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-Signaling Pathway in Macrophages at Different Stages of Maturation1
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, WI 53706
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. This difference is controlled at the transcriptional level and
results from a specific inability of the less mature WEHI-3 cells to
utilize either the IFN-stimulated response element or the -activated
sequence DNA regulatory element in response to stimulation with
IFN-, while other aspects of IFN- gene induction remain intact.
In the work described here, we examined the components of the IFN-
signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine
whether differences in pathway components or activity exist in WEHI-3
cells that could give rise to this difference in transcriptional
response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric
analyses indicated that the levels of IFN- receptor mRNA
accumulation and protein expression are comparable for RAW 264.7 and
WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the
principal components of this signaling pathway, including JAK1, JAK2,
and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However,
analysis of STAT1 DNA-binding activity by electrophoretic mobility
shift assay and of STAT1 phosphorylation by immunoblot revealed that
this DNA-binding factor is active in RAW 264.7, but not in WEHI-3,
cells after IFN- stimulation. These results demonstrate that the
components of the IFN- signal transduction pathway are intact in
WEHI-3 cells, but stimulation of these cells by IFN- does not result
in STAT1 activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which induce MP functional
activation. Recent work from a number of laboratories has confirmed
that such cytokine stimulation occurs as a result of specific
receptor-ligand interactions, which ultimately result in the induction
of proteins and molecules required for the expression of diverse MP
effector functions (2).
IFN- is one of the most potent MP-activating factors. Recent
investigation of IFN- signaling has elucidated the mechanism of
IFN--mediated gene induction (3). Upon binding its ligand, the
IFN-R dimerizes and allows transphosphorylation of
receptor-associated tyrosine kinases, JAK1 and JAK2. The activation of
these kinases induces phosphorylation of the cytoplasmic tail of the
receptor itself, which lacks intrinsic kinase activity. The cytosolic
molecule STAT1 is then recruited to the activated receptor complex and
phosphorylated. Upon phosphorylation, STAT1 proteins form a homodimer
and translocate to the nucleus to enhance transcription via binding of
the -activated site (GAS) in the promoter of IFN--induced genes.
Diverse members of the large family of cytokine receptors trigger
similar signal transduction pathways, although the specific
combinations of pathway components and target DNA elements differ in
each case (2). These recent studies have highlighted the components of
receptor-mediated signal transduction required to transmit an effective
activation signal to IFN--stimulated cells.
Previous work by our laboratory and others has shown that the murine MP
cell lines RAW 264.7 and WEHI-3 differ in effector capability and in
the population of genes induced by IFN- (4, 5). In addition,
previous studies have shown that these lines represent different stages
of maturation, with RAW 264.7 phenotypically and functionally
resembling a mature elicited MP, and WEHI-3 resembling a less mature,
monocyte-like cell (5, 6, 7). Recently, we showed that two genetic
regulatory elements, the IFN-stimulated response element (ISRE) and the
GAS element, are differentially utilized in RAW 264.7 vs WEHI-3 cells,
with both elements transcriptionally active in RAW 264.7 cells and
inactive in WEHI-3 cells (5). These results suggested that differential
promoter element utilization underlies the distinct patterns of
IFN--mediated gene induction seen in these two cell lines and
pointed to possible differences in the IFN- signal transduction
pathways expressed in the two cell populations.
In the studies described here, we examined the elements of the
IFN--signaling pathway in RAW 264.7 and WEHI-3 cells to test the
hypothesis that an absence or lack of activity of the JAK/STAT
molecules involved in the IFN- signaling pathway is responsible for
the restricted response to IFN- in the less mature WEHI-3 cell line.
