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Janus Kinase-STAT Signaling and Gene Induction by Down-Regulation of the IFN- Receptor1
Departments of
*
Medical Microbiology and Immunology and
Microbiology, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible genes and activation of Janus Kinase (JAK)-STAT
pathway in Mycobacterium avium-infected macrophages.
Reduced expression of IFN--inducible genes—MHC class II gene Eß;
MHC class II transactivator; IFN regulatory factor-1; and Mg21, a gene
coding for a GTP-binding protein—was observed in M.
avium-infected macrophages. Decreased tyrosine phosphorylation
and DNA binding activity of STAT1 in M. avium-infected
macrophages stimulated with IFN- was observed. Tyrosine
phosphorylation of JAK1, JAK2, and IFN-R
was also reduced in
infected cells. Northern and Western blot analyses showed that a
down-regulation of IFN-R - and ß-chain mRNA and protein
occurred in M. avium-infected macrophages. The
down-regulation of IFN-R and inhibition of STAT1 activation were
time dependent and required 4 h of infection for down-regulation
of the IFN-R and 8 h for STAT1 inhibition. These findings
suggest that M. avium infection inhibits induction of
IFN--inducible genes in mouse macrophages by down-regulating
IFN-R, resulting in reduced phosphorylation of IFN-R, JAK1,
JAK2, and STAT1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mycobacteria are taken up by macrophages by phagocytosis and reside
within phagosomes of these cells (1, 2, 3). Infected
macrophages initiate a cell-mediated immune response by processing and
presenting Ag to T cells in context of MHC cell-surface molecules. The
activated T cells then secrete IFN-, which activates macrophages and
increases expression of MHC class II molecules and other costimulatory
molecules on the cell surface (6, 7, 8, 9).
Stimulation with IFN- results in activation of the Janus kinase
(JAK)3 /STAT signal
transduction pathway (10, 11). IFN- binds to its
cell-surface receptor consisting of two heterodimeric subunits,
IFN-R and IFN-Rß, which are associated with Janus kinases,
JAK1 and JAK2, respectively (11, 12, 13, 14). IFN- binding
results in receptor dimerization/oligomerization and phosphorylation of
JAK1 and JAK2. Phosphorylated JAK1 and JAK2 are responsible for
phosphorylation of IFN-R, recruitment of STAT1, and its
phosphorylation (10, 11, 12, 13, 15, 16). Phosphorylated STAT1
dimerizes and translocates to the nucleus, where it binds to
activation site (GAS) of IFN--inducible genes, including class II
transactivator (CIITA) and IFN regulatory factor-1 (IRF-1) genes
(15, 16, 17, 18).
Virulent mycobacteria are able to survive and multiply within
macrophages for extended periods of time. The ability of mycobacteria
to evade being killed within phagocytic cells contributes to its
success as a pathogen. Macrophages infected with mycobacteria are poor
responders to IFN-. Expression of MHC class II molecules is
decreased in macrophages infected with mycobacteria (9, 19, 20, 21). Infected macrophages also have decreased ability to
present Ags and to inhibit microbial and tumor growth (8, 22, 23). However, the mechanism by which M avium
infection affects the expression of IFN--inducible genes remains
unknown.
The purpose of this study was to investigate the mechanism by which
M. avium infection of macrophages results in
unresponsiveness to IFN-. Our results indicate that inhibition of
expression of IFN--inducible genes in M. avium-infected
macrophages is the result of down-regulation of IFN-R and disruption
of the IFN- signaling pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c mice were purchased from Charles River Breeding Laboratories (Wilmington, MA) at 5–6 weeks of age. The mice were housed in groups of five in isolation cages (Lab Products, Maywood, NJ) and were provided food and water ad libitum.
