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*
Lymphocyte Cell Biology Section,
Clinical Research Group, and
Genetics Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892;
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Experimental Immunology Branch and
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Dermatology Branch, National Cancer Institute, Bethesda, MD 20892; and
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Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and LPS-treated monocytes and dendritic cells express
high levels of Stat4. Monocyte-expressed Stat4 in humans is
phosphorylated in response to IFN-
, but not IL-12. In contrast, the
Th2 cytokines, IL-4 and IL-10, specifically down-regulate Stat4
expression in activated monocytes, while having little effect on Stat6
expression. Moreover, macrophages in synovial tissue obtained from
patients with rheumatoid arthritis express Stat4 in vivo, suggesting a
potential role in a prototypical Th1-mediated human disease.
IFN--induced Stat4 activation in human monocytes represents a
previously unrecognized signaling pathway at sites of Th1
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. IL-12 and
IFN- then act to drive expansion of a subset of
CD4+ T cells (Th1 cells) characterized by
production of IFN- and lymphotoxin (1, 2, 3). Stat4 is a
key molecule in the intracellular signal transduction cascade activated
in response to IL-12 stimulation. It is phosphorylated in response to
IL-12, resulting in the translocation of Stat4 to the nucleus and
transactivation of genes that presumably promote Th1 differentiation
(4, 5). Stat4-deficient mice highlight the central role of
Stat4 in mediating IL-12 signals, as they have major defects in IFN-
production, NK cell activity, IL-12-mediated proliferation, and Th1
differentiation (6, 7).
Yet, in
humans, Stat4 is not only activated by IL-12. In human lymphoid cells,
unlike in murine cells, it is also phosphorylated by IFN-,
demonstrating that the use of Stat4-dependent Th1 differentiation
pathways in humans may not be under the exclusive control of IL-12
(8, 9). IFN- also acts to enhance the Th1 inflammatory
response by increasing NK activity and promoting Th1 differentiation of
T cells (9, 10). Although the exact role of Stat4 in the
positive regulation of Th1 inflammation by IFN- has not been
defined, the possibility exists that, in addition to IL-12, IFN-
modulates cellular immunity via Stat4.
Stat4 was initially reported to be expressed in the thymus, spleen, and testes. However, its role in testicular development is evidently inconsequential; only T cell and NK cell abnormalities were reported in Stat4 knockout mice. Immature myeloid cell lines were also shown to express Stat4, but it was reported that differentiated myeloid cells lacked Stat4 (11). Thus, all subsequent studies focused on T cells and NK cells. However, peripheral blood monocytes, macrophages, and DC play pivotal roles in cell-mediated immunity. We therefore asked whether Stat4, a key transcription factor that promotes cellular immunity, was expressed and functional in these cells of monocyte lineage. Stat4 expression in T cells is greatly influenced by their state of activation. Human peripheral blood T cells do not have Stat4 in their basal state, but its expression is markedly induced following stimulation (4). Based on this result, we hypothesized that other immune cells, including differentiated cells of monocyte lineage, may only express Stat4 upon activation.
We demonstrate in this paper for the first time that Stat4 is present
in activated monocytes and DC. Monocyte-expressed Stat4 is activated in
response to IFN-, but not to IL-12. We further show that Stat4 is
abundantly expressed in synovial macrophages in patients with
rheumatoid arthritis (RA), an autoimmune disease characterized by Th1
inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes were purified from the peripheral blood of normal volunteers by elutriation (Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD). In selected experiments, these monocytes were further purified by depleting the nonmonocytic population with commercially available immunomagnet beads using the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). Isolated monocytes were stained with fluorochrome-conjugated CD3, CD14, CD16, CD19, and CD56 Abs, or the respective conjugate-matched, isotype-matched irrelevant control Abs (Becton Dickinson, San Jose, CA), following Fc receptor blockade with FCS and human Ig. The resulting cells were consistently >99% CD14+ monocytes with <1% contamination with B cell, T cells, or NK cells by flow cytometry.