These studies revealed that the known components of the
IFN--signaling pathway are expressed in both RAW 264.7 and WEHI-3
cells at comparable levels. However, the critical transcription factor
STAT1 fails to be tyrosine phosphorylated in WEHI-3 cells following
IFN- stimulation and does not acquire the ability to undergo nuclear
translocation or to bind to the GAS of the IFN consensus
sequence-binding protein (ICSBP) gene. These results
demonstrate that although the components of the IFN- signal
transduction pathway, including STAT1 protein, are present in WEHI-3,
the activation of STAT1 does not occur in this less mature cell line.
Such differences in regulation of the IFN--signaling pathway during
MP development, and the resulting differences in IFN--induced gene
expression, may contribute to the MP functional heterogeneity seen
in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MP cell lines RAW 264.7 and WEHI-3 (H-2d haplotype)
were obtained from the American Type Culture Collection (ATCC,
Rockville, MD). Mycoplasma-free cell lines were
maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY)
supplemented with 2 mM glutamine, 1 mM pyruvate, 50 U/ml penicillin, 50
µg/ml streptomycin, 2 g/L sodium bicarbonate (all from Sigma, St.
Louis, MO), plus 10% FBS (Life Technologies), as described previously
(9). For activation, the cells were stimulated with 20 U/ml murine
rIFN- (Schering, Bloomfield, NJ; sp. act. 1.7 x
106 U/mg; LPS levels <0.1 EU/ml by Limulus
amebocyte lysate assay (provided by the American Cancer Society,
Atlanta, GA)) for various times up to 24 h, or mock stimulated
with medium alone, as we have described (5).
Flow cytometry
Flow cytometry was performed as described previously (8). For
these studies, 2 x 106 RAW 264.7 or WEHI-3 cells were
analyzed for IFN-R expression by indirect staining, using the rat
anti-mouse anti-IFN-R Ab GR20 (9) (hybridoma obtained from
ATCC), or an isotype control Ab (5D4, produced in our laboratory),
followed by FITC-conjugated RG7/9.1 (anti-rat 
-chain) to detect
bound Ab. Cells were fixed in 2% paraformaldehyde before analysis.
Samples were analyzed on a Becton Dickinson FACScan flow cytometer with
a logarithmic scale (Becton Dickinson, Bedford, MA); 104
cells were analyzed to generate staining histograms.
RNA isolation
Isolation of RNA from RAW 264.7 and WEHI-3 MP was performed as
described previously (5). Total RNA was isolated using 2 ml of RNA
STAT-60 (Tel-Test "B," Friendswood, TX) according to the
manufacturer’s instructions. To ensure that there was no genomic DNA
contamination in the RNA to be used for reverse transcriptase-PCR
(RT-PCR), the RNA preparation was then treated with DNase I (Sigma) in
the presence of prime inhibitor (5' 
3', Boulder, CO). The RNA was
then repurified with RNA-STAT 60. Removal of genomic DNA was confirmed
by lack of amplification of the constitutive gene G3PDH from
this RNA preparation, as described below.
RT-PCR
Reverse transcription and PCR assays were performed as previously described (5). Briefly, cDNA was synthesized from 10 µg purified RNA after priming with oligo(dT) (Boehringer Mannheim, Indianapolis, IN). The final reaction volume was diluted to 200 µl, and 4 µl of each cDNA sample was used as a template for each gene-specific PCR amplification.
PCR amplifications were performed in 50-µl volumes in a 96-well thermocycler (MJ Research, Watertown, MA). To verify that equal amounts of cDNA were added to each PCR, G3PDH gene expression was assessed. The PCR products were separated in a 1% agarose gel and visualized by ethidium bromide staining.
