By
From the * Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland 20892-1674; the Division of Cell and Gene
Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda,
Maryland 20892; the § Ministry of Agriculture and Forestry (Verification Agency), Te Kuiti, New
Zealand; and the ¶ Dairy Science Group, AgResearch, Ruakura, Hamilton, New Zealand
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Abstract |
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We have analyzed the immune system in Stat5-deficient mice. Although Stat5a/
splenocytes
have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b
/
splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained
by diminished expression of the IL-2 receptor
chain (IL-2R
), which is a component of the
receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect
in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b
/
mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a
decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the
diminished proliferation, basal as well as IL-2- and IL-15-mediated boosting of NK cytolytic
activity was also greatly diminished. These data indicate an essential nonredundant role for
Stat5b for potent NK cell-mediated proliferation and cytolytic activity.
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Introduction |
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Signal transducers and activators of transcription (STAT)1 proteins are cytosolic latent transcription factors that are rapidly activated after cellular exposure to interferons, cytokines, or growth factors (1). A considerable amount of investigation has centered on the specificity of the signals transduced by each of the seven mammalian STAT proteins. For three of these STATs (Stat1, Stat4, and Stat6), knockout mice reveal specific defects limited to the immune system consistent with defective signaling in response to interferons (4, 5), IL-12 (6, 7), or IL-4/IL-13 (8- 10), respectively. In contrast, Stat3 knockout mice exhibit fetal lethality (11), a finding consistent with the activation of Stat3 by many important cytokines, including but not limited to the IL-6 family of cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin 1; reference 3).
Stat5 was discovered as a mammary gland factor induced
by prolactin (12). Subsequently, a number of studies revealed the existence of two closely related Stat5-like proteins with >90% amino acid identity, now denoted as
Stat5a and Stat5b (13). The genes encoding Stat5a and
Stat5b are closely linked on human chromosome 17 (17)
and mouse chromosome 11 (18). Both Stat5a and Stat5b
are activated not only by prolactin but also by a very wide
range of other cytokines, including growth hormone,
erythropoietin, thrombopoietin, hematopoietic cytokines
(IL-3, GM-CSF, and IL-5), and cytokines more specific for
lymphocytes (IL-2, IL-7, IL-9, and IL-15; reference 3).
Despite their similarities, Stat5a and Stat5b differ in their
COOH-terminal transactivation domains (19), and relatively specific actions have been attributed to Stat5a and
Stat5b. For example, mice lacking Stat5a exhibit defective lactation with impaired lobuloalveolar proliferation of the
mammary epithelium during pregnancy, and reduced expression of whey acidic protein (20), indicative of defective
prolactin signaling. Mice lacking Stat5b exhibit a loss of
sexually dimorphic growth, which results from sex differences in the pattern of growth hormone secretion from the
pituitary (21). A recent study analyzing mice in which the
two Stat5 genes were deleted individually or together (by
retargeting Stat5a+/ embryonic stem cells with a Stat5b
targeting construct) confirmed these findings and additionally showed a greater defect in growth in mice lacking both
genes (22). Thus, Stat5a and Stat5b have distinctive roles but
appear also to have partially redundant roles (22). Interestingly, the double knockout mice also exhibited a significant
decrease in expression of Stat3 (which results in fetal lethality
when expression is totally eliminated; reference 11), raising
the possibility that diminished Stat3 levels contributed to
some of the abnormalities in these mice, such as the diminished response of bone marrow cells to G-CSF (22).
In Stat5a/
mice, bone marrow-derived macrophages
exhibit defective GM-CSF-induced proliferation and gene
expression (23); moreover, these mice also exhibit several
immunological defects, including reduced expansion of T
cells in vivo (24). This latter defect is associated with diminished IL-2-mediated induction of the IL-2R
chain,
although TCR-mediated IL-2R
induction appears to be
relatively normal; thus, impaired IL-2 signaling is believed to at least partially explain the defects in these mice.
We have now analyzed the immunological properties in
Stat5b/
mice and shown that they have a modest decrease in both thymic and splenic cellularity. Moreover,
Stat5b
/
splenocytes exhibit a marked decrease in IL-2-
mediated proliferation and a more moderate decrease in the
number of NK cells. Although Stat5a
/
mice also exhibit
a partial decrease in the number of NK cells, only the
Stat5b
/
mice have a dramatic defect in NK cell function.
The implications of these findings are discussed below.
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Materials and Methods |
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Mice.
The Stat5aFlow Cytometric Analysis.