Purified monocytes were cultured in complete RPMI (c-RPMI; RPMI
supplemented with 10% heat-inactivated FCS (Biofluids, Rockville,
MD)), 2 mM L-glutamine (Biofluids) and 100 U/ml Pen-Strep
(Biofluids) in 5 ml cultures in 6 well polystyrene tissue culture
plates (Becton Dickinson). In some cases, monocytes were activated with
1000 U/ml IFN- (Biological Resource Bank, National Cancer Institute,
Frederick, MD), 200 ng/ml LPS (Sigma, St. Louis, MO), and/or 1000 U/ml
IFN- (Biological Resource Bank, National Cancer Institute). As
indicated, cytokine neutralizations were performed using goat
polyclonal Abs (R&D Systems, Minneapolis, MN) as follows: 20 µg/ml
anti-lL-4, 20 µg/ml anti-lL-10, and 10 µg/ml anti-lL-12.
Additionally, conditioned medium was obtained from monocytes stimulated
with LPS and IFN- for 36 h. This conditioned medium was added
to cultures in the indicated experiments to examine the role of soluble
factors in Stat4 induction.
DC were generated by culturing purified monocytes (2 x
106 cells/ml) for 7 days in c-RPMI supplemented
with IL-4 and GM-CSF (1000 U/ml and 5 ng/ml, respectively; R&D
Systems). A portion of these cells was evaluated by flow cytometry for
purity and found to be >99% CD14- (Becton
Dickinson) and predominantly CD1a+ (PharMingen,
San Diego, CA). The cells were then cultured for an additional 24
h, with selected wells stimulated with IFN- and LPS, as described
above.
RNase protection assays
RNA from unstimulated and LPS and/or IFN--stimulated
monocytes (107) was prepared using the RNA
STAT-60 kit (Tel-Test, Friendswood, TX). The GAPDH and L32 housekeeping
control probes were obtained from a commercial source (PharMingen).
Twenty percent of the RNA obtained from the preparations described
previously was used for each RNase protection assay, performed using
the PharMingen protocol. The Stat4 probe used in this assay was
generated by cloning a Stat4 RT-PCR product into the pCR 2.1-TOPO TA
cloning vector (Invitrogen, Carlsbad, CA). Reverse transcription was
performed using a commercial kit (Boehringer Mannheim, Indianapolis,
IN). The PCR was conducted with a 60°C
annealing temperature and primers used were as follows:
5'-GAGGCAGCTTCTAACAATGAAAC-3' and 5'-CTCTGATCCATTGTCTGAATTG-3'.
Immunoprecipitation and Western blot analysis
Following incubation, the monocyte culture plates were placed on ice for 25 min. The monocytes were removed from the plate by gentle scraping, sedimented, and lysed in buffer containing Triton X-100. Stat4 immunoprecipitation and Western blotting using polyclonal rabbit anti-huStat4 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-huStat6 (1:1000, Santa Cruz Biotechnology), or anti-phosphotyrosine (4G10, 1:1000; Upstate Biotechnology, Lake Placid, NY) were performed as described (12).
Immunofluorescent staining
Elutriated monocytes adhered to coverslips inserted in the wells
of a 24-well plate at a density of 5 x 105
cells/ml and were stimulated with 200 ng/ml LPS and 1000 U/ml IFN-
for 24 h. The coverslips were washed twice with PBS, fixed for 2
min with 50%/50% ice-cold methanol/acetone, washed three times with
PBS, and blocked with PBS supplemented with 1% BSA (Sigma), 5% goat
serum (Life Technologies, Grand Island, NY), and 100 µg/ml of human
gamma globulin (Sigma). Primary and secondary staining was conducted at
room temperature for 1 h in PBS supplemented with 1% BSA, 5%
goat serum, and 20 µg/ml human gamma globulin. The following Abs were
used for primary staining at 1 µg/ml: polyclonal Stat4 Ab (as above)
and monoclonal anti-CD14 (mAb 63d3, provided by Dr. Steven Shaw,
National Cancer Institute). As a negative control for the polyclonal
Ab, purified rabbit IgG (Jackson ImmunoResearch, West Grove, PA) was
used at 5 µg/ml, while UCHT1 (anti-CD3, IgG1, provided by Dr.