G3PDH primers were purchased from Clontech Laboratories
(Palo Alto, CA). The primers for the IFN- receptor 
and ß,
JAK1, JAK2, and STAT1 genes were
designed in our laboratory using the Oligo 4.0 Macintosh program
(National Biosciences, Plymouth, MN) and were synthesized by Eppendorf
(Madison, WI). The STAT1 primer sequences were described previously
(5); the remaining primers had the following sequences:
IFN-R sense: 5'-CGGTCGAAAAAGAAGAGTGTA-3'; antisense:
5'-TCGGGAGTGATAGGCGGTGAG-3'; IFN-Rß sense:
5'-TACACTTCTCCCCTCCCTTTG-3'; antisense:
3'-ACATCATCTCGCTCCTTTTCT-3'; JAK1 sense:
5'-ATGGAAGACGGAGGCAATGGT-3'; antisense: 5'-GGAACTTTAGAGGCAGAATAC-3';
JAK2 sense: 5'-TGGAGATGTGCCGCTATGACC-3'; and antisense:
5'-TCTCGATGTATGTGAAAAGTT-3'.
Immunoblot analysis
SDS-PAGE was performed essentially as described (10). Briefly,
RAW 264.7 or WEHI-3 cells, stimulated with IFN- or mock stimulated,
were lysed in Nonidet P-40 detergent lysis buffer and the protein
concentration was determined by spectrophotometry (Bio-Rad, Hercules,
CA). Protein was incubated at 100°C for 1 min in Laemmli buffer, and
20 µg of total protein was loaded in each lane of a 12%
SDS-polyacrylamide gel. After electrophoresis, the proteins were
transferred electrophoretically onto nitrocellulose (Micron
Separations, Westborough, MA). Following protein transfer, the
nitrocellulose was incubated in PBS with 0.5% Tween-20 (PBS-Tween;
Sigma) and 5% nonfat dry milk for 1 to 12 h at room temperature,
rinsed with PBS-Tween, and incubated with the primary Ab for 1 to
2 h. The nitrocellulose membrane was washed three times in
PBS-Tween and incubated with PBS-Tween plus anti-rabbit IgG
secondary Ab conjugated to horseradish peroxidase (Bio-Rad). The blot
was washed again and developed using LumiGLO reagents according to the
manufacturer’s instructions (Kirkegaard & Perry Laboratories,
Gaithersburg, MD). The concentration and source of the primary Abs used
were: anti-JAK1, 1:1000 (Transduction Laboratories,
Lexington, KY); anti-JAK2, 1:5000 (Upstate
Biotechnology, Lake Placid, NY); anti-STAT1, 1:1000 (Santa Cruz
Biotechnology, Santa Cruz, CA); and anti-actin, 1:500 (Sigma).
Densitometry was performed using a Zeineh model SL-2D scanning
densitometer and accompanying software (Biomed Instruments,
Fullerton, CA).
Analysis of STAT1 phosphorylation
The phosphorylation status of STAT1 was assessed by
immunoprecipitation followed by immunoblot analysis. For these studies,
STAT1 molecules were specifically immunoprecipitated from serum-starved
WEHI-3 or RAW 264.7 cells, either mock-stimulated or following IFN-
stimulation. Cells were lysed in ice-cold immunoprecipitation buffer
(50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 7.4), 1 mM
Na3 VO4, 20 mM ß-glycerol phosphate, 1 mM
PMSF, 1 µg/ml leupeptin, and 2 µg/ml aprotinin plus 1% Nonidet
P-40, 0.25% deoxycholate, and 0.1% SDS). STAT1 molecules were
immunoprecipitated by incubation of lysates with protein A-Sepharose
beads (Pharmacia-LKB, Piscatawy, NJ) followed by a polyclonal Ab to
STAT1 (Santa Cruz Biotechnology). Precipitated proteins were released
from the beads by boiling in SDS sample buffer and then separated by
SDS-PAGE on a 10% acrylamide resolving gel. Immunoblot analysis using
antiphosphotyrosine Abs was done essentially as described above, with
some modifications. Briefly, following electrophoresis, proteins were
transferred to polyvinylidene difluoride membranes (Immobilon-P,
Millipore, Bedford, MA) and probed with a mixture of Abs specific for
phosphotyrosine residues (4G10, Upstate Biotechnology; PY20,
Transduction Laboratories) diluted according to the manufacturer’s
suggestion in Tris-buffered saline containing 0.25% gelatin (Sigma).
