# M-CSF and GM-CSF Regulation of STAT5 Activation and  DNA Binding in Myeloid Cell Differentiation is Disrupted in Nonobese Diabetic Mice

Authors: B. Rumore-Maton; J. Elf; N. Belkin; B. Stutevoss; F. Seydel; E. Garrigan; S. A. Litherland
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Defects in macrophage colony-stimulating factor (M-CSF) signaling disrupt myeloid cell differentiation in nonobese diabetic (NOD) mice, blocking myeloid maturation into tolerogenic antigen-presenting cells (APCs). In the absence of M-CSF signaling, NOD myeloid cells have abnormally high granulocyte macrophage colony-stimulating factor (GM-CSF) expression, and as a result, persistent activation of signal transducer/activator of transcription 5 (STAT5). Persistent STAT5 phosphorylation found in NOD macrophages is not affected by inhibiting GM-CSF. However, STAT5 phosphorylation in NOD bone marrow cells is diminished if GM-CSF signaling is blocked. Moreover, if M-CSF signaling is inhibited, GM-CSF stimulationin vitro can promote STAT5 phosphorylation in nonautoimmune C57BL/6 mouse bone marrow cultures to levels seen in the NOD. These findings suggest that excessive GM-CSF production in the NOD bone marrow may interfere with the temporal sequence of GM-CSF and M-CSF signaling needed to mediate normal STAT5 function in myeloid cell differentiation gene regulation.


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## Body

## 1. Introduction

Myeloid cell differentiation gives rise
to 3 populations of professional antigen-presenting cells (APC) in the immune system:monocytes, macrophages, and dendritic cells (DC).  Sequential signaling from interleukin-3 (IL-3), granulocyte-macrophage-,  macrophage-, and granulocyte-colony
stimulating factors (GM-, M-, and G-CSF, respectively) sets the microenvironment
necessary for bone marrow myeloid precursor cells to differentiate and function
[1]. The timing and level of each cytokine in myeloid cell microenvironmentare tightly controlled in order for myeloid cells differentiate to functional APC, and to prevent the premature activation of genes involved in macrophage activation, such as cyclooxygenase/prostaglandin synthase 2 (COX
2/PGS2), which could promote deleterious inflammation.The cytokine GM-CSF has a unique dual influence in myeloid cells, acting first as a differentiation factor in myeloid hematopoiesis, and then later as an activation stimulus in mature monocytes and macrophages. Both overexpression and knockout deletion of GM-CSF in mice can lead to dysregulation of myeloid differentiation and
autoimmune disease [2], suggesting that GM-CSF influence exerts discrete temporal and quantitative regulatory effects in myeloid cell differentiation and mature cell activation. This switch in GM-CSF function relies heavily on a change in responsiveness of cells to GM-CSF before and after M-CSF stimulation [3], and on the ability of GM-CSF to activate STAT5 phosphorylation at different stages of myeloid cell maturation [4, 5].Work by Piazza et al. [4] suggests that
STAT5 proteins can act as key cytokine and growth factor-inducible regulatory “switches”
for gene expression during myeloid differentiation and activation. In early
myeloid differentiation stages, IL-3 and GM-CSF can induce signaling through
both STAT5A (94-96kD) and B (94-92kD) isoforms which act as adaptor molecules
for histone acetylases and deacetylases [4–7]. Subsequent signaling
through M-CSF and G-CSF signals in matured and activated cells also activates
STAT5 [4, 8, 9]. Thus, during cytokine-induced differentiation, STAT5 can act
to promote both gene transcriptional regulation through epigenetic chromatin dynamics [4, 6, 7].Incomplete myeloid differentiation is emerging as a common component of multiple
autoimmune pathologies, both in mice and man. Serreze et al. (1993) [10] found
that myeloid antigen presenting cell (APC) differentiation is impaired in NOD
mouse bone marrow due to a lack of responsiveness to M-CSF. This
nonresponsiveness was not linked to any defect in M-CSF expression or defect in
its receptor, but to unknown problems with M-CSF intracellular signaling. Morin
et al. (2003) [11] also noted that high GM-CSF can skew NOD myeloid
differentiation away from macrophage and DC development, and toward an excess of
granulocyte production. Several laboratories have reported disproportion of
immature DC compared to mature DC in the diabetic NOD, a process dependent on
regulation of GM-CSF signaling [10–13]. These dysfunctional
APC phenotypes suggest that NOD myeloid cell differentiation and activation are
defective at a point or points, where the determination of specific cell lineage decisions is made based on the
cytokine microenvironment [1, 4]. Similar defective myeloid cell phenotypes
have been seen in human autoimmune diseases such as T1D, SLE, and autoimmune
thyroid diseases (ATD) [10–15]. The result
of these defective myeloid cell properties is a loss of professional APC that
can efficiently promote tolerance and regulate appropriate macrophage
inflammatory responses [12, 16, 17]. Loss of these functions sets up an immune
microenvironment conducive to immunopathogenesis.In our studies of APC dysfunction in
autoimmunity, we have found that both NOD and autoimmune human myeloid cells
express abnormally high levels of GM-CSF and have prolonged STAT5 activation
after even brief exposure to GM-CSF [18–20].In these studies, we test the potential
connection of GM-CSF signaling and its activation of STAT5 isoforms with the
defect in M-CSF signaling in NOD myeloid differentiation in vitro. We find that both GM-CSF
overproduction and persistent STAT5 phosphorylation phenotypes are amplified in
autoimmune NOD bone marrow cells, but can be overridden by in vitro inhibition of endogenous
GM-CSF signaling. Furthermore, the persistent STAT5 phosphorylation phenotype
seen in NOD myeloid cells can be recreated in non-autoimmune C57BL/6 bone
marrow cultures by blocking M-CSF while stimulating with NOD level GM-CSF. These
findings in vitro suggest that
the defect in NOD M-CSF signaling in
vivo may occur
during the switch of influence between GM-CSF to M-CSF during myeloid
differentiation, and indicate that prolonged expression of GM-CSF promotes the
dysregulation of STAT5, which may interfere with M-CSF signaling during NOD myeloid
cell differentiation.