Reagents
Phosphatase inhibitors sodium orthovanadate
(Na3VO4) and sodium
fluoride (NaF), protease inhibitors aprotinin and PMSF, and latex beads
were purchased from Sigma (St. Louis, MO). Protease inhibitors
leupeptin and pepstatin were obtained from Boehringer Mannheim
(Indianapolis, IN). STAT1 mAb was purchased from Transduction
Laboratories (Lexington, KY). Phospho-specific STAT1 Ab was from New
England Biolabs (Beverly, MA). Anti-phosphotyrosine mAb 4G10, rabbit
anti-human JAK1, and rabbit anti-mouse JAK2 were obtained from
Upstate Biotechnology (Lake Placid, NY). Affinity-purified IFN-R
polyclonal Abs were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Monoclonal Abs to IFN-Rß MOB-47 and MOB-55 were a gift from
Dr. R. D. Schreiber (Washington University, St. Louis, MO).
Peroxidase-conjugated affinity-purified goat anti-hamster IgG was
purchased from Accurate Chemical & Scientific (Westbury, NY).
Peroxidase-linked donkey anti-rabbit and sheep anti-mouse Abs
were obtained from Amersham (Arlington Heights, IL). Culture media and
all supplements were purchased from Life Technologies
(Gaithersburg, MD).
Macrophages
The RAW 264.7 mouse macrophage cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA; TIB 71). Peritoneal macrophages were obtained by lavage of mice that had been injected with 4% thioglycollate broth (Difco, Detroit, MI) 4 days previously. Both peritoneal macrophages and RAW 264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 5 mM sodium pyruvate, and penicillin-streptomycin at 37°C in 5% CO2. Peritoneal macrophages were purified by adherence in six-well tissue culture plates at 5 x 106 cells/well. After 6 h of culture, nonadherent cells were removed by gentle washing in HBSS. The resultant adherent cells were >90% macrophages as determined by nonspecific esterase staining.
Mycobacteria
M. avium (35713; ATCC) was grown in Middlebrook 7H9 broth supplemented with oleic acid-albumin-dextrose complex (Difco) at 37°C in 5% CO2 until mid-log phase. Bacteria were frozen in 1-ml aliquots in 10% glycerol at -80°C at the concentration of 2.38 x 108 cfu/ml. Frozen aliquots were thawed and briefly sonicated before each use. Heat-killed bacteria were prepared by autoclaving at 121°C for 20 min.
Mycobacterium infection and IFN- stimulation
The expression of IFN--inducible genes was examined by
Northern blot hybridization. RAW 264.7 cells or peritoneal macrophages
were cultured in six-well tissue culture plates for 6 h at 5
x 106 cells per well. The nonadherent cells were
removed by washing with HBSS, and the adherent cells were infected
overnight with M. avium at 10:1 bacteria-to-macrophage
ratio in complete DMEM without antibiotics and FBS. At this
bacteria-to-macrophage ratio, 60% of the cells are infected with an
average of five bacteria per cell as determined by carbol fuchsin
staining and microscopic examination. Identical results were obtained
when macrophages were infected in the presence of FBS. In some
experiments, higher ratios of infection (25:1 and 50:1) were used as
described in the figure legends. Nonphagocytized bacteria were removed
by washing with HBSS, and fresh antibiotic-free DMEM with 10% FBS was
added to the cells. The macrophages were stimulated with IFN- (100
U/ml) for 20 h before isolation of RNA by the acid guanidinium
isothiocyanate phenol chloroform extraction method of Chomczynski and
Sacchi (24). For EMSA and Western blotting, cells infected
overnight were stimulated with IFN- (100–500 U/ml) for the times
indicated in each experiment.
Northern blot hybridization
RNA (10–25 µg/lane) was size fractionated in 1% formaldehyde
agarose gel and transferred by capillary blotting onto
Hybond-N+ membranes (Amersham). RNA ladder
(0.24–9.5 Kb; Life Technologies) was included in each gel and stained
with ethidium bromide for RNA size determination. Northern blot
hybridization were performed as described previously (25).
