruitment into the airways by inhibiting
CD4+ T-cell infiltration.8 Taken together,
these observations suggest that antigen-induced eosinophil recruitment
into the airways is regulated by the balance between T helper 1 (Th1)
and T helper 2 (Th2) cells, in which Th2 cells up-regulate but Th1
cells down-regulate the eosinophil recruitment. Although it is apparent
that IL-5 plays an important role for antigen-induced eosinophil
recruitment into the tissue, it remains unclear which signaling
molecules mediate antigen-induced, IL-5-dependent eosinophil
recruitment into the tissue.
IL-5 has been reported to activate a number of kinases, including Jak2,
Lyn, Syk, and Raf-1, as well as the phosphatase, Shp2.9-12 Among these molecules, Jak2 has been shown to be essential for eosinophil development13 and for the prevention of mature
eosinophil apoptosis.10 In addition, administration of Jak2
inhibitor, AG490,14 prevents antigen-induced eosinophil
recruitment into the airways (Kumano et al, in preparation), suggesting
that Jak2 activation is required for IL-5-induced tissue eosinophilia.
However, the interpretation of these experiments with AG490 must be
evaluated with caution because AG490 inhibits Jak3 as well as
Jak2.15
In the IL-5 signaling pathway, the most well-studied signaling
molecules downstream of Jak2 are Stat5a and Stat5b, 2 highly related
signal transducers and activators of transcription (STAT proteins).16-18 STAT proteins are cytosolic latent
transcription factors that are rapidly activated after cellular
exposure to interferons, cytokines, or growth factors.16-18
Stat5 was originally identified as a mammary gland factor
(MGF)-induced by prolactin.19 Subsequently, this protein
was renamed Stat5a when a second, homologous gene, denoted Stat5b, was
identified.18 Both Stat5a and Stat5b are activated not only
by prolactin, but also by a very wide range of other cytokines,
including IL-5.20 Although Stat5a and Stat5b are highly
homologous, the specificities of their actions are demonstrated by the
observations that Stat5a-deficient (Stat5a/)
mice exhibit defective prolactin-related functions, with impaired lobuloalveolar outgrowth of mammary epithelium during pregnancy, resulting in defective lactation,21 whereas
Stat5b-deficient (Stat5b/) mice exhibit
defective growth similar to that found in Laron dwarfism.22
In addition, Stat5a/ and
Stat5b/ mice are also immunologically
different each other. For example, basal as well as IL-2- and
IL-15-mediated boosting of NK cytolytic activity is greatly diminished
in Stat5b/ mice but only partially diminished
in Stat5a/ mice.23 Moreover,
although Stat5a/ splenocytes exhibit only a
partial defect in anti-CD3-induced proliferation that can be largely
overcome by high-dose IL-2,24 defective proliferation in
Stat5b/ splenocytes cannot be corrected by
this treatment.23 These different phenotypes underscore the
distinctive roles of Stat5a and Stat5b. However, Stat5a and
Stat5b may also have overlapping functions because Stat5a/Stat5b
double-deficient mice exhibit a severe defect in T-cell
proliferation.25
Although IL-5 is essential for antigen-induced eosinophil
recruitment into the airways6,7 and IL-5 activates
Stat5a and Stat5b,20 the role of Stat5a and Stat5b in
allergic inflammation remains unclear. Therefore, we have now analyzed
the allergic properties of Stat5a/ and
Stat5b/ mice. Given the role of IL-5 in
eosinophil development7,26,27 and allergic
inflammation,6,7 we were particularly interested in
analyzing whether the allergic inflammation is normally induced and
whether IL-5 induces eosinophilopoiesis in
Stat5a/ mice and
Stat5b/ mice. We found that antigen-induced
eosinophil and CD4+ T-cell recruitment was severely
impaired in Stat5a/ mice and
Stat5b/ mice. Furthermore, IL-5-induced
eosinophilopoiesis was also impaired in
Stat5a/ mice and
Stat5b/ mice. The implications of these
findings are discussed.