Immunoblots were then reacted with an anti-mouse Ig Ab conjugated
to horseradish peroxidase and developed using the LumiGLO reagents
(Kirkegaard & Perry Laboratories), as described above. Following
phosphotyrosine analysis, peroxidase activity was quenched by
incubation in 1.5% H2O2 for 15 to 30 min at
room temperature and the blots were reprobed with an Ab to STAT1, as
described above.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as described (11). RAW 264.7 or
WEHI-3 cells were stimulated with IFN- or were mock stimulated for
30 min before lysis. A 25-bp double-stranded oligonucleotide
corresponding to the GAS from the ICSBP promoter was used as a probe
(sequence: 5'-GATCAGTGATTTCTCGGAAAGAGAG-3'). EMSA was
performed essentially as described (11, 12). For supershifts, 1 µg
anti-STAT1 Ab (Santa Cruz Biotechnology) or an irrelevant control
Ab was added after the addition of labeled probe to nuclear extracts,
and all samples were incubated 1 h at 4°C before
electrophoresis. Shifted complexes were visualized directly by
autoradiography after drying the gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor in RAW 264.7 and
WEHI-3 cells
To determine whether differences in the IFN- response between
RAW 264.7 and WEHI-3 cells could be due to the level of IFN-R
expression, we evaluated receptor expression at the mRNA and protein
levels in RAW 264.7 and WEHI-3 cells. Total RNA was isolated from both
cell lines following treatment for 24 h with 20 U/ml IFN- or
with medium alone, and RT-PCR was performed to assess levels of mRNA
encoding the IFN-R -chain, required for IFN- binding, and the
ß-chain, required for signaling (13). Both genes were constitutively
expressed in RAW 264.7 and WEHI-3 cells, and mRNA accumulation was not
substantially altered by IFN- stimulation (Fig. 1
A).
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R expression by flow cytometry,
using GR20, a mAb directed against the IFN-R -chain (9). As shown
in Figure 1
B, the cell lines expressed comparable levels of
IFN-R. Taken together, these data demonstrate no substantial
differences in IFN-R expression between RAW 264.7 and WEHI-3
cells. Analysis of expression of JAK family kinases
The receptor-associated protein tyrosine kinases JAK1
and JAK2 have been shown to be necessary for signaling
through the IFN-R (14, 15). To determine whether differences in
expression of these two proteins between RAW 264.7 and WEHI-3 MP could
explain the distinct IFN- responses observed in these cells, we
assessed production of JAK1 and JAK2 at both the
mRNA and protein levels. RT-PCR analysis on RNA isolated from both cell
lines showed that JAK1 and JAK2 mRNA was readily
detectable in each cell line, with or without IFN- treatment (Fig. 2A). Immunoblot analysis of
protein from each cell line then was performed, as described in
Materials and Methods, using Abs to JAK1 and
JAK2. Anti-actin Abs were included as a positive control for
protein preparations, gel loading, and transfer differences. Figure 2B shows that both RAW 264.7 and WEHI-3 expressed
JAK1 and JAK2 in comparable amounts before and
after stimulation with IFN-. Thus, both cell lines express similar
levels of mRNA and protein for the tyrosine kinases JAK1
and JAK2.