Single-cell suspensions from the thymus and spleen were stained and analyzed using a FACSort® (Becton Dickinson, San Jose, CA) with CELLQuest software (Becton Dickinson). The following antibodies were purchased from PharMingen (San Diego, CA): PE-conjugated anti-TCR-RNA Analysis.
To evaluate IL-2-induced IL-2RWestern Blotting.
Whole cell extracts (15 µg/sample) were fractionated on 8% SDS polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Blots were processed as previously described (24). In brief, after blocking, blots were incubated with antisera specific for Stat5a or Stat5b (17), followed by the incubation with anti-rabbit IgG conjugated with horseradish peroxidase (Nycomed Amersham, Little Chalfont, Buckinghamshire, UK). Blots were washed and developed with an enhanced chemiluminescent substrate (Pierce Chemical Co., Rockford, IL). To examine Stat6 expression, anti-Stat6 antibody (Transduction Laboratories, Lexington, KY) was used.Proliferation Assay.
Fresh splenocytes (2 × 105 cells/well) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics with or without 2 nM IL-2 or 100 ng/ml (7.75 nM) IL-15 (Peprotech, Rocky Hill, NJ) for 48 h and pulsed with 1 µCi of [3H]thymidine (6.7 Ci/mmol, NEN, Boston, MA) for the last 12 h of culture. For proliferation of anti-CD3-stimulated splenocytes, cells (105/well) were cultured for 24 h in the presence or absence of 1 nM IL-2 with a 1 µCi [3H]thymidine pulse for the last 10 h. Because IL-4 alone cannot potently stimulate splenocytes, we stimulated splenocytes with a combination of PMA (1 ng/ml) and murine IL-4 (1,000 U/ml) for 48 h and pulsed them with 1 µCi of [3H]thymidine for the last 12 h of culture in order to evaluate the ability of IL-4 to induce proliferation in Stat5-deficient mice.Cytotoxicity Assay.
51Cr-release assays were performed by standard methods in final volumes of 200 µl. YAC-1 (H-2A), an NK-sensitive cell, was used to assess NK activity with effector/ target ratios of 100, 50, 25, and 12.5:1 and 40, 10, 2.5, and 0.6:1, respectively. 10 nM IL-2 or 15.5 nM IL-15 were added to wells. Data are expressed as lytic units (LU) in 107 cells (1 LU corresponds to the number of effector cells lysing 20% of the targets) and obtained by fitting the titration curves to scale families of curves, as previously described (27). ![]() |
Results and Discussion |
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The majority of mice lacking Stat5a have normal levels
of Stat5b in thymus and spleen (24). Correspondingly,
Stat5b/
mice express relatively normal levels of Stat5a.
Thus, in both Stat5a
/
and Stat5b
/
mice, the loss of expression of one Stat5 gene did not typically affect the expression of the other Stat5 gene in either thymus or spleen,
although occasional variations were seen (Fig. 1 A, reference 24, and data not shown). Furthermore, both Stat5a
/
(24) and Stat5b
/
mice had normal levels of Stat3 (data
not shown).
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We first analyzed lymphoid development in Stat5b/
mice. In these mice, thymocyte numbers were modestly
diminished (P < 0.01; Fig. 1 B). Flow cytometric analysis
of thymocytes from these mice revealed that all populations
were represented, with almost normal numbers of CD4
and CD8 single positive and CD4+CD8+ double positive
thymocytes (Fig. 1 C). However, among the double negative cells there was a moderate decrease in the percentage of CD44+CD25
cells and increase in the percentage of
CD44
CD25+ cells (Fig. 1 D).
In the periphery, Stat5b/
mice tended to have decreased numbers of splenocytes (P < 0.001; Fig. 1 E), as is
observed in Stat5a
/
mice (24). The T/B cell ratio was
slightly decreased (Fig. 1 F). Flow cytometric analysis revealed diminished numbers of single positive splenocytes;
the number of CD4
CD8+ splenocytes was substantially
reduced, whereas the number of CD4+CD8
cells was at
most only slightly affected (Fig. 1 G). Interestingly, whereas
Stat5a
/
mice have decreased numbers of TCR-
/
cells
in the spleen (24), this was not the case for Stat5b
/
mice
(Fig. 1 H).