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Materials and methods |
Mice and genetic analysis
As the magnitude of antigen-induced eosinophil recruitment into the
airways differs depending on a genetic background of mice (our
unpublished data), Stat5a-deficient mice
(Stat5a/, ref. 21) and Stat5b-deficient mice
(Stat5b/, ref. 22) were back-crossed to
BALB/c mice (Charles River Laboratories, Atsugi, Japan) for 4 generations. All mice were H-2d/d and littermate wild-type
mice were used as a control. In preliminary experiments, we found that
the littermate control wild-type mice exhibited indistinguishable
responses to those seen with BALB/c mice in our assays; antigen-induced
eosinophil and T-cell recruitment into the airways, titer of
antigen-specific IgE antibody, and in vitro cytokine production and
proliferation of splenocytes (data not shown). Mice were housed in
microisolator cages under pathogen-free conditions. All experiments
were performed according to the guidelines of Chiba University. The
mice were genotyped by PCR of tail DNA, using the following
primer pairs: To detect Stat5a wild-type gene,
5'-CTTTATTGATAACGATCTATCCCTCACCC-3' and 5'-CTCCACTCTGCAGAGTCTATGGAATCC-3'; to detect
Stat5a-deficient gene, 5'-GCTGACAGCCGGAACACGGCGG-3' and
5'-GTGCAATCCATCTTGTTCAATGGCCG-3'; to detect Stat5b
wild-type gene, 5'-CAGGAGGGATCCAGTGCCAGC-3' and 5'-TGGCTCTACAGTGAGTTTGGT-3'; to detect Stat5b-deficient
gene, 5'-GACTTGGAGATTGCCAACCCATATCTAAGT-3' and
5'-TGAGCCGAAGGTGTAGTCGGAGTTTGCATT 3'.
Immunization
Mice (age 7-8 weeks) were immunized intraperitoneally twice with 4 µg of ovalbumin (OVA) (Sigma Chemical Co, St. Louis, MO) in 4 mg of
aluminum hydroxide at a 2-week interval. Twelve to 14 days after the
second immunization, the sensitized mice were challenged with
aerosolized OVA as described below.
Antigen-induced eosinophil and T-cell infiltration in mouse
airways
The eosinophil infiltration into the airways was induced by the
inhalation of antigen in sensitized mice, and the number of eosinophils
infiltrating into the submucosal tissue of trachea was evaluated as
described previously.6 Briefly, the sensitized mice were
given aerosolized OVA (50 mg/mL) dissolved in 0.9% saline by a
DeVilbiss 646 nebulizer (DeVilbiss Corp, Somerset, PA) for 20 minutes.
As a control, 0.9% saline alone was administered by the nebulizer. At
24 or 48 h after the inhalation, the tracheas were excised, fixed in
10% buffered-formalin, and embedded in paraffin. The specimens (3 µm
thick) were stained with Luna solution and hematoxylin-eosin solution.
The number of eosinophils in the submucosal tissue of trachea was
counted in Luna-stained sections and expressed as the number of
eosinophils per the length of the basement membrane of trachea, which
was measured with a digital curvimeter.
The eosinophil and T-cell infiltration into the bronchoalveolar lavage
fluid (BALF) was also evaluated as described previously.28 In short, bronchoalveolar lavage was performed with 1.2 mL of phosphate-buffered saline (PBS) at 24 or 36 hours after saline or OVA
inhalation. BALF was centrifuged at 400g for 5 minutes at
4°C, and cell differentials were determined by counting 500 cells
stained with Wright-Giemsa solution. A fraction of the cells were
subjected to a flow cytometric analysis for the lymphocyte surface
phenotyping of CD4 and CD8, as described below.
IL-5 levels in BALF
The BALF was centrifuged at 400g for 5 minutes at 4°C,
and the amount of IL-5 in the supernatant was measured by the enzyme immunoassay, according to the manufacturer's instruction (PharMingen, San Diego, CA). The assays were performed in duplicate.