## 2. Methods and Materials

### 2.1. Mouse Strains Used

Eight to twelve-week old NOD and C57BL/6
female mice (The Jackson Laboratory, Bar Harbor, Mich, USA)
were used for all studies. These strains were maintained as breeding stock in
our Pathology Department SPF colony, in microisolator cages with food and water
ad libium. All procedures were conduced to maintain humane treatment of all
animals according to IACUC approved protocols B083 and D754, and humanely
euthanized by cervical dislocation while over-anesthetized. All tissue collections were
done postmortem. A minimum of 3 animals per strain was used per treatment for each run of each
experiment.


### 2.2. Bone Marrow Differentiation Culture and Sample Preparation

Long bones from the legs were collected postmortem into cold RPMI +?10%FCS medium (Cellgro, MediaTech, Manassas, Va, USA)
and the marrow flushed into a sterile
tube by cold medium injected into the bone through a 30-gauge needle. The
marrow samples were washed with cold media by centrifugation and red blood
cells in samples lysed by incubation in nonisotonic 0.84% ammonium chloride buffer
(Fisher Scientific, Pittsburgh, Pa, USA).
The remaining bone marrow cells are then plated at 5–10 million
cells/well on tissue culture dishes and fed with fresh sterile medium alone or with
medium containing 1000?U/mL of GM-CSF (In vitrogen-Biosource, Carlsbad, Calif,
USA) +?2?µg/mL
anti-M-CSF blocking antibodies (Pierce-Endogen, Rockford, Ill, USA),or medium containing 500?U/mL M-CSF (In
vitrogen) + anti-GM-CSF blocking antibodies (Pierce-Endogen). Cultures were
maintained for 24 hours at 37C°/5%CO2, then washed, and resupplemented for an additional 24 hours in culture at 37C°/5%CO2. Cells were then analyzed
for myeloid phenotypic identification and STAT5 phosphorylation by flow
cytometry and deconvolution microscopy.


### 2.3. Flow Cytometric and Deconvolution Microscopic Image Analysis

Flow cytometric analysis of fluorescently conjugated antibody
surface and intracellular binding was used to phenotypically identify myeloid cells and quantify
the level of STAT5 tyrosine phosphorylation (STAT5Ptyr) in cellsex vivo and after in vitro stimulation. Cells were
first labeled with anti-CD11b antibody FITC or PE conjugates (BD Biosciences, Calif, USA) to identify
monocytes and macrophages in the sample, then fixed with 2% (v/v?Cf)
formaldehyde (Sigma-Aldrich, St. Louis, Mo, USA) and permeabilized using 0.5% saponin
(Sigma-Aldrich) in a high protein, isotonic FACS buffer [14]. Intracellular staining
of STAT5Ptyr was done using FITC or APC-conjugated anti-STAT5Ptyr-specific
monoclonal antibodies  (Millipore-Upstate monoclonal antibody conjugated FITC (Millipore-Upstate) or with APC using labeling kit, Prozyme, San Leandro, Calif,
USA). Data were analyzed as the percentage of positive myeloid cells (%STATPtyr+/CD11b+) to
assess the number of cells in the  myeloid population with activated STAT5.After flow analysis,
the labeled cells are centrifuged onto slides and counterstained for chromatin (DAPI, Invitrogen-Molecular
Probes, Carlsbad, Calif, USA).
These cells were used for imaging by deconvolution microscopy as previously
described [19, 20] for 3-dimensional projection rotational reconstruction image
analysis to identify subcellular location of activated STAT5. Statistical
analyses of flow cytometric data were performed using Prism 5 software
(GraphPad, San Diego, Calif,
USA), with pairwise comparisons being done using Student’s t-test or Mann-Whitney U
analyses and multiple group analyses done using one-way ANOVA.