Steven Shaw) was used as monoclonal control at 1 µg/ml. This was
followed by a total of five washes with PBS supplemented with 1% BSA.
Affinity purified goat anti-rabbit IgG-FITC and goat
anti-mouse-IgG-Cy5 (Jackson ImmunoResearch) were used at 5 µg/ml
as secondary Abs. The coverslips were washed as before and air-dried.
After the coverslips were mounted on slides using "Prolong"
(Molecular Probes, Eugene OR), staining was analyzed on a Zeiss LSM 410
laser scanning confocal microscope with a x63, planapochromat
objective (N.A. 1.4), using 488 nm krypton/argon and 633 nm helium/neon
laser excitation. Fluorescence was collected with two photomultiplier
tubes using 510–540 nm and 670–810 nm band pass emission filters for
FITC and CY5, respectively. Positive samples and negative controls were
viewed using the same contrast and brightness settings.
Immunohistochemical staining of tissue samples
Synovial tissue was obtained at joint replacement surgery from three patients who met 1987 American College of Rheumatology criteria for RA. Appropriate patient consent was obtained. Frozen tissue sections were cut in 8-micron sections, affixed to slides, and fixed in chilled acetone for 15 min. Immunohistochemical methods were employed utilizing the following Abs: polyclonal rabbit anti-huStat4 (0.4 µg/ml), monoclonal mouse anti-huCD14 (2.5 µg/ml), and rabbit IgG (0.4 µg/ml). Tissue sections were rehydrated in Tris-buffered saline (TBS), then blocked for 10 min with 0.03% hydrogen peroxide blocking solution. Following a brief wash, a blocking solution (25% normal goat serum and 25% pooled human serum in TBS) was applied, and slides were incubated in a humidified slide chamber for 30 min at room temperature. The blocking solution was removed and 100 µl of the diluted primary mAb was applied. Slides were then incubated overnight at 4°C in the slide chamber. Next, slides were washed twice in TBS, and 100 µl of the appropriate HRP-conjugated secondary Ab was applied to each slide for an incubation period of 60 min at room temperature. Following a final wash, the color substrate (Liquid DAB; Dako, Carpinteria, CA) was applied with color development allowed up to 10 min. The slides were washed in distilled water, counterstained with hematoxylin solution, and evaluated by light microscopy. Polyclonal rabbit anti-huStat4 Ab at 0.4 µg/ml was incubated with Stat4 control peptide (Santa Cruz Biotechnology) at a ratio of 1:10 (Ab:Ag) for 1 h at 37°C before application to each tissue section in neutralization studies. In double-staining experiments, tissue preparation and staining with rabbit anti-huStat4 Ab (0.6 µg/ml) was performed as described above, and color was developed using diaminobenzidine substrate. Slides were then washed twice, and mouse anti-huCD14 Ab was applied (2.5 µg/ml) for 2 h at room temperature in a humidified slide chamber. After two washes, a biotinylated goat anti-mouse Ab (Dako; used at 2.8 µg/ml) was applied for 1 h. Streptavidin alkaline phosphatase (Dako; used at 4 µg/ml) was applied to each slide again after two washes for a 1-h incubation. After a final wash, another substrate (New Fuchsin; Dako) was applied for color development. Slides were again washed in distilled water and evaluated as before.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments to examine Stat4 expression in cells of
monocyte lineage, we analyzed highly purified human monocytes (<1% T
cell, NK, or B cell contamination), first measuring Stat4 mRNA by RNase
protection assay. As shown in Fig. 1
A, Stat4 message was
negligible in unstimulated monocytes (lanes 1 and
2). IFN- is a well-characterized activator of monocytes
and a promoter of cell-mediated immunity (13). We
therefore treated monocytes with IFN- to determine whether Stat4
could be induced. Interestingly, stimulation of purified monocytes with
IFN- alone induced low level Stat4 expression (lane
3). LPS, another activator of macrophage function, also stimulated
low level induction of Stat4 message (lane 4). Much
higher levels of Stat4 expression were detected in monocytes cultured
with both IFN- and LPS (lane 5), an effect seen
with many other monocytic genes. Indeed, the level of Stat4 in
activated monocytes was equivalent to that of PHA-stimulated PBMC
(lane 6), which are predominantly lymphocytes and
known to express high levels of Stat4 (4).