Antigen-induced cytokine production and proliferation in
splenocytes
The spleen was removed from OVA-sensitized mice and a single cell
suspension of splenocytes was prepared. Splenocytes
(2 × 105) were then suspended in 200 µL of RPMI
1640 medium supplemented with 10% FCS (GIBCO BRL, Rockville, MD), 10 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin and
were cultured in triplicate in the absence or presence of OVA (200 µg/mL) in a 96-well microtiter plate for 72 hours. In some
experiments, splenocytes (2 × 105) were also
cultured for 72 hours in a 96-well microtiter plate coated with 5 µg/mL of anti-CD3 mAb (145-2C11, PharMingen). The culture
supernatant was collected and the amounts of IL-4, IL-5, and IFN-
were determined by enzyme-linked immunosorbent assay (ELISA) according
to the manufacturer's instructions (PharMingen). For proliferation
assays, splenocytes (2 × 105) were cultured in the
same conditions as described previously, with 1 µCi (0.037 MBq)
3H] thymidine added for the final 16 hours.
Determination of antigen-specific IgE antibody in serum
Two weeks after the second immunization, the titer of OVA-specific
IgE antibody in mouse serum was assessed by a 24-hour passive cutaneous
anaphylaxis (PCA) reaction as described by Ovary.29
Determination of antigen-specific IgG1 and IgG2a antibodies in
serum
The amount of OVA-specific IgG1 and IgG2a in serum was measured by
ELISA as described elsewhere.30 In brief, ELISA plates were
coated with OVA (250 µg/mL), washed 3 times with PBS containing 0.05% Tween20 (PBST), and blocked with blocking buffer (PBS containing 2% bovine serum albumin [BSA; Sigma Chemical Co]). Serum samples were added to the wells after 1:1000 or 1:3000 dilution in blocking buffer. As a control, serial dilution of pooled serum from
OVA-sensitized wild-type mice were analyzed in each plate. After 1-hour
incubation, wells were washed with PBST, added either biotinylated
antimouse IgG1 (PharMingen) or biotinylated antimouse IgG2a
(PharMingen) at 2 µg/mL in blocking buffer, and incubated for 1 hour.
After washing, wells were incubated with 100 µL of ExtrAvidin
alkaline phosphatase (1:2000 dilution, Sigma Chemical Co, St Louis, MO) for 45 minutes, washing with PBST, and the reaction was developed with
pNPP (Sigma Chemical Co).
IL-5-induced eosinophilopoiesis
IL-5-induced eosinophilopoiesis was analyzed as described
previously.26 In brief, mice were injected
intraperitoneally with 20 000 U of recombinant murine IL-5
(rmIL-5)26 daily for 4 days. Forty-eight hours after the
fourth administration of rmIL-5, mice were killed and cell
differentials in bone marrow, peripheral blood, and peritoneal cavity
were determined by counting 500 cells stained with Wright-Giemsa
solution. A part of the cells in the peritoneal cavity were subjected
to a flow cytometric analysis for the surface phenotyping of VLA-4 and
Gr-1 as described below.
Flow cytometric analysis
Cells from the BALF and peritoneal cavity were stained and analyzed
on a FACScaliber (Becton Dickinson, San Jose, CA) with CELLQuest
software.24 For direct staining, the following
conjugated antibodies were purchased from PharMingen: anti-CD4 FITC
(H129.19), anti-CD8 APC, 53-6.7 anti-VLA-4 FITC (R1-2), and anti-Gr-1
APC (RB6-8C5). Before staining, Fc receptors were blocked with
anti-CD16/32 antibody (2.4G2, PharMingen).
Data analysis
Data are summarized as mean ± SD. The statistical analysis of
the results was performed by the unpaired t test. P
values < .05 were considered significant.