Probes to IFN-R - and ß-chain genes, CIITA and IRF-1, were
derived by RT-PCR of IFN--stimulated macrophages using the following
primers: IFN-R: sense, 5'-GGTTCCTGGACTGATTCCTGCACC-3';
anti-sense, 5'-AGTTCTTCCTGTTCTGCTGCTTCGG-3'; IFN-Rß, sense,
5'-TACACTTCTCCCCTCCCTTTG-3'; anti-sense,
5'-ACATCATCTCGCTCCTTTTCT-3'; CIITA: sense,
5'-CAAGTCCCTGAAGGATGTGGA-3'; anti-sense,
5'-ACGTCCATCACCCGGAGGGAC-3'; IRF-1: sense, 5'-CCAAGAGGAAGCTGTGTGGAG-3';
anti-sense, 5'-CAGCAGGCTGTCCATCCACATG-3'. The IFN-R
-chain primers were designed using PC/GENE (IntelliGenetics,
Mountain View, CA). The IFN-Rß, IRF-1, and CIITA primer sequences
were described previously (26, 27, 28). All the
primers were synthesized by Life Technologies. Identity of each probe
was confirmed by DNA sequencing. cDNA inserts of Mg21, MHC class II
gene Eß, and G3PDH were isolated from a subtraction library of
IFN--stimulated macrophages. The probes were labeled with
[32P]dCTP by high-prime DNA labeling system
(Boehringer Mannheim).
Nuclear extraction and EMSA
Nuclear extracts were prepared as described previously (29). Macrophages (10 x 106 cells per treatment) were washed twice with ice-cold PBS and incubated on ice for 15 min in 400 µl hypotonic buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.10 mM EDTA, 1 mM DTT, 0.50 mM PMSF, 1 µg/ml aprotinin, 50 mM NaF, and 1 mM Na3VO4. The cells were then lysed by adding 25 µl of 10% Nonidet P-40 and brief vortexing. Nuclei were pelleted and extracted on ice for 15 min in 100 µl of buffer containing 20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, 50 mM NaF, and 1 mM Na3VO4. Nuclear extracts were recovered from supernatants after centrifugation at 10,000 x g for 15 min. Protein concentration was determined by Bradford method using Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA). The extracts were assayed immediately for STAT1 activity or stored at -80°C until further use.
EMSA were performed in 20 µl binding reactions containing 3 µg of nuclear extract, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM DTT, 5 mM MgCl2, 10% glycerol, 0.20% Nonidet P-40, 1 µg poly(dI-dC), and 70,000 cpm of [32P]dCTP-labeled GAS probe radiolabeled by filling with klenow DNA polymerase. The GAS probe (5'-AGCCATTTCCAGGAATCGAAA-3') was derived from sequence of the Mg21 promoter (W.P.L., unpublished observations) and contains a GAS site identical with the optimum GAS sequence (TTCCSGGAA) for STAT1 binding (30). Binding reactions were incubated for 20 min at room temperature and then subjected to electrophoresis on 5% polyacrylamide gels in 0.5x TBE. The gels were dried and analyzed by autoradiography. In competition assays, 100x unlabeled GAS or IFN-stimulated response element (ISRE) probes were added along with radiolabeled GAS probe. The ISRE oligonucleotide (5'-GATCGGGAAAGGGAAACCGAACTGAAGC-3') was derived from the sequence of the ISG15 promoter (31). In supershift assays, 1 µg of STAT1 mAb was incubated with binding reactions for 20 min before the addition of the radiolabeled GAS probe.
Immunoprecipitation
Immunoprecipitation of IFN-R (- and ß-chains) and Janus
kinases (JAK1 and JAK2) were performed as previously described with
slight modifications (12, 28). For IFN-R
immunoprecipitation, 20 x 106 RAW 264.7
cells were lysed on ice in lysing buffer consisting of 0.5% Nonident
P-40, 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 10% glycerol, 0.1 mM EDTA,
1 mM DTT, 1 mM Na3VO4, 1 mM
PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 3 µg/ml
aprotinin. The immunoprecipitation buffer for JAK kinases consisted of
1% Triton X-100, 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 50 mM NaF, 5 mM
sodium pyrophosphate plus phosphatase and protease inhibitors as
described above. The samples were centrifuged at 10,000 x
g for 10 min. The supernatants were cleared for 2–6 h with
recombinant protein G agarose (Life Technologies) preincubated with
normal rabbit serum, at 4°C. After removal of protein G agarose by
centrifugation, the lysates were incubated with Abs indicated in each
experiment and protein G agarose for 6–18 h at 4°C. The protein G
agarose was then collected by brief centrifugation and washed four
times with lysis buffer. The immunoprecipitated proteins were then
removed by boiling with SDS sample buffer. For Western blot analysis of
STAT1 protein, 10 x 106 cells per treatment
were lysed in buffer containing 1% Triton X-100, 20 mM Tris-HCl, pH
8.0, 137 mM NaCl, 10% glycerol, and phosophatase and protease
inhibitors at 4°C.