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B).
Elutriated monocytes activated with IFN- and LPS
(CD14+, red, middle left and
lower left panels) showed Stat4 staining (green, upper
left and lower left panels). Specificity was confirmed
by the absence of staining with control IgG under the same conditions
(right panels). Finally, Stat4 production was demonstrated
at the protein level by Western blotting in elutriated monocytes in a
time course experiment (Fig. 1C). Although no Stat4 was
detected at rest (lane 2), the protein was detected
following 24 h of stimulation with IFN- and LPS
(lane 6).
Furthermore, the synergy between IFN- and LPS was observed at the
protein level as well (Fig. 2A). Following 2 days of
culture, only very low level induction was seen in IFN--stimulated
(lane 3) or LPS-stimulated (lane 4)
cultures of purified monocytes compared with unstimulated controls
(lane 2). However, very high levels of Stat4 were
present in monocytes stimulated with both IFN- and LPS
(lane 5). For comparison, lysate from an NK cell line
that expresses abundant levels of Stat4 is shown in lane 1.
Taken together, our data provide strong and unambiguous evidence that
activated monocytes express Stat4 at levels comparable to activated
lymphocytes; the potent monocyte activators LPS and IFN-
synergistically induce the expression of this transcription factor.
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and LPS, or whether secondary factors secreted by monocytes were
involved in this process. The induction of Stat4 protein production was
indirect, at least partially, as 36-h conditioned medium from LPS- and
IFN--treated monocytes accelerated the Stat4 induction kinetics
in monocytes (Fig. 1D). Monocytes stimulated in the
presence of conditioned medium (right lanes) showed at least
an 8 h acceleration in Stat4 production kinetics compared with
those cultured with LPS and IFN- alone (left
lanes). Because levels of LPS and IFN- were present at
saturating levels, this acceleration could not be accounted for by
small differences in exogenous LPS and IFN- present in the cultures,
implying a role for soluble, monocyte-derived factors in the
up-regulation of Stat4 protein. One such factor produced by monocytes
in response to combined LPS and IFN- stimulation is IL-12 (104 pg/ml
by ELISA; data not shown). Blockade of IL-12 did result in a small
reduction in the induction of Stat4 by LPS and IFN- (Fig. 1E, lane 6 vs lane 4), while control
Ig had no effect (lane 5), suggesting the fascinating
possibility of autocrine regulation of Stat4. Th2 cytokines specifically down-regulate Stat4 expression in activated monocytes
In contrast to IFN- and LPS, cytokines such as IL-4 and IL-10
promote allergic responses and inhibit cell-mediated immunity
(1). We therefore examined whether incubation of monocytes
with either of these cytokines could regulate Stat4 expression. As
shown in Fig. 2A, both IL-4 (lane 6) and
IL-10 (lane 9) blocked the induction of Stat4
production by IFN- and LPS (lane 5). The
Th2-derived cytokine effect on Stat4 induction was specific, as
neutralizing Abs to IL-4 (lane 7) and IL-10
(lane 10), but not control IgG (lanes
8 and 11), reversed the blockade on Stat4 induction.
For comparison, we examined regulation of Stat6 production in the same
culture conditions (Fig. 2A, lower panel). The
production of Stat6, a molecule critical for the Th2 immune response,
also was inducible and up-regulated synergistically by IFN- and LPS
(lane 5). However, Stat6 expression was unaffected by
IL-4, and only modestly decreased by IL-10 (lanes 6
and 9), indicating that the blockade of Stat4 expression by
these Th2-derived cytokines is relatively specific.