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The expression of the critical transcription factor STAT1 was
analyzed by both RT-PCR and immunoblotting in RAW 264.7 and WEHI-3
cells. Figure 3 shows the results of
RT-PCR analysis of STAT1 and the constitutively expressed
gene G3PDH. STAT1 mRNA was expressed in RAW 264.7 and WEHI-3 cells both
with and without IFN- stimulation (Fig. 3), as we have previously
described (5). Visual assessment of the level of STAT1 mRNA suggested
it was up-regulated in both cell lines following IFN- treatment,
although the RT-PCR analysis did not allow quantitation of STAT1
levels. We then performed immunoblot analysis of RAW 264.7 and WEHI-3
cell lysates using STAT1 Ab. STAT1 protein was constitutively expressed
in both cell lines, and protein levels appeared to be up-regulated
following IFN- treatment, in a manner similar to that observed for
mRNA (Fig. 4A). To determine
more accurately the level of protein induction seen after IFN-
treatment, we compared the level of STAT1 protein expression in control
and IFN--treated cells by densitometry, using actin expression to
normalize the data. This quantitation revealed that STAT1 protein
levels are induced to similar levels by IFN- in both cell
populations (Fig. 4B). These data demonstrate that RAW 264.7
and WEHI-3 cells express readily detectable levels of constitutive and
inducible STAT1 mRNA and protein.
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Signaling through the IFN-R ultimately effects gene
transcription through utilization of GAS elements in the promoters of
IFN--inducible genes (18, 19, 20). We used the GAS from the promoter of
the ICSBP gene in an EMSA to detect GAS-binding proteins in
the two cell lines. As is shown in Figure 5, both cell lines possessed nuclear
proteins that bound to the GAS element. Some differences in the pattern
of protein binding were detectable between the two cell lines, as we
have observed previously (12). This binding was specific, since
competition with excess unlabeled GAS oligonucleotide effectively
competed for this binding (Fig. 5). To determine whether the STAT1
protein we detected by immunoblot in both RAW 264.7 and WEHI-3 cells
was active and capable of DNA binding in both cell lines, we incubated
the EMSA reaction mixture with Ab to STAT1 or a control Ab, as
described in Materials and Methods. Addition of the STAT1 Ab
resulted in formation of a supershifted complex, visualized most
readily by concomitant disappearance of the original STAT1-containing
band, in the RAW 264.7 cells after treatment with IFN- (Fig. 5).
However, no changes were detected using nuclear extracts from
IFN-stimulated WEHI-3 cells or following the addition of the irrelevant
(control) Ab. These results demonstrate that while STAT1 undergoes
nuclear translocation following IFN- stimulation in RAW 264.7 cells,
and is capable of binding specifically to the ICSBP GAS element, this
factor is not productively activated in IFN--stimulated WEHI-3
cells.
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stimulation
The absence of detectable STAT1-binding activity in nuclear
extracts of WEHI-3 cells raised the question of whether STAT1 is
phosphorylated in these cells in response to IFN-. We therefore
analyzed the phosphorylation status of STAT1 by immunoblot analysis in
both cell lines, before and after IFN- treatment. For these
experiments, STAT1 was immunoprecipitated from cell lysates, separated
by electrophoresis, and probed with a mixture of Abs specific for
phosphorylated tyrosine residues, as described in Materials and
Methods. As shown in Figure 6,
phosphorylated STAT1 molecules were detected only in RAW 264.7 cells
treated with IFN- (Fig. 6A). The identity of the
phosphorylated band was confirmed by subsequent probing of the blots
with a polyclonal Ab against STAT1, as described above (Fig. 6B). These results demonstrate that STAT1 is effectively
phosphorylated in response to IFN- stimulation in RAW 264.7, but not
WEHI-3, cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and capable of
up-regulating some genes after IFN- stimulation, this response does
not include expression of genes that rely primarily on the ISRE or GAS
elements (5). Thus, the IFN- response in WEHI-3 cells is modulated
via the regulation of activation of a critical signaling protein. This
regulation leads to the selective pattern of IFN--induced gene
expression observed in these less mature MP.
The IFN--signaling pathway has been the target of considerable study
since it was first outlined in a series of reports by Darnell et al.