### 2.4. Double Chromatin Immunoprecipitation (dbChIP) of Chromatin-Bound Phospho-STAT5 Protein

Five million bone marrow cells
were cultured with or without 100?µM Na vanadate (in DMSO, Sigma-Aldrich) for 30 minutes at 378C°/5%CO2. Half of the cultures +/- vanadate were then supplemented with 1000?U/mL
GM-CSF, and all were incubated for an additional 90 minutes at 378C°/5%CO2. The cells were then fixed in situ, with 1% formaldehyde in 1xPBS (methanol-free,
Sigma-Aldrich),
for 10 minutes at 37C° prior to lysis with high SDS lysis buffer (Millipore-Upstate). The
fixed lysates were sonicated to disrupt membranes and shear chromatin to
approximately 1000?bp fragments then frozen at -80C° until analyzed. The sample
was split into five
1×106 cell aliquots for use in anti-STAT5Ptyr/anti-Histone H3
mediated double ChIP (dbChIP) protein isolations for Western blot analysis of
STAT5 associated with histone/chromatin complexes. Aliquots used for dbChIP were
precleared with salmon sperm DNA Protein A or G agarose beads (Millipore-Upstate), then
incubated overnight at 4C° with antityrosine phosphorylated STAT5 (STAT5Ptyr)
antibodies (Millipore-Upstate). After incubation, the antibody-bound chromatin
complexes were precipitated using salmon sperm DNA Protein A agarose beads, and
washed extensively with a series of increasing stringency
buffers (low salt, high salt, LiCl, TE; ChIP reagent kit, Millipore-Upstate).
A nonspecific antibody control ChIP (mouse IgG, Millipore-Upstate) and a sham IP containing no
extract were used as negative controls.ChIP extract aliquots dissociated from the beads in 1%SDS, 0.1?M bicarbonate
buffer (Fisher Scientific).
To both unprecipitated (total) extracts
and ChIP samples, NaCl was added to a final concentration of 500?mM and the
samples were
incubated for 4 hours
at 65C° to reverse the formaldehyde cross-links. The released anti-STAT5Ptyr-selected
complexes were neutralized and reprecipitated (“double ChIP”, dbChIP) overnight
at 4C° using precoupled anti-Histone H3-Protein G Salmon sperm DNA-coated beads
(Millipore-Upstate).
The dbChIP complexes were then washed with the same sequence of buffers as used
in the first ChIP, and then boiled in 1x Leammili buffer (BioRad, Hercules, Calif, USA) for Western
blot analysis. Western blots were probed with anti-STAT5 Ptyr (Millipore-Upstate)
antibodies and cross-linked and uncoupled proteins were visualized using
Amersham ECL plus chemilumenescence reagents (GE Healthcare/Amersham, Piscataway, NJ, USA).
Densitometric analysis using BioRad
Imager and Quantity One Software was used to estimate molecular weight of
cross-linked dimer complexes and freed monomeric isoforms in STAT5Ptyr positive
bands.


## 2.1. Mouse Strains Used

Eight to twelve-week old NOD and C57BL/6
female mice (The Jackson Laboratory, Bar Harbor, Mich, USA)
were used for all studies. These strains were maintained as breeding stock in
our Pathology Department SPF colony, in microisolator cages with food and water
ad libium. All procedures were conduced to maintain humane treatment of all
animals according to IACUC approved protocols B083 and D754, and humanely
euthanized by cervical dislocation while over-anesthetized. All tissue collections were
done postmortem. A minimum of 3 animals per strain was used per treatment for each run of each
experiment.


## 2.2. Bone Marrow Differentiation Culture and Sample Preparation

Long bones from the legs were collected postmortem into cold RPMI +?10%FCS medium (Cellgro, MediaTech, Manassas, Va, USA)
and the marrow flushed into a sterile
tube by cold medium injected into the bone through a 30-gauge needle. The
marrow samples were washed with cold media by centrifugation and red blood
cells in samples lysed by incubation in nonisotonic 0.84% ammonium chloride buffer
(Fisher Scientific, Pittsburgh, Pa, USA).
The remaining bone marrow cells are then plated at 5–10 million
cells/well on tissue culture dishes and fed with fresh sterile medium alone or with
medium containing 1000?U/mL of GM-CSF (In vitrogen-Biosource, Carlsbad, Calif,
USA) +?2?µg/mL
anti-M-CSF blocking antibodies (Pierce-Endogen, Rockford, Ill, USA),or medium containing 500?U/mL M-CSF (In
vitrogen) + anti-GM-CSF blocking antibodies (Pierce-Endogen). Cultures were
maintained for 24 hours at 37C°/5%CO2, then washed, and resupplemented for an additional 24 hours in culture at 37C°/5%CO2. Cells were then analyzed
for myeloid phenotypic identification and STAT5 phosphorylation by flow
cytometry and deconvolution microscopy.


## 2.3. Flow Cytometric and Deconvolution Microscopic Image Analysis

Flow cytometric analysis of fluorescently conjugated antibody
surface and intracellular binding was used to phenotypically identify myeloid cells and quantify
the level of STAT5 tyrosine phosphorylation (STAT5Ptyr) in cellsex vivo and after in vitro stimulation. Cells were
first labeled with anti-CD11b antibody FITC or PE conjugates (BD Biosciences, Calif, USA) to identify
monocytes and macrophages in the sample, then fixed with 2% (v/v?Cf)
formaldehyde (Sigma-Aldrich, St. Louis, Mo, USA) and permeabilized using 0.5% saponin
(Sigma-Aldrich) in a high protein, isotonic FACS buffer [14]. Intracellular staining
of STAT5Ptyr was done using FITC or APC-conjugated anti-STAT5Ptyr-specific
monoclonal antibodies  (Millipore-Upstate monoclonal antibody conjugated FITC (Millipore-Upstate) or with APC using labeling kit, Prozyme, San Leandro, Calif,
USA). Data were analyzed as the percentage of positive myeloid cells (%STATPtyr+/CD11b+) to
assess the number of cells in the  myeloid population with activated STAT5.After flow analysis,
the labeled cells are centrifuged onto slides and counterstained for chromatin (DAPI, Invitrogen-Molecular
Probes, Carlsbad, Calif, USA).
These cells were used for imaging by deconvolution microscopy as previously
described [19, 20] for 3-dimensional projection rotational reconstruction image
analysis to identify subcellular location of activated STAT5. Statistical
analyses of flow cytometric data were performed using Prism 5 software
(GraphPad, San Diego, Calif,
USA), with pairwise comparisons being done using Student’s t-test or Mann-Whitney U
analyses and multiple group analyses done using one-way ANOVA.