DC express Stat4 protein following activation
DC are an important component of Th1 responses and are major
producers of IL-12 (14). IL-4, used in combination with
GM-CSF, is a standard means to differentiate monocytes into immature DC
(15). Based on the finding that treatment of monocytes
with IL-4 inhibited Stat4 expression, we postulated IL-4-differentiated
DC might also lack Stat4. Peripheral blood monocytes were cultured with
IL-4 and GM-CSF for 7 days, at which time they developed the phenotype
of DC by flow cytometry (CD14-,
CD1a+). Importantly, DC produced by this standard
protocol did not express Stat4 (Fig. 2A, lane
13). We therefore asked if DC could still be activated to express
Stat4. Consequently, we activated DC with the same stimuli that induced
Stat4 in monocytes, namely IFN- and LPS. DC activated by treatment
with IFN- and LPS, like monocytes, were induced to express Stat4
(Fig. 2A, lane 14). Shown as a positive control
for Stat4 expression is lysate from the NK 3.3 cell line
(lane 12).
Stat4 in monocytes is phosphorylated in response to IFN-
Stat-mediated transcriptional activation is dependent upon
phosphorylation of a conserved C-terminal tyrosine residue.
Dimerization then occurs through reciprocal phosphotyrosine/Src
homology 2 (SH2) interaction, a requirement for subsequent DNA binding
and gene induction (16, 17). Having shown that Stat4 was
expressed in activated monocytes, we next investigated what conditions
would result in its activation. Because IL-12 is the classical cytokine
activating Stat4, we first sought to determine whether this cytokine
could activate monocyte-expressed Stat4. As activated monocytes produce
IL-12, this would provide an autocrine means of promoting cell-mediated
immune responses. Nevertheless, although activated monocytes expressed
detectable IL-12R ß1 protein, no IL-12R ß2 protein was detected by
flow cytometry under any condition tested. Not surprisingly, therefore,
we found no detectable IL-12-induced Stat4 phosphorylation in monocytes
under these conditions (data not shown). However, because blockade of
IL-12 reduced monocyte production of Stat4 (Fig. 1E), there
exists the possibility that there may be a short-lived period of IL-12R
ß2 production during monocyte activation which allows this signaling
to occur. Alternatively, high-affinity IL-12R could be present below
the level of detection of FACS, but still at sufficient levels to allow
for some signaling to occur.
Although IL-12 is a major activator of Stat4, it is not the only factor
to induce Stat4 phosphorylation in humans; Stat4 has been shown to be
phosphorylated in response to IFN- in human lymphoid cells (8, 9). We therefore investigated whether IFN- stimulation
initiates phosphorylation of Stat4 in activated monocytes. Purified
monocytes were pretreated with LPS (Fig. 2B, lanes
1–6) for 24 h to induce Stat4 production before stimulation
of selected cultures with IFN- for various periods of time. No
phosphorylation of Stat4 was detected in unstimulated monocytes
(lane 2), but monocytes stimulated with IFN-
showed phosphorylation of Stat4 at 30 min that declined over 4 h
(lanes 3–5). Similarly, in cultures pretreated with
LPS and IFN- to induce even higher levels of Stat4 production
(lanes 7 and 8), phosphorylated Stat4 was
detected following 2 h of IFN- stimulations as well, although
not increased when compared with LPS pretreatment alone. Within each
group representing the two prestimulation conditions, equivalent levels
of Stat4 measured by Western blot were immunoprecipitated from
unstimulated cultures or those stimulated with IFN- (Fig. 2B, bottom). The slower migrating band in the
Stat4 reblot corresponded to phosphorylated Stat4.
Stat4 is expressed by macrophages in rheumatoid synovium
Although Stat4 was present and functional in activated monocytes
in vitro, we wanted to determine whether this was relevant in vivo.