Western blot analysis
The samples were resolved by 8% SDS-PAGE, transferred with the
transblot semidry transfer cell (Bio-Rad) to polyvinlyidene difluoride
membranes (Bio-Rad). The membranes were then blocked in 3% BSA in TBS
containing 0.5% Tween-20. The membranes were washed and incubated with
primary Abs STAT1 (1:2000), phospho-STAT1 (1:500), IFN-R
(1:1000), IFN-Rß (1:1000), JAK1 and JAK2 (1:4000), and
phosphotyrosine 4G10 (1:2000) followed by 1:5000 dilution of
anti-mouse, anti-rabbit or anti-hamster HRP-conjugated IgG.
The blots were developed using chemiluminescence kit (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible genes in mouse macrophages
We examined the expression of a panel of IFN--inducible genes
including MHC class II gene Eß, CIITA, IRF-1, and Mg21, a gene coding
for an intracellular protein with a GTP-binding motif
(25). IFN--stimulation of macrophages infected with
M. avium in the absence of serum resulted in the reduced
expression of IFN--inducible genes when compared with mock-infected
cells. M. avium infection inhibited the expression of
IFN--inducible genes in both RAW 264.7 cells and peritoneal
macrophages (Fig. 1
). Similar results
were obtained when macrophages were infected with M. avium
in media containing FBS (data not shown). We found that the inhibitory
effect of M. avium infection on expression of
IFN--inducible genes was dose dependent. A 10:1
mycobacteria-to-macrophage ratio was sufficient to inhibit the
expression of IFN--inducible genes in RAW 264.7 macrophages (Fig. 2). However, a higher
mycobacteria-to-macrophage ratio of infection (50:1) was required to
inhibit expression of IFN--inducible genes in peritoneal macrophages
(Fig. 1).
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-inducible genes
We also examined the effect of live vs heat-killed M.
avium on the expression of IFN--inducible genes. Heat-killed
bacteria were as effective as viable mycobacteria in inhibiting the
expression of IFN--inducible genes (Fig. 3A). To rule out the
possibility that phagocytosis alone might be responsible for the
inhibitory effect, we incubated RAW 264.7 cells with sterile latex
beads before stimulating the cells with IFN-. Phagocytosis of latex
beads did not inhibit expression of IFN--inducible genes (Fig. 3B).
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Binding of IFN- to its cell-surface receptor activates the
JAK/STAT cell signaling pathway that leads to expression of
IFN--inducible genes (10, 11, 17). We examined the
effect of M. avium infection on STAT1 activation and
phosphorylation. Nuclear extracts prepared from mock-infected cells and
M. avium-infected RAW 264.7 cells were analyzed for STAT1
activation by EMSA gels (Fig. 4A). Reduced STAT1 binding to
the GAS element was observed in M. avium-infected cells. The
specificity of the GAS-STAT1 complex was confirmed by supershift
analysis with STAT1 mAb and by competition with 100x unlabeled GAS
probe. A nonspecific competitor, ISRE, did not compete for binding. We
also examined STAT1 activation in macrophages incubated with
heat-killed M. avium. We found that heat-killed bacteria
equally inhibited binding of STAT1 to the GAS element (data not shown).
Experiments using thioglycollate-elicited mouse peritoneal macrophages
gave similar results.
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B).