We next evaluated immune function in the periphery in
Stat5b/
mice and found a defect in the responsiveness of
splenocytes to mitogenic stimulation. Previously, we had
found that Stat5a
/
splenocytes exhibited a defect in IL-2-
induced proliferation of splenocytes that had been preactivated for 48 h with anti-CD3. However, the diminished
proliferation in Stat5a
/
splenocytes could be overcome
with high concentrations of IL-2 (24), suggesting that this
defect primarily related to impaired IL-2-mediated induction of IL-2R
(24), a protein that is required for formation of high-affinity IL-2 receptors (28). In Stat5b
/
splenocytes, we observed a similar defect in IL-2-induced
IL-2R
expression, both at the protein (Fig. 2 A) and
mRNA (Fig. 2, B and C) levels. This defect in IL-2-
induced IL-2R
expression seen in both Stat5a
/
and
Stat5b
/
mice can be explained by the presence of an IL-2
response element in the IL-2R
gene that is dependent on
Stat5 proteins (29). However, in contrast to the ability
of high doses of IL-2 (sufficient to titrate intermediate affinity receptors) to normalize proliferation in Stat5a
/
mice (reference 24 and data not shown), this was not the
case for Stat5b
/
mice (Fig. 2 D). The diminished expression of IL-2R
(Fig. 2, B and C) presumably contributed
to this effect. Thus, although diminished proliferation in
Stat5a
/
mice is primarily related to diminished IL-2R
expression (24), the Stat5b
/
mice appeared to have a
more defective IL-2-signaling pathway involving but not
necessarily limited to defective IL-2R
and IL-2R
expression. Indeed, additional defects are indicated given that
the Stat5b
/
splenocytes also exhibited defective proliferation to a combination of PMA and IL-4 (Fig. 2 E), even
though IL-4 activates Stat6 rather than Stat5 proteins. Because Stat6
/
mice exhibit diminished IL-4-dependent
proliferation (8), we confirmed that Stat6 expression
was normal in Stat5b
/
splenocytes (Fig. 2 F). These data
are consistent with a more general defect or perhaps multiple defects that together diminish but not abrogate T cell
proliferation.
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Considering the above partial defect in proliferation of
preactivated T cells, we next tested the ability of freshly isolated splenocytes to proliferate in response to IL-2 and IL-15. Proliferation of Stat5a/
splenocytes was at most
slightly diminished, whereas IL-2- and IL-15-induced proliferation of Stat5b
/
splenocytes was greatly diminished
(Fig. 3 A). We speculated that this defect in proliferation
might result from a decrease in the number and/or function of NK cells, given that NK cells represent a prominent
IL-2-responsive cell population in murine spleens and that
IL-2
/
mice exhibit essentially normal T and B cell responses but compromised NK cell responses (32). Indeed,
flow cytometric analysis revealed that the percentage of
NK (CD3
DX5+) cells was uniformly diminished in
Stat5b
/
mice (~55% of the percentage seen in wild-type
mice; Fig. 3 B). Interestingly, the percentage of NK cells in
Stat5a
/
mice was also diminished, although to a lesser extent (~70% of the percentage seen in wild-type mice).
However, the much greater defect in proliferation in
Stat5b
/
compared with Stat5a
/
mice indicated that the
defect was not proportional to the number of NK cells but
rather reflected a greater functional defect in Stat5b
/
mice.
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Corresponding to the greater defect in proliferation in
freshly isolated splenocytes from Stat5b/
versus Stat5a
/
mice (Fig. 3 A), NK cytolytic activity was much more impaired in Stat5b
/
than in Stat5a
/
splenocytes (Fig. 3 C).
The defect in Stat5b
/
mice was even more evident when
either IL-2 (Fig. 3 D) or IL-15 (Fig. 3 E) was added during
the NK assay (note the difference in the scale on the ordinate in Fig. 3, C-E). Thus, Stat5b
/
mice exhibited substantial defects in both proliferative and cytolytic activities.
Because of these IL-2 and IL-15 signaling defects, and
because the IL-2 and IL-15 receptors both contain IL-2R
and
c as critical receptor components (33, 34), we evaluated expression of these proteins in NK cells. No defect in
c expression was observed in either Stat5a
/
or Stat5b
/
mice (data not shown). Interestingly, however, the number
of NK cells expressing IL-2R
expression was uniformly
decreased in all Stat5b
/
mice examined (n = 17; Fig. 3 F);
in contrast, in Stat5a
/
NK cells, IL-2R
expression was
typically normal, although 2 out of 14 mice examined had
a significantly diminished percentage of IL-2R
-expressing NK cells. Overall, the decreased number of IL-2R
+
NK cells in Stat5b
/
mice generally correlated with the
decreased proliferative response to IL-2 and IL-15 (Fig. 3 A)
as well as the marked decrease in IL-2- and IL-15-mediated boosting of NK cytolytic activity (Fig. 3, D and E).