That is, the question still remained whether monocyte-derived cells
express Stat4 in vivo at sites of inflammation in human subjects. To
address this question, we obtained tissue samples from actively
inflamed joints of RA patients, a disease process marked by
Th1-dominated inflammation. In sections of synovial membrane from
affected RA patients, Stat4 staining was observed in the synovial
lining cells (brown, Fig. 3A).
Parallel sections stained for CD14 showed that the cells in this area
were predominantly type A synoviocytes (macrophages) characteristic of
rheumatoid synovial membrane (pink, Fig. 3B)
(18). This finding was confirmed in parallel sections
double-stained for both Stat4 and CD14, in which the cells clearly
showed staining for both molecules (Fig. 3C). No Stat4
staining was observed in experiments including preincubation of the
anti-Stat4 Ab with its cognate peptide (Fig. 3D),
indicating that the observed Stat4 staining was specific. These data
demonstrate that Stat4 expression occurs at sites of inflammation in
humans, and is not simply an in vitro phenomenon.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, but
not by IL-12.
Taken together, our data indicate that the Stat4 production and
phosphorylation are highly regulated in both myeloid and lymphoid
cells. The Stat4 signal transduction pathway is not functional in
unactivated cells of monocyte lineage, because Stat4 is not expressed.
Only after activation is Stat4 present and able to transduce signals in
cells of monocyte lineage, limiting the availability of this signal
transduction pathway to sites of inflammation. Furthermore, because
Stat4 production is negatively regulated by Th2 cytokines,
Stat4-dependent IFN- signaling may be restricted to inflammation
characterized by Th1 predominance. Macrophages are known to develop
different phenotypes and costimulatory functions depending on the
balance between Th1 and Th2 cytokines present during culture
(19). Our data demonstrate that the ability to transduce
Stat4 signals likewise distinguishes monocytes grown in Th1 vs Th2
conditions. Moreover, our results may now explain why Stat4 has been
reported not to be present in unactivated DC in other systems
(14).
Interestingly, murine bone marrow-derived macrophages and DC have been
reported to produce IFN- in response to IL-12 in the same
manner as lymphoid cells (20, 21). Indeed, Stat4 is
phosphorylated in response to lL-12 in murine macrophages and DC.
Moreover, lL-12-dependent IFN- production in these cells is
abrogated in Stat4-deficient mice (unpublished results). In contrast to
murine DC, however, we did not observe IL-12R ß2 protein in human
monocytes, making Stat4-dependent IL-12 signaling unlikely.
Nevertheless, other conditions may exist under which the IL-12 receptor
components and Stat4 are coexpressed in cells of monocyte lineage. In
addition, we have detected only very small levels of IFN- production
by purified peripheral blood monocytes and macrophages. Moreover, we
did not detect IFN- production by human bone marrow-derived
macrophages stimulated with IL-12 and IL-18, which is in stark contrast
to levels produced by murine bone marrow-derived macrophages
(unpublished results).
Naturally, the following question arises: Why do monocytes make Stat4
upon activation? The fact that IFN- induces phosphorylation of Stat4
may address this question. IFN- clearly has been demonstrated to
promote cell-mediated immunity and can drive Th1 differentiation even
in the absence of IL-12 (9, 10). It is possible that some
of these functions require Stat4. Unfortunately, murine knockout models
will have some limitations in assessing the function of
monocyte-derived Stat4 in humans, as Stat4 is not activated in response
to IFN- in the mouse (9). Given the interspecies
differences in IFN- signaling, this issue will need to be addressed
in other ways. Samples from human patients with Stat4 mutations, if
they exist, will be useful for this purpose.
We speculate that Stat4 expression reflects a point of human monocyte
differentiation, facilitating a new signal transduction pathway in the
response to IFN-. In the absence of IL-12-dependent Stat4
phosphorylation, IFN- signaling via Stat4 may occur as part of the
inflammatory process in RA. Indeed it appears that a majority of the
macrophages present in the synovial lining layer of joints affected by
RA express Stat4. Interestingly, a RA disease is reported in patients
receiving IFN- as therapy for malignancies (22).