M. avium infection results in reduced
phosphorylation of IFN-R and Janus kinases
STAT1 activation requires phosphorylation of upstream components
of the IFN- signal transduction pathway including IFN-R, JAK1,
and JAK2 (10, 11, 12, 13). A reduction of tyrosine phosphorylation
of IFN-R, JAK1, and JAK2 proteins in RAW 264.7 cells infected
with M. avium for 16 h was observed (Fig. 5A). This suggests that
reduced phosphorylation of STAT1 was the result of reduced
phosphorylation of upstream JAK kinases and IFN-R required for
STAT1 phosphorylation.
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R
protein and mRNA expression
Western blot analysis of protein showed that levels of JAK1 and
JAK2 did not change in infected cells (Fig. 5B). However, we
found that the expression of IFN-R - and ß-chains was reduced
in infected cells (Fig. 5, B and C). Northern
blot analysis also indicated a decreased expression of IFN-R -
and ß-chain mRNA in M. avium-infected macrophages
beginning 4 h after infection (Fig. 6)
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R down regulation and STAT1 activation
in M. avium-infected macrophages
IFN-R expression and STAT1 tyrosine phosphorylation and
activation by IFN- was examined in RAW 264.7 macrophages infected
with M. avium in time course experiments. These experiments
show that at least 4 h of M. avium infection was
required to reduce IFN-R protein expression (Fig. 7A) and 8 h of infection
to reduce STAT1 activity as determined by EMSA gels (Fig. 7B) and Western blotting with Abs to STAT1 and pTyr-STAT1
(Fig. 7C). In contrast, analysis of cytoplasmic extracts
from the same cells showed an increase in STAT1 expression at 8 and
16 h (Fig. 7D). This increase in cytoplasmic STAT1 is
probably due to increased gene expression and not due to accumulation
of pTyr-STAT1 in the cytoplasm, because we were unable to detect any
pTyr-STAT1 in the cytoplasmic extract (not shown). These results are
consistent with the results of time course experiments of IFN-R mRNA
expression in M. avium-infected cells (Fig. 6) and indicate
that down-regulation of IFN-R mRNA and protein expression begins
first at 4 h followed by reduced ability of the infected cell to
activate STAT1 at 8 and 16 h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible genes by
down-regulation of the IFN-R required for activation of the JAK-STAT
signal transduction pathway (11, 12, 13). This down-regulation
of the IFN-R by M. avium was accompanied by a global
inhibition of IFN--induced gene expression. However, the expression
of the IFN--induced genes varied in the extent to which they were
inhibited by M. avium. We found the expression of MHC class
II gene Eß to be more sensitive to inhibition by M. avium
than IRF-1 and Mg21. This is most likely a consequence of the Eß gene
requiring synthesis of transcription factors for induction by IFN-,
while IRF-1 and Mg21 genes do not require protein synthesis for IFN-
induction (25). Thus, the effect of M. avium
infection on Eß expression was probably a cumulative effect because
expression of IFN--inducible genes CIITA and IRF-1, required for the
induction of MHC class II gene Eß, are inhibited by M.
avium infection. CIITA is required for MHC class II genes
transcription (32), and a binding site for IRF-1 in the
promoter of CIITA has been described recently (17). A GAS
sequence required for IFN- induction is also present in the
promoters of both IRF-1 and CIITA (17, 18).
The global inhibition of IFN--inducible genes by M. avium
infection suggests that the inhibition may lie somewhere in the IFN-
cell signaling pathway. IFN-R (- and ß-chains), JAK kinases
(JAK1 and JAK2), and STAT1 are the key components of this pathway
(10, 11, 12, 13, 15, 16). IFN- binds to IFN-R -chain but
signaling only occurs in presence of an intact ß-chain
(33). The -chain has been shown to exist in macrophages
both on the cell surface and in a large intracellular pool
(13, 14, 15). IFN- binding to cell surface receptors
results in internalization of receptor-ligand complex. This complex
then enters an acidified compartment where the complex dissociates and
free IFN- is trafficked to the lysosomes for degradation
(34). The uncoupled receptor -chain enters a large
intracellular pool of -subunits and eventually recycles back to the
cell surface (11, 14). Using Western blot analysis, we
showed that there was a decrease in IFN-R - and ß-chain protein
in M. avium-infected macrophages. It is possible that
M. avium infection of macrophages may interfere with
recycling of IFN-R or enhance its degradation. We also found that
M. avium infection reduces mRNA levels of both IFN-R -
and ß-chains. Thus, infection may also result in altered
transcription of IFN-R genes or in the altered stability of their
mRNA,s.