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Results |
Antigen-induced eosinophil recruitment into the airways is
diminished in Stat5a/ mice and
Stat5b/ mice
Because Stat5 is activated in response to IL-520 and
IL-5 is known to be vital for antigen-induced eosinophil recruitment into the airways,6 we first determined whether
antigen-induced eosinophil recruitment into the airways was normal in
Stat5a-deficient (Stat5a/) mice and in
Stat5b-deficient (Stat5b/) mice. As shown in
Figure 1A, antigen-induced eosinophil
infiltration into the trachea at 24 hours after antigen inhalation was
severely diminished in both Stat5a/ mice and
Stat5b/ mice by 85% and 84%, respectively
(wild-type mice 17.2 ± 5.2, Stat5a/
mice 2.5 ± 1.4, and Stat5b/ mice
2.8 ± 1.0 eosinophils/mm, mean ± SD, n = 8 mice in each group, P < .001). Antigen-induced eosinophil infiltration
into the trachea at 48 hours was also significantly diminished in
Stat5a/ mice and
Stat5b/ mice (data not shown). Consistent
with diminished antigen-induced eosinophil infiltration in the trachea,
the number of eosinophils recovered in BALF at 36 hours after antigen
inhalation was also significantly decreased in
Stat5a/ mice and
Stat5b/ mice by 70% and 74%, respectively
(n = 5, P < .005) (Figure 1B).
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| Fig 1.
Antigen-induced eosinophil infiltration into the airways
is diminished in both Stat5a/ mice and
Stat5b/ mice.
(A) OVA-sensitized Stat5a/ mice,
Stat5b/ mice, and littermate wild-type mice
were challenged with the inhalation of OVA or saline (as control), and
the number of eosinophils infiltrating into the submucosal tissue of
trachea was evaluated at 24 hours after the inhalation. Data are means ± SD for 8 mice in each group. The mean values in
Stat5a/ mice and
Stat5b/ mice are significantly different from
the mean value of wild-type mice, *P < .001. (B) Similar to
A, the number of eosinophils in BALF was evaluated at 36 hours after
the inhalation. Data are means ± SD for 5 mice in each group.
*P < .005.
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Antigen-induced T-cell recruitment into the airways is diminished in
Stat5a/ mice and
Stat5b/ mice
FACS analysis of BALF cells revealed that antigen-induced
CD4+ T-cell infiltration into the airways at 24 hours was
also decreased in both Stat5a/ mice and
Stat5b/ mice by 64% and 61%, respectively
(n = 5, P < .005) (Figure 2). In addition, consistent with diminished CD4+ T-cell
infiltration in the airways, IL-5 levels in the BALF at 24 hours after
antigen challenge were decreased in Stat5a/
mice and Stat5b/ mice by 57% and
59%, respectively (wild-type mice 549 ± 98,
Stat5a/ mice 239 ± 90, and
Stat5b/ mice 227 ± 86 pg/mL, n = 5,
P < .005) (Figure 3). Taken
together, these results suggest that both Stat5a and Stat5b are
required for antigen-induced eosinophil and T-cell recruitment into the airways and that diminished antigen-induced eosinophil recruitment in
Stat5a/ mice and
Stat5b/ mice results in part from impaired
IL-5 production in the airways.
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| Fig 2.
Antigen-induced T-cell infiltration into the airways is
diminished in Stat5a/ mice and
Stat5b/ mice.
OVA-sensitized Stat5a/ mice,
Stat5b/ mice, and littermate wild-type mice
were challenged with OVA or saline as described in Figure 1. The T-cell
infiltration in BALF was examined at 24 hours after OVA or saline
inhalation. Shown are representative FACS profiles of CD4 versus CD8
staining on BALF cells using anti-CD4-FITC and anti-CD8-APC
(n = 5 mice in each group).
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| Fig 3.
Antigen-induced IL-5 production in BALF is decreased in
Stat5a/ mice and Stat5b/ mice.