## 2.4. Double Chromatin Immunoprecipitation (dbChIP) of Chromatin-Bound Phospho-STAT5 Protein

Five million bone marrow cells
were cultured with or without 100?µM Na vanadate (in DMSO, Sigma-Aldrich) for 30 minutes at 378C°/5%CO2. Half of the cultures +/- vanadate were then supplemented with 1000?U/mL
GM-CSF, and all were incubated for an additional 90 minutes at 378C°/5%CO2. The cells were then fixed in situ, with 1% formaldehyde in 1xPBS (methanol-free,
Sigma-Aldrich),
for 10 minutes at 37C° prior to lysis with high SDS lysis buffer (Millipore-Upstate). The
fixed lysates were sonicated to disrupt membranes and shear chromatin to
approximately 1000?bp fragments then frozen at -80C° until analyzed. The sample
was split into five
1×106 cell aliquots for use in anti-STAT5Ptyr/anti-Histone H3
mediated double ChIP (dbChIP) protein isolations for Western blot analysis of
STAT5 associated with histone/chromatin complexes. Aliquots used for dbChIP were
precleared with salmon sperm DNA Protein A or G agarose beads (Millipore-Upstate), then
incubated overnight at 4C° with antityrosine phosphorylated STAT5 (STAT5Ptyr)
antibodies (Millipore-Upstate). After incubation, the antibody-bound chromatin
complexes were precipitated using salmon sperm DNA Protein A agarose beads, and
washed extensively with a series of increasing stringency
buffers (low salt, high salt, LiCl, TE; ChIP reagent kit, Millipore-Upstate).
A nonspecific antibody control ChIP (mouse IgG, Millipore-Upstate) and a sham IP containing no
extract were used as negative controls.ChIP extract aliquots dissociated from the beads in 1%SDS, 0.1?M bicarbonate
buffer (Fisher Scientific).
To both unprecipitated (total) extracts
and ChIP samples, NaCl was added to a final concentration of 500?mM and the
samples were
incubated for 4 hours
at 65C° to reverse the formaldehyde cross-links. The released anti-STAT5Ptyr-selected
complexes were neutralized and reprecipitated (“double ChIP”, dbChIP) overnight
at 4C° using precoupled anti-Histone H3-Protein G Salmon sperm DNA-coated beads
(Millipore-Upstate).
The dbChIP complexes were then washed with the same sequence of buffers as used
in the first ChIP, and then boiled in 1x Leammili buffer (BioRad, Hercules, Calif, USA) for Western
blot analysis. Western blots were probed with anti-STAT5 Ptyr (Millipore-Upstate)
antibodies and cross-linked and uncoupled proteins were visualized using
Amersham ECL plus chemilumenescence reagents (GE Healthcare/Amersham, Piscataway, NJ, USA).
Densitometric analysis using BioRad
Imager and Quantity One Software was used to estimate molecular weight of
cross-linked dimer complexes and freed monomeric isoforms in STAT5Ptyr positive
bands.