(reviewed in 3 . These studies revealed that IFN--induced
tyrosine phosphorylation and DNA-binding activity of the 91-kDa
component of the transcription factor ISGF3, later renamed STAT1,
was critical for initiation of gene expression in several cell types
(16). They and others also showed that the target of this binding
factor generally is the GAS element found in the promoters of many
IFN--inducible genes (17, 18, 19, 20). However, recent studies in MP suggest
that these cells may have additional complexity in terms of their
IFN- response. For example, studies in our laboratory demonstrated
that some genes containing GAS regulatory elements, including FcRI
and class II transactivator, are induced by IFN- in WEHI-3 cells,
while others, like ICSBP, remain unexpressed (Ref. 5 and our
unpublished observations). These observations suggest that MP may
differentially utilize specific GAS elements at different stages of
development.
In addition to differences dependent on the nature of the responding cell, the level of serine phosphorylation on STAT1 may affect its ability to effectively induce gene transcription (22). Although serine phosphorylation is not required for STAT1 homodimer binding to DNA in vitro (23), it appears to act to promote homodimer formation and transcriptional activation (24). This may be an important site of regulation in MP populations since studies in the human monocytic U937 cell line indicated that serine phosphorylation increased as U937 differentiated in vitro, while levels of tyrosine phosphorylation remained the same (24).
Finally, recent analyses of the regulation of the murine
guanylate-binding proteins have demonstrated that IFN--mediated
induction of these genes occurs through the use of a DNA response
element, the ISRE, and transcription factors, including IRF-1, which
are distinct from those typically described for GAS-mediated response
described in other IFN-activated genes (12, 25). Taken together, these
results suggest that the IFN--signaling pathway in MP may be
regulated at several levels.
Our previous studies demonstrated that MP cell lines that differ in
maturational state have distinct responses to IFN- in both the
acquisition of effector function (4) and the population of genes
induced (5, 21). These findings echo those reported by others for
primary MP cell populations, particularly the observation that monocyte
differentiation is accompanied by acquisition of numerous effector
functions including Ag presentation and tumoricidal activity (26, 27, 28).
The studies reported here using homogeneous MP cell lines provide a
molecular explanation of the heterogeneous IFN- response routinely
observed in primary MP. Thus, the outcome of the IFN- response in
individual MP may vary due to differential STAT1 phosphorylation as a
result of differentiation stage, ultimately influencing the biologic
response of activated cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Fred Hutchinson Cancer Research Center,
Seattle, WA 98104.
3 J.E.S.D. is supported by a predoctoral fellowship
awarded from the Howard Hughes Medical Institute, Chevy Chase,
MD.
4 Address correspondence and reprint requests to Dr. Donna Paulnock, Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706. E-mail address:
5 Abbreviations used in this paper: MP, macrophages; ISRE, IFN-stimulated response element; GAS, -activated site; ICSBP, interferon consensus sequence-binding protein; EMSA, electrophoretic mobility shift assay.
Received for publication June 4, 1997. Accepted for publication January 7, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Cell. Immunol. 120:401.[Medline]
responsive elements in two maturationally distinct MP cell lines. J. Immunol. 155:4933.[Abstract]
receptor. J. Leukocyte Biol. 51:507.[Abstract]
and IFN and is likely to autoregulate the p91 gene. EMBO J. 13:158.[Medline]
signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain. Proc. Natl. Acad. Sci. USA 92:5401. signal transduction pathway. Nature 366:166.[Medline]
/ß and - signal transduction. Nature 366:129.[Medline]
/ß and requires a DNA element to which a tyrosine-phosphorylated 91 kD protein binds. Proc. Natl. Acad. Sci. USA 90:6806. responsive element to which an ISGF3 subunit (or like) molecule binds. Mol. Cell. Biol. 13:3951. induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kD subunit of transcription factor ISGF3. Proc. Natl. Acad. Sci. USA 90:4314. expression during the differentiation of bone marrow-derived macrophages. J. Immunol. 142:153.[Abstract]
/ß by CSF-1 or GM-CSF-derived macrophages. J. Leukocyte Biol. 48:43.[Abstract]
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