OVA-sensitized mice were challenged with inhaled OVA. At 24 hours after
OVA inhalation, IL-5 levels in the BALF were determined by ELISA. Data
are means ± SD for 5 mice in each group. IL-5 in the BALF was
undetectable in mice that were challenged with inhaled saline (data not
shown). *Significantly different from the mean value of wild-type mice,
*P < .005.
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Antigen-induced IL-4 and IL-5 production is decreased in
Stat5a/ mice and
Stat5b/ mice
We then examined the Th1 cell and Th2 cell development in
Stat5a/ mice and
Stat5b/ mice. As shown in Table
1, IL-4 and IL-5 production from antigen (OVA)-stimulated splenocytes was undetectable in
Stat5a/ mice and
Stat5b/ mice, whereas antigen-stimulated
wild-type splenocytes produced substantial amounts of IL-4 and IL-5
(n = 5, P < .01). Similarly, IFN- production from
antigen-stimulated splenocytes was undetectable in
Stat5a/ mice and
Stat5b/ mice but was readily detected in
antigen-stimulated wild-type splenocytes (n = 5, P < .01)
(Table 1).
Although similar numbers of T cells were found in splenocytes from
wild-type mice, Stat5a/ mice, and
Stat5b/ mice (data not shown), it was
possible that antigen-specific T cells were decreased in
Stat5a/ mice and
Stat5b/ mice. To determine whether the
defects in cytokine production from antigen-stimulated splenocytes in
Stat5a/ mice and
Stat5b/ mice resulted from the impaired
generation of an antigen-specific T-cell pool, we analyzed cytokine
production from anti-CD3-stimulated splenocytes. IL-4 production from
anti-CD3-stimulated splenocytes was approximately 90% reduced in both
Stat5a/ mice and
Stat5b/ mice, compared with IL-4 production
in wild-type mice (n = 5, P < .001) (Table 1). IL-5
production from anti-CD3-stimulated splenocytes was also similarly
decreased in Stat5a/ mice and
Stat5b/ mice by approximately 80% (n = 5,
P < .001) (Table 1). In contrast, IFN- production from
anti-CD3-stimulated splenocytes was normal in
Stat5a/ mice and but moderately diminished in
Stat5b/ mice by 41% (n = 5,
P < .01) (Table 1).
Antigen-induced T-cell proliferation is decreased in
Stat5a/ mice and
Stat5b/ mice
Given the importance of IL-2 in T-cell proliferation31
and the ability of IL-2 to potently activate Stat5a and
Stat5b,32-34 we next examined whether antigen-induced
proliferation of splenocytes was normal in these mice. Antigen-induced
proliferation of splenocytes was substantially observed in wild-type
mice, whereas antigen-induced proliferation of splenocytes was
significantly decreased in Stat5a/ mice and
Stat5b/ mice by 79% and 81%, respectively
(n = 5, P < .005) (Figure
4A). This defect may account for the
diminished antigen-induced T-cell infiltration into the airways (Figure
2). In contrast to the diminished antigen-induced proliferation,
anti-CD3-induced proliferation of splenocytes was normal in
Stat5a/ mice and slightly decreased (but not
statistically significant) in Stat5b/ mice
(n = 5) (Figure 4B). These results suggest that Stat5a and Stat5b
might be required for the generation of antigen-specific T-cell pools
in the periphery.
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| Fig 4.
Diminished antigen-induced proliferation of splenocytes
in Stat5a/ mice and Stat5b/ mice.
(A) Splenocytes from OVA-sensitized mice were cultured in the presence
or absence of 200 µg/mL of OVA for 72 hours. Data are means ± SD
for 5 mice in each group. *Significantly different from the mean value
of wild-type mice, *P < .005. (B) Splenocytes from
OVA-sensitized mice were stimulated with or without plate-coated
anti-CD3 mAb for 72 hours. Data are means ± SD for 5 mice in each
group.