## 3. Results

### 3.1. GM-CSF and M-CSF Signaling Interplay to Regulate STAT5 Activation

Mouse bone marrow
cells can differentiate to mature different macrophage phenotypes within 4 days
culture in M-CSF or 4–7 days in GM-CSF
[3, 21, 22]. Our experiments attempted to examine myeloid differentiation at
stages prior to complete maturation, at 2 to 48 hours in culture. For our in vitro myeloid cell differentiation
experiments, NOD and C57BL/6 mouse bone marrow cells were stimulated with one
cytokine (1000?U/mL GM-CSF or 500?U/mL M-CSF) in the presence of blocking
antibodies for the other cytokine (anti-GM-CSF or anti-M-CSF, 2?µg/mL) for 48 hours at 37C°/5%CO2.In earlier
studies, we found that blocking GM-CSF signaling by anti-GM-CSF antibodies or
with the Jak2/3 inhibitor AG490 in mature monocytes and macrophages did not
affect the persistence of phosphorylated STAT5 [19]. In contrast, blocking endogenous GM-CSF or
M-CSF stimulation by specific antibodies significantly diminished STAT5
phosphorylation in NOD bone marrow cells (p*=.0240, ANOVA, Figure 1(a)).Flow cytometric analysis of STAT5 phosphorylation inin vitro myeloid differentiation. (a) Bone marrow cultures (1 million cells/mL)
from NOD and C57BL/6 control mice were differentiated in culture with either, (b)
1000?U/mL GM-CSF plus 2µg/mL
anti-M-CSF blocking antibodies a-M),
or (c) with 500?U/mL M-CSF with 2µg/mL
anti-GM-CSF blocking antibodies a-GM),
for 48 hours at 3C°/5CO2.
Cultures treated with medium only (“media
only” in (a), “0” in (b) and (c))
or with medium supplemented only with antibodies to block M-CSF or GM-CSF (ANTI-M-CSF, ANTI-GM-CSF, respectively, in (a)) were run in parallel with the
cytokine-stimulated cultures. In addition, separate aliquots of bone marrow
cells were cultured in M-CSF or GM-CSF alone without blocking antibodies (“no” in (b) and (c)). At 48 hours, cells were
stained with anti-STAT5Ptyr-FITC and anti-CD11b-PE antibodies for analysis by
intracellular immune-histochemical flow cytometry. Strain and treatment
regiments for each pair of cultures are listed on the X-axis. The percentage of
STAT5Ptyr+ in CD11b+ cells is given on the Y-axis. Graphs are representative of
3–9 separate runs
of each treatment. The p-values indicated were derived from Mann-Whitney
U test, Student’s t-test, or one-way ANOVA analyses, as appropriate, for
the sample group comparisons indicated by brackets.
(a)(b)Deconvolution
microscopic image analysis (see Figure2) suggested that we could recreate the NOD STAT5 phosphorylation
phenotype in non-autoimmune C57BL/6 cells by blocking M-CSF signaling while
increasing the GM-CSF stimulation to the level seen in the NOD [19, 20]. Flow
cytometric analysis also indicated that blocking endogenous M-CSF while stimulating with GM-CSF promoted
STAT5 phosphorylation in C57BL/6 48-hour bone marrow cultures (p*=.0167, Mann-Whitney U test, Figure 1(b)).
However, when we stimulated bone marrow cultures with M-CSF in vitro in the presence of blocking
antibodies to GM-CSF, STAT5 phosphorylation was also stimulated in C57BL/6
cultures, and remained statistically unchanged in NOD bone marrow cells (p*=.0336
and .5556, respectively, Mann-Whitney U test, Figure 2(c)).Figure 2
GM-CSF/M-CSF
cytokine-induced myeloid differentiationin
vitro. Bone marrow cultures
from NOD and C57BL/6 control mice were differentiated in culture using regiment
of cytokine and anticytokine blocking antibodies. Cells were treated with
either 1000?U/mL GM-CSF plus 2µg/mL anti-M-CSF blocking antibodies,
with 500?U/mL M-CSF plus 2µg/mL anti-GM-CSF blocking antibodies, or
with media alone for 48 hours at 3C°/5CO2. Cells were stained
with anti-STAT5Ptyr-FITC (green) and anti-CD11b-PE (red) antibodies for
analysis by flow cytometry (summarized in Figure 1) and deconvolution microscopy (shown here) for the
development of cells with myeloid phenotype (CD11b+) and intracellular
expression of tyrosine phosphorylated STAT5. Background staining for
anti-STAT5Ptyr-FITC antibody was determined by parallel sample stained with a
nonspecific mouse IgG isotype control (bottom most panels). The images are composites
of 3D projection of 20 optical sections (0.2 micron each) showing both immunohistochemical
labels as well as DAPI (blue) poststaining of chromatin within the cells. Accompanying
panels show the STAT5Ptyr-FITC staining alone (green) or CD11b-PE and DAPI
staining (red/blue). Treatment regiment and the cell source strain for each
pair of cultures are listed to the left of the images. Images are representative of 5
random fields observed per sample treatment and 3 separate runs of the
experiment.Taken together, these
findings suggest that the interplay M-CSF and GM-CSF signaling is required to
properly regulate STAT5 function, and support the role of temporally sequenced
GM-CSF and M-CSF signaling on controlling STAT5 activation during myeloid cell
differentiation. Furthermore, this interplay is modulated by the timing and
concentration of GM-CSF stimulation early in differentiation, which appears to
set the stage for STAT5 activation in later stages, regardless of further
stimulation by GM-CSF or M-CSF. Because the NOD cells have higher
endogenous GM-CSF production [19], this temporal signaling pattern is disrupted
and leads to aberrant cell responses to GM-CSF.