Because monocytes and their descendents are central to the
granulomatous response to intracellular infections and in certain
autoimmune diseases, our data suggest the intriguing possibility that
Stat4 signaling in monocytes may be relevant in other disease
processes. Stat4 gene targets could be predicted to overlap those
involved in Th1 function of lymphocytes, but may also include genes
specific only to myeloid cells. Therefore, determining the gene targets
of Stat4 will be essential in ascertaining its role in myeloid cells.
Although the function of Stat4 in murine myeloid cells is becoming
clearer, its function in human myeloid cells is yet to be defined.
We demonstrate that upon activation, in Th1-dominated sites of
inflammation, Stat4 is present in monocytes and available for
phosphorylation by IFN-. Although the role of Stat4 in the
IL-12-dependent activation and differentiation of T cells and NK cells
is well described, Stat4 expression in cells of monocyte lineage was
previously unrecognized. Our data demonstrate that IFN- signaling
via Stat4 is a transduction pathway common to both lymphocytes and
monocytes and complements the action of IL-12 signaling where IL-12 or
its receptor are unavailable. In this manner, it may represent an
important pathway in the early steps of the cellular immune response
and is a potential target for therapeutic intervention.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David M. Frucht, Building 10, Room 9N262, 9000 Rockville Pike, Bethesda, MD 20892.
3 Abbreviations used in this paper: DC, dendritic cells; hu, human; RA, rheumatoid arthritis.
Received for publication September 24, 1999. Accepted for publication February 16, 2000.
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interferon activation site-binding protein expressed in early myeloid differentiation. Mol. Cell. Biol. 14:4342.. Annu. Rev. Immunol. 15:749.[Medline]
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A. Suto, H. Nakajima, N. Tokumasa, H. Takatori, S.-i. Kagami, K. Suzuki, and I. Iwamoto Murine Plasmacytoid Dendritic Cells Produce IFN-{gamma} upon IL-4 Stimulation J. Immunol., November 1, 2005; 175(9): 5681 - 5689. [Abstract] [Full Text] [PDF]
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 T. R. Kataoka, N. Komazawa, E. Morii, K. Oboki, and T. Nakano Involvement of connective tissue-type mast cells in Th1 immune responses via Stat4 expression Blood, February 1, 2005; 105(3): 1016 - 1020. [Abstract] [Full Text] [PDF]
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J. C. Deng, X. Zeng, M. Newstead, T. A. Moore, W. C. Tsai, V. J. Thannickal, and T. J. Standiford STAT4 Is a Critical Mediator of Early Innate Immune Responses against Pulmonary Klebsiella Infection J. Immunol., September 15, 2004; 173(6): 4075 - 4083. [Abstract] [Full Text] [PDF]
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 A. Di Stefano, G. Caramori, A. Capelli, I. Gnemmi, F.L. Ricciardolo, T. Oates, C.F. Donner, K.F. Chung, P.J. Barnes, and I.M. Adcock STAT4 activation in smokers and patients with chronic obstructive pulmonary disease Eur. Respir. J., July 1, 2004; 24(1): 78 - 85. [Abstract] [Full Text] [PDF]
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 N. Torpey, S. E. Maher, A. L. M. Bothwell, and J. S. Pober Interferon {alpha} but Not Interleukin 12 Activates STAT4 Signaling in Human Vascular Endothelial Cells J. Biol. Chem., June 18, 2004; 279(25): 26789 - 26796. [Abstract] [Full Text] [PDF]
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C. M. U. Hilkens, J. F. Schlaak, and I. M. Kerr Differential Responses to IFN-{alpha} Subtypes in Human T Cells and Dendritic Cells J. Immunol., November 15, 2003; 171(10): 5255 - 5263. [Abstract] [Full Text] [PDF]