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Antigen-specific IgE production is diminished in
Stat5a/ mice but not in
Stat5b/ mice.
It has been shown that antigen-specific IgE production is a marker
of systemic Th2 responses,35 but it is not essential for
antigen-induced airway eosinophilia in mice36,37 (and our unpublished data). Therefore, we examined antigen-specific IgE production in Stat5a/ mice and
Stat5b/ mice at 2 weeks after the second
immunization. Interestingly, Stat5a/ mice
exhibited decreased levels of antigen-specific IgE production (n = 10, P < .005), whereas antigen-specific IgE
production was normal in Stat5b/ mice (Figure
5A). In addition, antigen-specific IgG1
production was also diminished in Stat5a/
mice (n = 10, P < .01) (Figure 5B), suggesting that
systemic Th2 response was diminished in
Stat5a/ mice but not in
Stat5b/ mice. In contrast to the diminished
IgE and IgG1 production in Stat5a/ mice,
antigen-specific IgG2a production was increased in
Stat5a/ mice (n = 10, P < .005)
(Figure 5C), suggesting the Th1-polarized immune responses in
Stat5a/ mice.
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| Fig 5.
Antigen-specific IgE production is diminished in
Stat5a/ mice but not in Stat5b/
mice.
(A) The titer of anti-OVA IgE antibody in sera was assessed at 2 weeks
after the second immunization by a 24-hour passive cutaneous
anaphylaxis (PCA) reaction. Data are means ± SD for 10 mice in each
group. *P < .005. (B-C) Anti-OVA IgG1 (B) and IgG2a (C)
antibodies in sera were assessed at 2 weeks after the second
immunization by ELISA as described in "Materials and Methods."
Data are means ± SD for 10 mice in each group.
*P < .01, **P < .005.
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IL-5-induced eosinophilopoiesis is diminished in
Stat5a/ mice and
Stat5b/ mice.
Although the diminished antigen-induced eosinophil recruitment into
the airways in Stat5a/ mice and
Stat5b/ mice (Figure 1) presumably results in
part from diminished IL-5 production in the airways (Figure 3), the
defect in eosinophil recruitment into the airways appeared more severe
than the defect in IL-5 production. We therefore investigated whether
IL-5-dependent eosinophilopoiesis was normal in
Stat5a/ mice and
Stat5b/ mice. To address this issue,
Stat5a/ mice and
Stat5b/ mice were injected intraperitoneally
with recombinant murine IL-5 (rmIL-5; 20 000 U/d) for 4 days and the
eosinophil numbers in the bone marrow, peripheral blood, and peritoneal
cavity were determined at 48 hours after the last injection. The
administration of rmIL-5 significantly increased eosinophil numbers in
the bone marrow in wild-type mice (n = 5) (Figure
6A). In Stat5a/
mice and Stat5b/ mice; however, the magnitude
of the IL-5-induced eosinophilia in the bone marrow was approximately
half of that in rmIL-5-treated wild-type mice (n = 5,
P < .005) (Figure 6A). These results indicate that both
Stat5a and Stat5b play a role in IL-5-dependent eosinophilopoiesis.
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| Fig 6.
IL-5-induced eosinophilopoiesis is diminished in
Stat5a/ mice and Stat5b/ mice.
Stat5a/ mice,
Stat5b/ mice, and wild-type mice were
injected intraperitoneally daily for 4 days with 20 000 U of
recombinant murine IL-5. Forty-eight hours after the last injection,
mice were killed and the numbers of eosinophils in bone marrow (A),
peripheral blood (B), and peritoneal cavity (C) were evaluated. Data
are means ± SD for 5 mice in each group. *Significantly different
from the mean value of wild-type mice, *P < .005.