### 3.2. NOD Bone Marrow Cell DNA Binding of STAT5 Is Increased by GM-CSF

To evaluate the possibility that
increased STAT5 phosphorylation could lead to increased chromatin binding in
response to GM-CSF, we performed double ChIP analyses on NOD and C57BL/6 bone
marrow cells that had been cultured with GM-CSF for 2 hours, and looked forin situ assembly of
chromatin-associated protein complexes which contain both STAT5 and Histone H3 (Figure 3). We controlled for the
possibility of the prolonged phosphorylation of cytoplasmic DNA-unbound STAT5
masking a more transient phosphorylation of DNA-bound STAT5 isoforms, by
culturing these cells in the presence and absence of the phosphatase inhibitor,
sodium vanadate. In these dbChIP analyses, we detected bands containing
phosphorylated STAT5 only in GM-CSF-treated NOD cells (Figure 3). The presence or absence of the phosphatase inhibitor
did not alter the banding pattern in either NOD or C57BL/6 cell extracts. Higher
molecular weight STAT5-containing complexes were also detected in both NOD and C57BL/6 cell dbChIP
isolates, though at markedly different levels. The level of these
complexes in C57BL/6 dbChIP isolates did not significantly change with GM-CSF
and/or vanadate treatment. These higher molecular weight bands may be an
artifact or represent complexes containing formaldehyde cross-linked dimers of
STAT5 in multiple size isoforms (i.e., STAT5A and STAT5B homo- and heterodimers,
either in full-length or truncated isoforms).
No bands at any of the sizes predicted for monomeric STAT5A, STAT5B, or
truncated isoforms were seen associated with histone H3 in any of the
treatments used in C57BL/6 cell cultures. These findings suggest that even
though STAT5 can be activated in both mouse strain bone marrow cells by GM-CSF
if M-CSF signaling is blocked, functional STAT5 binding on chromatin is
inducible in NOD but not C57BL/6 bone marrow cells under these conditions.Figure 3
STAT5 binding to chromatin increases after GM-CSF stimulation in NOD but not C57BL/6 bone marrow cells. Five million bone marrow cells were cultured
with or without 100µM Na vanadate for 30 minutes at 37C°/5CO2.
Half of the cultures+/- vanadate were then supplemented with 1000?U/mL GM-CSF, and all were
incubated for an additional 90 minutes at 37C°/5CO2. The cells were then fixed in situ, extracted, and sonicated.
The sample was split into five 1×106 cell aliquots for use in
anti-STAT5Ptyr/anti-Histone H3-mediated double ChIP protein isolations for Western
blot analysis of STAT5 associated with histone/chromatin complexes. Protein
isolated from the precipitated chromatin complexes and analyzed by Western blot
probed with anti-STAT5Ptyr antibody. Densitometric/Rf-value anti-STAT5Ptyr Western blot-detected bands separated on 4-20% SDS-PAGE were used to give the approximate
size and location of the STAT5 protein complexes and monomers (approximate
molecular weights indicated on the left of the figure). Higher molecular weight
bands that bound the anti-STAT5Ptyr antibody had Rf values suggesting that they
may represent a mix of formaldehyde-cross-linked dimer complexes containing mixed isoforms of STAT5 (STAT5A (A
96?K), STAT5B (B 92?K), and truncated (T 80?K, 77?K)), both in homodimers (A-A
192?K, B-B 184?K, T-T) and heterodimers (A-B, A-T, B-T, T-T) complexes. Only
monomeric STAT5A size bands (96?K) are detected in the GM-CSF-induced NOD cell
cultures at this early time point. The positions of the precipitating
antibodies heavy chain (50?K) and light chain (25?K), which acts as an internal
protein loading standards, were determined from the extract minus sham control
(sh) and size standards
(indicated but not shown). Treatment/lane key: 0 = untreated, V =
vanadate alone, G = GM-CSF
alone, VG = vanadate and GM-CSF-supplemented
2-hour cultures. Data represents 3 runs
of the double ChIP analysis.