Again, however, the defects in IL-2- and IL-15-induced proliferation and cytolytic activity were more severe than
would have been predicted based solely on the decreased
number of CD3
DX5+IL-2R
+ cells, suggesting that
Stat5b plays an additional important and specific role(s) in
IL-2- and IL-15-induced signaling in NK cells.
To further investigate the basis for defective NK function, we examined 2-integrin (CD18) and perforin expression. CD18 is known to be important for recognition
of target cells by NK cells (35), and perforin is known to be
one of the key molecules for NK cell-mediated cytolysis
(36). Interestingly, although cell surface CD18 expression
was not diminished (data not shown), induction of perforin
mRNA by IL-2 and IL-15 was markedly decreased in
Stat5b
/
splenocytes (Fig. 4).
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The mechanism of NK cell development has been an
area of active investigation. NK cells do not develop in several different knockout mice, including those lacking IL-2R (37),
c (38, 39), or Jak3 (40), which are receptor
or signaling components for both IL-2 and IL-15. Interestingly, although IL-2 potently augments NK cytolytic activity, the fact that IL-2
/
mice have NK cells indicates that
IL-2 is not absolutely required for NK cell development.
Instead, IL-15 has been implicated as playing an important
role in NK cell development (43). Supporting this concept, the absence of NK cells in IRF1
/
mice has been
correlated with defective IL-15 production (47). Our data
now reveal a partial decrease in NK cells in both Stat5a
/
and Stat5b
/
mice, indicating that Stat5 activation is vital
for normal NK cell development.
Interestingly, although diminished NK cells were seen in
both Stat5a/
and Stat5b
/
mice, it was only the NK cells
from Stat5b
/
mice that exhibited a dramatic defect in
their ability to respond to IL-2 and IL-15 (Fig. 3, D and E),
and only Stat5b
/
mice that had a lower percentage of
NK cells expressing IL-2R
(Fig. 3 F). The basis for this
decrease in IL-2R
+ NK cells remains unclear; although a
number of transcription factors including early growth response (Egr)-1, Ets-1, and GABP have been shown to be
important for IL-2R
promoter activity (48, 49), a Stat5b-dependent response element for this gene has not been
reported. The decrease in perforin mRNA in IL-2- and
IL-15-treated Stat5b
/
splenocytes is consistent with perforin being a Stat5b-regulated gene.
In conclusion, just as Stat4 and Stat6, respectively, are
important for normal Th1 and Th2 cell development and
function (6), our results reveal that both Stat5a and
Stat5b contribute to normal NK cell development with a
greater defect being seen particularly for NK function in
Stat5b/
mice. Although Stat5a and Stat5b have similar
(but not identical) binding activities in vitro and can both
drive expression of similar reporter constructs in transfected
cells, the data presented here are consistent with published
studies suggesting differential roles for these proteins in
nonlymphoid settings in vivo (20). It will be vital to
further clarify the range of target genes that are controlled
by Stat5a and Stat5b. We have shown that IL-2R
is defective in the absence of either Stat5 protein, whereas IL-2R
and perforin appear more dependent on Stat5b. Moreover,
by demonstrating that Stat5b plays a particularly critical role
for proliferation and cytolytic activity of NK cells, we now
provide a key example of different biological roles for these
two Stat5 proteins within the immune system.
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Footnotes |
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Address correspondence to Warren J. Leonard, Bldg. 10, Rm. 7N252, NIH, Bethesda, MD 20892-1674. Phone: 301-496-0098; Fax: 301-402-0971; E-mail: wjl{at}helix.nih.gov
Received for publication 23 July 1998 and in revised form 29 September 1998.
K. Imada was supported in part by a Japanese Society for the Promotion of Science research fellowship for Japanese biomedical and behavioral researchers at NIH and by the Sankyo Foundation of Life Science.We thank Drs. Lothar Hennighausen, Xiuwen Liu, and Anthony Wynshaw-Boris (National Institutes of
Health [NIH]) for Stat5a/
mice, and Dr. Alex Grinberg (NIH) for thawing and implanting Stat5b
/
frozen embryos. We thank Dr. Thomas R. Malek (University of Miami, Miami, FL) for the murine IL-2R
cDNA; Drs. Grace Ju and John Hakimi (Hoffmann-La Roche) for recombinant IL-2; Dr. James E. Darnell,
Jr. (The Rockefeller University, New York, NY) for pHe7 cDNA; Dr. William E. Paul (NIH) for murine
IL-4; and Drs. Dana P. Ascherman and Robert D. Schreiber for valuable discussions/critical comments.
Abbreviation used in this paper STAT, signal transducers and activators of transcription.
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References |
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