|
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The eosinophil numbers in the peripheral blood and the peritoneal
cavity were also significantly increased by the intraperitoneal injection of rmIL-5 in wild-type mice (Figure 6B and C). Although eosinophil numbers in the peripheral blood were similarly increased by
the administration of rmIL-5 among the 3 groups of mice (Figure 6B),
however, the eosinophil numbers in the peritoneal cavity were
significantly decreased in Stat5a/ mice and
Stat5b/ mice by 59% and 57%, respectively
(n = 5, P < .005) (Figure 6C). Consistent with increased
eosinophils in the peritoneal cavity in rmIL-5-treated wild-type mice,
FACS analysis revealed that the Gr-1mid VLA-4+
population (R1 region), which most likely represents
eosinophils,38 was selectively increased in the peritoneal
cavity after rmIL-5 administration (Figure
7). In contrast, the Gr-1high
VLA-4 population (R2 region), which most likely
represents neutrophils,38 was not increased in the
peritoneal cavity on rmIL-5 administration (Figure 7). Consistent with
the diminished eosinophils in the peritoneal cavity of rmIL-5-treated
Stat5a/ mice or
Stat5b/ mice, the fraction of
Gr-1mid VLA-4+ cells in the peritoneal cavity
was also diminished in Stat5a/ and
Stat5b/ mice (Figure 7). These results
suggest that, in addition to the role in IL-5-induced
eosinophilopoiesis, Stat5a and Stat5b may be necessary for
IL-5-induced chemotaxis of eosinophils.
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| Fig 7.
Gr-1mid VLA-4+ population is
diminished in IL-5-treated Stat5a/ mice and
Stat5b/ mice.
Mice were injected intraperitoneally with rmIL-5 as described in Figure
6. Forty-eight hours after the last injection, mice were
killed and cells in the peritoneal cavity were stained with anti-VLA-4
FITC and anti-Gr-1 APC. Shown are representative FACS profiles from 5 mice in each group.
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| |
Discussion |
It has been shown that antigen-induced eosinophil
recruitment into the airways of sensitized mice is mediated by the Th2
subset of CD4+ T cells and subsequent secretion of
IL-5.6 In this study, we showed that both Stat5a and Stat5b
were required for antigen-induced eosinophil and T-cell recruitment
into the airways. We found that antigen-induced eosinophil infiltration
in the airways and antigen-induced CD4+ T-cell infiltration
and IL-5 production in the airways were diminished in both
Stat5a/ mice and
Stat5b/ mice. Moreover, we found that
antigen-induced proliferation of splenocytes was diminished in
Stat5a/ mice and
Stat5b/ mice, suggesting that the generation
of antigen-primed T cells might be compromised in
Stat5a/ mice and
Stat5b/ mice. This defect may be due to
diminished IL-2-induced proliferation of T cells in
Stat5a/ mice24 and
Stat5b/ mice23 and may account
for the diminished antigen-induced T-cell infiltration into the
airways. Finally, we found that IL-5-dependent eosinophilopoiesis was
impaired in Stat5a/ mice and
Stat5b/ mice. Therefore, these results
indicate that the diminished antigen-induced eosinophil recruitment
into the airways in Stat5a/ mice and
Stat5b/ mice results from both impaired IL-5
production in the airways and impaired IL-5 responsiveness of eosinophils.