## 3.1. GM-CSF and M-CSF Signaling Interplay to Regulate STAT5 Activation

Mouse bone marrow
cells can differentiate to mature different macrophage phenotypes within 4 days
culture in M-CSF or 4–7 days in GM-CSF
[3, 21, 22]. Our experiments attempted to examine myeloid differentiation at
stages prior to complete maturation, at 2 to 48 hours in culture. For our in vitro myeloid cell differentiation
experiments, NOD and C57BL/6 mouse bone marrow cells were stimulated with one
cytokine (1000?U/mL GM-CSF or 500?U/mL M-CSF) in the presence of blocking
antibodies for the other cytokine (anti-GM-CSF or anti-M-CSF, 2?µg/mL) for 48 hours at 37C°/5%CO2.In earlier
studies, we found that blocking GM-CSF signaling by anti-GM-CSF antibodies or
with the Jak2/3 inhibitor AG490 in mature monocytes and macrophages did not
affect the persistence of phosphorylated STAT5 [19]. In contrast, blocking endogenous GM-CSF or
M-CSF stimulation by specific antibodies significantly diminished STAT5
phosphorylation in NOD bone marrow cells (p*=.0240, ANOVA, Figure 1(a)).Flow cytometric analysis of STAT5 phosphorylation inin vitro myeloid differentiation. (a) Bone marrow cultures (1 million cells/mL)
from NOD and C57BL/6 control mice were differentiated in culture with either, (b)
1000?U/mL GM-CSF plus 2µg/mL
anti-M-CSF blocking antibodies a-M),
or (c) with 500?U/mL M-CSF with 2µg/mL
anti-GM-CSF blocking antibodies a-GM),
for 48 hours at 3C°/5CO2.
Cultures treated with medium only (“media
only” in (a), “0” in (b) and (c))
or with medium supplemented only with antibodies to block M-CSF or GM-CSF (ANTI-M-CSF, ANTI-GM-CSF, respectively, in (a)) were run in parallel with the
cytokine-stimulated cultures. In addition, separate aliquots of bone marrow
cells were cultured in M-CSF or GM-CSF alone without blocking antibodies (“no” in (b) and (c)). At 48 hours, cells were
stained with anti-STAT5Ptyr-FITC and anti-CD11b-PE antibodies for analysis by
intracellular immune-histochemical flow cytometry. Strain and treatment
regiments for each pair of cultures are listed on the X-axis. The percentage of
STAT5Ptyr+ in CD11b+ cells is given on the Y-axis. Graphs are representative of
3–9 separate runs
of each treatment. The p-values indicated were derived from Mann-Whitney
U test, Student’s t-test, or one-way ANOVA analyses, as appropriate, for
the sample group comparisons indicated by brackets.
(a)(b)Deconvolution
microscopic image analysis (see Figure2) suggested that we could recreate the NOD STAT5 phosphorylation
phenotype in non-autoimmune C57BL/6 cells by blocking M-CSF signaling while
increasing the GM-CSF stimulation to the level seen in the NOD [19, 20]. Flow
cytometric analysis also indicated that blocking endogenous M-CSF while stimulating with GM-CSF promoted
STAT5 phosphorylation in C57BL/6 48-hour bone marrow cultures (p*=.0167, Mann-Whitney U test, Figure 1(b)).
However, when we stimulated bone marrow cultures with M-CSF in vitro in the presence of blocking
antibodies to GM-CSF, STAT5 phosphorylation was also stimulated in C57BL/6
cultures, and remained statistically unchanged in NOD bone marrow cells (p*=.0336
and .5556, respectively, Mann-Whitney U test, Figure 2(c)).Figure 2
GM-CSF/M-CSF
cytokine-induced myeloid differentiationin
vitro. Bone marrow cultures
from NOD and C57BL/6 control mice were differentiated in culture using regiment
of cytokine and anticytokine blocking antibodies. Cells were treated with
either 1000?U/mL GM-CSF plus 2µg/mL anti-M-CSF blocking antibodies,
with 500?U/mL M-CSF plus 2µg/mL anti-GM-CSF blocking antibodies, or
with media alone for 48 hours at 3C°/5CO2. Cells were stained
with anti-STAT5Ptyr-FITC (green) and anti-CD11b-PE (red) antibodies for
analysis by flow cytometry (summarized in Figure 1) and deconvolution microscopy (shown here) for the
development of cells with myeloid phenotype (CD11b+) and intracellular
expression of tyrosine phosphorylated STAT5. Background staining for
anti-STAT5Ptyr-FITC antibody was determined by parallel sample stained with a
nonspecific mouse IgG isotype control (bottom most panels). The images are composites
of 3D projection of 20 optical sections (0.2 micron each) showing both immunohistochemical
labels as well as DAPI (blue) poststaining of chromatin within the cells. Accompanying
panels show the STAT5Ptyr-FITC staining alone (green) or CD11b-PE and DAPI
staining (red/blue). Treatment regiment and the cell source strain for each
pair of cultures are listed to the left of the images. Images are representative of 5
random fields observed per sample treatment and 3 separate runs of the
experiment.Taken together, these
findings suggest that the interplay M-CSF and GM-CSF signaling is required to
properly regulate STAT5 function, and support the role of temporally sequenced
GM-CSF and M-CSF signaling on controlling STAT5 activation during myeloid cell
differentiation. Furthermore, this interplay is modulated by the timing and
concentration of GM-CSF stimulation early in differentiation, which appears to
set the stage for STAT5 activation in later stages, regardless of further
stimulation by GM-CSF or M-CSF. Because the NOD cells have higher
endogenous GM-CSF production [19], this temporal signaling pattern is disrupted
and leads to aberrant cell responses to GM-CSF.


## 3.2. NOD Bone Marrow Cell DNA Binding of STAT5 Is Increased by GM-CSF

To evaluate the possibility that
increased STAT5 phosphorylation could lead to increased chromatin binding in
response to GM-CSF, we performed double ChIP analyses on NOD and C57BL/6 bone
marrow cells that had been cultured with GM-CSF for 2 hours, and looked forin situ assembly of
chromatin-associated protein complexes which contain both STAT5 and Histone H3 (Figure 3). We controlled for the
possibility of the prolonged phosphorylation of cytoplasmic DNA-unbound STAT5
masking a more transient phosphorylation of DNA-bound STAT5 isoforms, by
culturing these cells in the presence and absence of the phosphatase inhibitor,
sodium vanadate. In these dbChIP analyses, we detected bands containing
phosphorylated STAT5 only in GM-CSF-treated NOD cells (Figure 3). The presence or absence of the phosphatase inhibitor
did not alter the banding pattern in either NOD or C57BL/6 cell extracts. Higher
molecular weight STAT5-containing complexes were also detected in both NOD and C57BL/6 cell dbChIP
isolates, though at markedly different levels. The level of these
complexes in C57BL/6 dbChIP isolates did not significantly change with GM-CSF
and/or vanadate treatment. These higher molecular weight bands may be an
artifact or represent complexes containing formaldehyde cross-linked dimers of
STAT5 in multiple size isoforms (i.e., STAT5A and STAT5B homo- and heterodimers,
either in full-length or truncated isoforms).
No bands at any of the sizes predicted for monomeric STAT5A, STAT5B, or
truncated isoforms were seen associated with histone H3 in any of the
treatments used in C57BL/6 cell cultures. These findings suggest that even
though STAT5 can be activated in both mouse strain bone marrow cells by GM-CSF
if M-CSF signaling is blocked, functional STAT5 binding on chromatin is
inducible in NOD but not C57BL/6 bone marrow cells under these conditions.Figure 3
STAT5 binding to chromatin increases after GM-CSF stimulation in NOD but not C57BL/6 bone marrow cells. Five million bone marrow cells were cultured
with or without 100µM Na vanadate for 30 minutes at 37C°/5CO2.
Half of the cultures+/- vanadate were then supplemented with 1000?U/mL GM-CSF, and all were
incubated for an additional 90 minutes at 37C°/5CO2. The cells were then fixed in situ, extracted, and sonicated.
The sample was split into five 1×106 cell aliquots for use in
anti-STAT5Ptyr/anti-Histone H3-mediated double ChIP protein isolations for Western
blot analysis of STAT5 associated with histone/chromatin complexes. Protein
isolated from the precipitated chromatin complexes and analyzed by Western blot
probed with anti-STAT5Ptyr antibody. Densitometric/Rf-value anti-STAT5Ptyr Western blot-detected bands separated on 4-20% SDS-PAGE were used to give the approximate
size and location of the STAT5 protein complexes and monomers (approximate
molecular weights indicated on the left of the figure). Higher molecular weight
bands that bound the anti-STAT5Ptyr antibody had Rf values suggesting that they
may represent a mix of formaldehyde-cross-linked dimer complexes containing mixed isoforms of STAT5 (STAT5A (A
96?K), STAT5B (B 92?K), and truncated (T 80?K, 77?K)), both in homodimers (A-A
192?K, B-B 184?K, T-T) and heterodimers (A-B, A-T, B-T, T-T) complexes. Only
monomeric STAT5A size bands (96?K) are detected in the GM-CSF-induced NOD cell
cultures at this early time point. The positions of the precipitating
antibodies heavy chain (50?K) and light chain (25?K), which acts as an internal
protein loading standards, were determined from the extract minus sham control
(sh) and size standards
(indicated but not shown). Treatment/lane key: 0 = untreated, V =
vanadate alone, G = GM-CSF
alone, VG = vanadate and GM-CSF-supplemented
2-hour cultures. Data represents 3 runs
of the double ChIP analysis.