JAK1 and JAK2 are cytoplasmic tyrosine kinases and are associated with
IFN-R cytoplasmic domains. The levels of tyrosine phosphorylation of
JAK1 and JAK2 decreased in M. avium-infected macrophages
concomitant with a down-regulation of the IFN-R. However, there was
no change in level of JAK1 and JAK2 expression. IFN- stimulates only
the autophosphorylation of JAK1 and JAK2, which is associated with
cytoplasmic domains of IFN-R. This observation is consistent with
the association of JAK1 and JAK2 with cytoplasmic domains of other
cytokines and growth factor receptors (35, 36), which may
not be affected by M. avium infection.
Our studies show that the effect of M. avium infection on
IFN- signaling is time dependent with inhibition of IFN-R mRNA
and protein expression beginning first at 4 h of infection
followed by inhibition of the signaling pathway at 8 and 16 h.
Because it has been shown that there is an large intracellular pool of
the IFN-R -chain (13, 14, 15), this difference between
receptor expression and inhibition of STAT1 activation is not
unexpected. STAT1 is also serine phosphorylated during activation. Wen
et al. (37) have shown that maximal activation by STAT1
requires both tyrosine and serine phosphorylation. Whether M.
avium infection also affects the serine phosphorylation of STAT1
is unknown.
There is very little information about negative regulation of the
IFN-R. Studies have shown that cytokines can alter the expression of
IFN-R in various cell types (38, 39, 40, 41, 42). IL-1, IL-6, and
TNF-, which are produced by infected macrophages and are involved in
controlling mycobacterial growth (43, 44, 45), have been shown
to enhance IFN-R expression on human monocytes
(38, 39, 40). However, macrophages have been shown to respond
to IFN- in the presence of these cytokines (46, 47, 48).
Mycobacteria-infected macrophages can also produce IFN-
(49). IFN- enhances the expression of IFN-R on the
U937 monocytic cell line (41) but suppresses the
expression of IFN-R ß-chain on T lymphocytes (50). A
reduction in expression of the IFN-R together with enhanced
production of IFN- has also been observed in PBMC of individuals
suffering from chronic renal disease (42). A similar
negative correlation in production of a cytokine (TNF-) and the
expression of its receptor on splenocytes from M.
avium-infected mice has also been observed (51).
Whether enhanced production of IFN- and TNF- are involved in
down-regulation of respective receptors is not known.
The production of TGF-ß and IL-10 by infected macrophages may account
for our observations (52, 53). TGF-ß has been shown to
reduce IFN- binding to macrophages (54). However, we
found that neutralization of TGF-ß and IL-10 with Abs did not
abrogate the inhibitory effect of M. avium infection on
macrophage gene expression (our unpublished observations). This finding
is consistent with those that showed that treatment of human U937
monocytic cells and rat astrocytes with recombinant TGF-ß did not
alter the phosphorylation of JAK1, JAK2, and STAT1 following treatment
with IFN- (28, 55). TGF-ß was shown in those studies
to completely inhibit the induction of CIITA and MHC class II mRNA
without affecting IFN- induction of guanylate-binding protein-1,
IRF-1, or ICAM-1 gene expression. The observation by Song et al.
(56) that treatment of human monocytes with rIL-10 does
not alter the phosphorylation of STAT1 supports our observation that
anti-IL-10 does not alter the effect of mycobacterial infection on
IFN--induced gene expression.