Given that Stat5a and Stat5b are highly homologous and exhibit
overlapping functions,18,25 it was surprising that
antigen-induced eosinophil and T-cell recruitment into the airways was
severely impaired in mice lacking either Stat5a or Stat5b. Because
Stat5b DNA binding activity is not dependent on the presence of
Stat5a,24 it is possible that the residual eosinophil
recruitment in Stat5a/ mice is regulated by
intact Stat5b. However, it is also possible that the residual
eosinophil recruitment in Stat5a/ mice or
Stat5b/ mice is regulated by
Stat5-independent signaling pathway(s). This can be further assessed
when Stat5a/Stat5b double-deficient mice in the BALB/c background are
available. Nevertheless, it may be predicted that antigen-induced
eosinophil recruitment into the airways, which is regulated by
CD4+ T cells,6 is greatly diminished in
Stat5a/Stat5b double-deficient mice, because TCR-mediated proliferation
of CD4+ T cells from Stat5a/Stat5b double-deficient mice is
defective.25
We also found that IL-5-induced in vivo eosinophilopoiesis was
diminished in Stat5a/ mice and
Stat5b/ mice. IL-5 acts on eosinophil
precursors, resulting in induction of proliferation and differentiation
into mature eosinophils.39 It has been reported that bone
marrow cells from IL-5R /
mice26 or IL-5/
mice7,27 fail to form eosinophilic colonies in
response to IL-5 or parasite infection. GM-CSF also plays an important
role in the production of eosinophils in bone marrow.40
IL-5, GM-CSF, and IL-3 all share common  chain (c) as a receptor
component41 and activate Jak2 and
Stat5a/Stat5b.16-18,20 Recently, Feldman et
al42 demonstrated that GM-CSF-induced proliferation of bone marrow-derived macrophages is diminished in
Stat5a/ mice, suggesting that Stat5a plays a
role in c-dependent proliferation. In contrast, it has been reported
that in vitro IL-5-induced colony formation from bone marrow cells is
normal in Stat5a/ mice and
Stat5b/ mice.43 At present, the
precise mechanism(s) underlying the discrepancy between the in vivo and
the in vitro requirement for Stat5a/Stat5b in IL-5-induced
eosinophilopoiesis is unclear. However, it is likely that the
compensatory role of Stat5a by Stat5b or Stat5b by Stat5a is not
sufficient for in vivo IL-5-induced eosinophilopoiesis, because
IL-5-dependent in vitro colony formation is diminished in
Stat5a/Stat5b double-deficient mice.43 In addition, our
finding of diminished eosinophil numbers in the peritoneal cavity after IL-5 administration in Stat5a/ mice and
Stat5b/ mice suggest that Stat5a and Stat5b
may also be necessary for IL-5-induced chemotaxis of
eosinophils.44
Interestingly, we found that IL-4 production from anti-CD3-stimulated
splenocytes was diminished in Stat5a/ mice.
We also found that antigen-specific IgE and IgG1 production was
diminished in Stat5a/ mice, whereas
antigen-specific IgG2a production was increased in
Stat5a/ mice. Therefore, in
Stat5a/ mice, the balance between Th1 and Th2
cell activation is biased toward a Th1 profile, suggesting that Stat5a
may be involved in the IL-4-dependent Th2 cell development. Consistent
with this finding, it has recently been shown that IL-4 signaling
activates Stat5 as well as Stat6.45 In contrast, in
Stat5b/ mice, the situation may be more
complicated. In these mice, although anti-CD3-induced IL-4 production
from splenocytes was diminished, antigen-specific IgE production in
vivo was normal. Because anti-CD3-induced IFN- production was also
slightly diminished in Stat5b/ mice, it is
possible that the balance between IL-4 and IFN- rather than the
amount of IL-4 may be critical for in vivo IgE production.
Alternatively, it is conceivable that the discrepancy may result from
compensatory role(s) of other cytokines such as IL-13.46
As discussed previously, diminished antigen-induced eosinophil
recruitment into the airways is likely to result from both impaired
IL-5 production by T cells and impaired IL-5 responsiveness of
eosinophils in Stat5a/ mice and
Stat5b/ mice. However, other cell populations
may also be involved in the diminished antigen-induced eosinophil
recruitment into the airways. Recently, it has been shown that  T
cells47 and NK cells48,49 play important
role(s) in allergic inflammation. Because T cells are diminished
in Stat5a/ mice24 and NK cell
function is particularly impaired in Stat5b/
mice,23 these defects may be responsible for the diminished antigen-induced eosinophil recruitment into the airways.
In this study, we provide the evidence that Stat5a and Stat5b are
important for allergic inflammation. Recently, several lines of
evidence have indicated that other STAT proteins besides Stat5 also
play important immunopath |
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Abstract
Introduction