## 4. Discussion

Our studies indicate that
overexpression of GM-CSF in both bone marrow myeloid precursor cells in NOD,
but not non-autoimmune mice, can prolong the activation of STAT5 and its
binding to chromatin. Furthermore,
treatment of non-autoimmune mouse bone marrow cells with anti-M-CSF antibodies
while stimulating them with GM-CSF at levels endogenously produced by the NOD
can recreate the activation of STAT5 but not its chromatin binding. The level
of STAT5 phosphorylation and function in NOD cells is independent of concurrent
phosphatase or kinase [19] activity in these cells. Therefore, these findings
indicate that the high level of GM-CSF produced by NOD bone marrow cells may be
promoting the aberrant STAT5 activation and its potential effects on myeloid
cell gene expression regulation.Blocking GM-CSF or M-CSF
signaling alone reversed the high STAT5 phosphorylation seen in the NOD, but did not
alter it in non-autoimmune bone marrow cells. In addition, M-CSF treatment only
significantly increased STAT5 activation in non-autoimmune cells, and only when
GM-CSF signaling was concurrently blocked. These findings suggest that the regulation
of STAT5 activation is affected by the interplay of GM-CSF and M-CSF signaling
in differentiation, and that this interplay is disrupted in NOD myeloid cells
by the persistence of GM-CSF influence on these cells.These data leave open the
question of how GM-CSF is aberrantly activated in NOD bone marrow myeloid cells
in the first place. GM-CSF autocrine stimulation of myeloid cells is a key
regulatory component in myeloid differentiation and activation responsiveness
in mature monocytes and macrophages and is under strict temporal regulation [2, 3]. GM-CSF gene expression can be induced in myeloid cells by cytokines such as
IL-6, IL-3, and IL-1ß,
which induce
epigenetic control modifications on its promoter [3, 21, 23, 24]. GM-CSF
downregulation prior to M-CSF activation of myeloid cells is primarily
regulated through GM-CSF’s own activation of IL-10 [3]. GM-CSF epigenetic
regulation is accomplished through the interaction of acetylases and
deacetylases at specific regions within the Csf2 gene promoter, which STAT proteins can mediate through their function as an
adaptor-DNA-binding proteins for these enzymes [6–25]. STAT5 can
act as an adaptor for both acetylases and deacetylases [6, 7, 25]. We have
found evidence that STAT5 can bind chromatin at sites previously identified as
epigenetic modification sites within the Csf2 gene promoter [26]. If such binding allows STAT5 to promote epigenetic regulation
of Csf2 gene expression, persistence
of its binding at the Csf2 promoter
could promote the aberrant GM-CSF expression seen in the NOD. Furthermore, our
data indicate that M-CSF signaling may be able to override NOD defects in the
activation/deactivation of STAT5 related to the overproduction of GM-CSF in
these cells, but its subsequent aberrant binding may be dependent on specific,
unique binding site polymorphisms found within the NOD genome, especially new
sites that allow STAT5 binding in the  promoter of the gene for GM-CSF itself ([26]
and Garrigan and Litherland, unpublished data).The autoimmune STAT5 dysfunction seen in the NOD may be best described as a “broken switch” in epigenetic control of cytokine signaling during myeloid differentiation.  In autoimmune cells, this switching mechanism is stuck in the “on” position; thereby, “shorting out” the activation of the normal sequence of cytokines and blocking their proper maturation. The incompletion of
myeloid cell maturation blocks the eventual functionality of this important
class of APC in the initiation and maintenance of self-tolerance, which has
been implicated as a major defect involved in immunopathogenesis.



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*Source: 769795-2009-01-20.xml*