Our observations that M. avium infection inhibits expression
of IFN--inducible genes is consistent with previous findings that
the expression of MHC class II molecules is reduced in
mycobacterium-infected macrophages (9, 19, 20, 21). However,
recently Hmama and coworkers (19) have shown that
opsonized Mycobacterium tuberculosis inhibited MHC cell
surface expression in the human monocytic cell line THP-1 by blocking
the transport and processing of class II molecules through the
endosomal/lysosomal system. In this study, the induction of MHC class
II and CIITA gene expression by IFN- and the activation of JAK-STAT
signaling by IFN- was not impaired in the M.
tuberculosis-infected cells. At present, we do not know if the
differences between this study and the present study results from
different cell lines being used, opsonized vs nonopsonized entry of the
mycobacterium into the macrophage, or a difference between M.
avium and M. tuberculosis. We have found in preliminary
studies that culture media from infected RAW 264.7 macrophages can also
inhibit IFN- signaling and IFN-R expression, suggesting that a
factor produced by the infected macrophages is responsible for the
inhibition. It is possible that the THP-1 cell line does not produce
this factor. It will also be particularly interesting to determine
whether M. tuberculosis infection of RAW 264.7 macrophages
inhibits IFN--inducible gene expression and JAK-STAT signaling.
Infection of macrophages by other intracellular pathogens has also been
shown to affect responses to IFN- (57, 58, 59, 60, 61, 62, 63).
Cryptococcus neoformans infection of mouse macrophages
results in inhibition of NO production following stimulation with
IFN- and LPS (57). Prior ingestion of heat-killed
Histoplasma capsulatum also renders macrophages unresponsive
to IFN--stimulation (58). A reduced expression of MHC
class I and MHC class II genes has been observed in Listeria
monocytogenes-infected mouse macrophages (59).
Leishmania donovani-infected monocytes show decreased
expression of MHC class II protein and reduced tyrosine phosphorylation
of STAT1 and JAK kinases (60, 61). A similar inhibitory
effect of IFN--induced tyrosine phosphorylation of STAT1 and JAK
kinases has been reported in Ehrlichia chaffeensis-infected
human monocytes (62). The inhibitory effect in that study
was immediate and did not require IFN-R down-regulation and
phagocytosis of the pathogen. Our findings indicate that the inhibition
of JAK-STAT signaling by M. avium infection was a result of
the decreased expression of IFN-R. Trypanosoma cruzi
infection of human PBMC has also been reported to induce a
down-regulation of IFN-R in B lymphocytes without affecting
expression of MHC class II Ag (63). The reduced expression
of the IFN-R was observed at 3 h and lasted at least for
24 h. These data are consistent with our observation that
down-regulation of IFN-R occurs by 4 h in M.
avium-infected macrophages and can be observed for at least
24 h.
Although down-regulation of IFN-R expression following M.
avium infection appears to be a major cause for the attenuation of
the JAK-STAT pathway, participation of phosphotyrosine phosphatases
cannot be ruled out. The protein tyrosine phosphatase (Src homology
protein-1) is associated with several cytokine receptors and has been
implicated with down-regulation of ligand-induced signaling through
dephosphorylation of the activated JAKs (64, 65). A recent
study by Knutson et al. (66) indicates that
lipoarabinomannan, a mycobacterial cell wall glycolipid, promotes
tyrosine dephosphorylation and inhibition of mitogen activated protein
kinase activity in human monocytes. This dephosphorylation of proteins
was the result of activation of Src homology protein-1.
Lipoarabinomannan has also been known to inhibit
IFN--mediated macrophage activation (67), but
its role in down-regulation of IFN-R has not been reported.
In conclusion, we have demonstrated that M. avium infection
inhibits expression of IFN--inducible genes in mouse macrophages.
This inhibitory effect is due in part to the down-regulation of the
IFN-R resulting in decreased JAK-STAT signaling. Our findings
represent one mechanism by which mycobacteria are capable of avoiding
immune surveillance and establishing chronic infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William P. Lafuse, Department of Medical Microbiology and Immunology, 333 West 10th Avenue, Columbus, OH 43210. E-mail address:
3 Abbreviations used in this paper: JAK, Janus kinase; GAS, -IFN activation site; CIITA, class II transactivator; IRF-1, IFN regulatory factor-1; ISRE, IFN stimulated response element.
Received for publication November 12, 1998. Accepted for publication May 27, 1999.
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