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 G. Lugo-Villarino, R. Maldonado-Lopez, R. Possemato, C. Penaranda, and L. H. Glimcher T-bet is required for optimal production of IFN-{gamma} and antigen-specific T cell activation by dendritic cells PNAS, June 24, 2003; 100(13): 7749 - 7754. [Abstract] [Full Text] [PDF]
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L. Kari, A. Loboda, M. Nebozhyn, A. H. Rook, E. C. Vonderheid, C. Nichols, D. Virok, C. Chang, W.-H. Horng, J. Johnston, et al. Classification and Prediction of Survival in Patients with the Leukemic Phase of Cutaneous T Cell Lymphoma J. Exp. Med., June 2, 2003; 197(11): 1477 - 1488. [Abstract] [Full Text] [PDF]
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K. Raman, M. H. Kaplan, C. M. Hogaboam, A. Berlin, and N. W. Lukacs STAT4 Signal Pathways Regulate Inflammation and Airway Physiology Changes in Allergic Airway Inflammation Locally Via Alteration of Chemokines J. Immunol., April 1, 2003; 170(7): 3859 - 3865. [Abstract] [Full Text] [PDF]
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J. J. Kohler, D. L. Tuttle, C. R. Coberley, J. W. Sleasman, and M. M. Goodenow Human immunodeficiency virus type 1 (HIV-1) induces activation of multiple STATs in CD4+ cells of lymphocyte or monocyte/macrophage lineages J. Leukoc. Biol., March 1, 2003; 73(3): 407 - 416. [Abstract] [Full Text] [PDF]
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 I. Sugawara, H. Yamada, and S. Mizuno Relative importance of STAT4 in murine tuberculosis J. Med. Microbiol., January 1, 2003; 52(1): 29 - 34. [Abstract] [Full Text] [PDF]
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E. Kuroda, T. Kito, and U. Yamashita Reduced Expression of STAT4 and IFN-{gamma} in Macrophages from BALB/c Mice J. Immunol., June 1, 2002; 168(11): 5477 - 5482. [Abstract] [Full Text] [PDF]
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 D. M. Frucht IL-23: A Cytokine That Acts on Memory T Cells Sci. Signal., January 8, 2002; 2002(114): pe1 - pe1. [Abstract] [Full Text] [PDF]
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 J. Maier, C. Kincaid, A. Pagenstecher, and I. L. Campbell Regulation of Signal Transducer and Activator of Transcription and Suppressor of Cytokine-Signaling Gene Expression in the Brain of Mice with Astrocyte-Targeted Production of Interleukin-12 or Experimental Autoimmune Encephalomyelitis Am. J. Pathol., January 1, 2002; 160(1): 271 - 288. [Abstract] [Full Text] [PDF]
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A. A. Lighvani, D. M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, B. D. Hissong, B. V. Nguyen, M. Gadina, A. Sher, W. E. Paul, et al. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells PNAS, December 18, 2001; 98(26): 15137 - 15142. [Abstract] [Full Text] [PDF]
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U. Grohmann, M. L. Belladonna, C. Vacca, R. Bianchi, F. Fallarino, C. Orabona, M. C. Fioretti, and P. Puccetti Positive Regulatory Role of IL-12 in Macrophages and Modulation by IFN-{{gamma}} J. Immunol., July 1, 2001; 167(1): 221 - 227. [Abstract] [Full Text] [PDF]
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T. Fukao, D. M. Frucht, G. Yap, M. Gadina, J. J. O'Shea, and S. Koyasu Inducible Expression of Stat4 in Dendritic Cells and Macrophages and Its Critical Role in Innate and Adaptive Immune Responses J. Immunol., April 1, 2001; 166(7): 4446 - 4455. [Abstract] [Full Text] [PDF]
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H. Schindler, M. B. Lutz, M. Rollinghoff, and C. Bogdan The Production of IFN-{{gamma}} by IL-12/IL-18-Activated Macrophages Requires STAT4 Signaling and Is Inhibited by IL-4 J. Immunol., March 1, 2001; 166(5): 3075 - 3082. [Abstract] [Full Text] [PDF]
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