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*
McGill University, Department of Experimental Medicine, Montreal General Hospital Research Institute, Montreal, Quebec, Canada; and
Central University of Venezuela, Faculty of Medicine, Institute of Immunology, Caracas, Venezuela
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced MHC class II expression in three types of macrophages:
those expressing a wild-type allele of the Nramp1 gene
(B10R and 129/M
), those carrying a susceptible form of the
Nramp1 gene (B10S), and those derived from
129-Nramp1-knockout mice (129/Nramp1-KO).
Previously, we published results showing that Ia protein expression is
significantly higher in the IFN--induced B10R macrophages, compared
with its susceptible counterpart. In this paper, we also show that the
higher expression of Ia protein in B10R cells is associated with higher
I-Aß mRNA expression, which correlates with a higher
level of IFN--induced phosphorylation of the STAT1-
protein and
subsequently with elevated expression of class II transactivator
(CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the
infection of macrophages with M. bovis BCG results in a
down-regulation of CIITA mRNA expression and, consequently, in the
inhibition of Ia induction. Therefore, our data explain, at least in
part, the molecular mechanism involved in the inhibition of
I-Aß gene expression in M. bovis
BCG-infected macrophages activated with IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, it has been shown that a single substitution of glycine with aspartic acid at position 169 of the predicted amino acid sequence of Nramp1 protein is responsible for loss of Nramp1 function in macrophages from bacillus Calmette-Guérin (BCG)-susceptible mice (7, 8, 9). Northern blot analysis has revealed that, in mice, the Nramp1 gene is expressed exclusively by macrophages. Although the exact function of the Nramp1 protein has not been clarified yet, an examination of the sequence of the gene indicates that Nramp1 is a transmembrane protein with a potential transport function (10). It has 12 highly hydrophobic regions that may form transmembrane domains, two potential N-linked glycosylation sites, several consensus protein kinase C (PKC) phosphorylation sequences, a putative intracytoplasmic consensus transport signature, and a proline/serine-rich putative Src homology 3 (SH3) binding domain (7, 11).
The evidence obtained from the analysis of Nramp1 homologues in yeasts SFM1 and SFM2 (from Saccharomyces cerevisiae) suggests that the Nramp1 protein may be involved in the transport of divalent cations, such as Mn2+, Zn2+, or Fe2+, across the membrane (12). The Nramp2 protein, the second member of Nramp family in mammals, has been shown to transport iron in many different tissues (13). Overall, the above evidence, together with the experiments showing the intracellular localization of the Nramp1 protein in the late phagosomal/lysosomal membrane (14), and an involvement of Nramp1 protein in phagosomal acidification (15) suggest that Nramp1 controls and/or regulates the intraphagosomal environment.
The Nramp1 gene has been shown to be directly involved in
determination of the resistance of mice to infections with several
unrelated pathogens, including Leishmania donovani,
Salmonella typhimurium, and Mycobacterium bovis
BCG (7, 11, 16). In the case of infection with M.
bovis BCG, a susceptible phenotype is characterized by the
inability of the host’s macrophages to control the growth of the
microorganism in the early phase of infection (7, 17, 18, 19, 20).
The inefficient bactericidal activity of macrophages derived from mice
susceptible to infection with M. bovis BCG seems to be
associated with an inadequate magnitude of activation
(21, 22, 23, 24, 25, 26, 27, 28). It has been demonstrated that Nramp1,
through a number of pleiotropic effects, influences the process of
macrophage activation. These effects include the differential
expression of IL-1ß, TNF-, and (IL-10-sensitive gene) KC,
inducible NO synthase (iNOS) resulting in variable production of
reactive nitrogen intermediates (RNI), the respiratory burst, as well
as MHC class II genes (27, 29, 30, 31, 32, 33).
It has been well documented that the expression of MHC class II genes
is essential for the development of an immune response
(34). The MHC class II molecule is encoded by two genes in
the mouse, denominated as I-A and I-E, which are located in two
subregions on murine chromosome 17. The glycoproteins encoded by the
I-A and I-E genes (called Ia surface proteins in mouse) are able to
bind and present foreign peptides to competent T cells. Both I-A and
I-E are expressed as heterodimers formed by the noncovalent association
of - and ß-chains on the surface of APC, including macrophages, B
cells, thymic epithelial cells, glial cells, and dendritic cells
(35). The expression of MHC II molecules can be either
constitutive or inducible, depending on the cellular type
(36). IFN- is a potent inducer of MHC class II
expression in macrophages. Although MHC II gene expression is under
strict and highly complex transcription regulation involving
interaction of several regulatory elements with specific transcription
factors (33, 36, 37), it seems that the entire process is
controlled by a single master regulator called class II transactivator
(CIITA) (38, 39, 40, 41). CIITA is necessary for both constitutive
and inducible expression of MHC II genes. It does not bind directly to
DNA, but rather interacts with transcription factors bound to the
promoter of the MHC genes perhaps via the N-terminal activation domain
(42, 43, 44). CIITA also contains a GTP-binding domain, which
is absolutely essential for its function (45), a
proline/serine/treonine-rich region of unknown function, as well as a
leucine-rich region and two leucine charged domains most likely
responsible for direct interaction with nuclear factors (41, 42).
The induction of CIITA mRNA expression by IFN- requires the presence
of the functional STAT1 protein (46, 47). STAT1 is
part of a well-described IFN- signal transduction pathway. The
binding of IFN- to IFN- receptors on the surface of macrophages
leads to the phosphorylation and activation of two tyrosine kinases
termed Jak1 and Jak2, which associate with the IFN-R (IFNGR-1)
and IFN-Rß (IFNGR-2) subunits, respectively (48). The
two Jak kinases rapidly induce the tyrosine phosphorylation of the
subunit of IFN- receptor providing docking site for STAT1
(49). The cooperation of the two Jak kinases results in
STAT1 protein phosphorylation, which is required for STAT1
release from the receptor, STAT1/STAT1 homodimer formation, and
translocation to the nucleus, where it can bind to DNA and modulate
gene expression (50).
The role of the Nramp1 protein in the regulation of the response of
macrophages to IFN- has not been elucidated yet. In this report, we
present evidence indicating that the higher level of the Ia Ag,
expressed by macrophages carrying the resistant allele of the
Nramp1 gene (B10R), compared with susceptible macrophages
(B10S), correlates with the higher level of CIITA expressed by those
macrophages. The difference in CIITA expression also correlates with
the superior capability of B10R macrophages to phosphorylate the
STAT1 protein in response to IFN- stimulation, compared with B10S
macrophages.
Furthermore, this study also shows the effect of M. bovis
BCG infection on I-Aß and CIITA mRNA expression
and, consequently, on Ia protein production in IFN--stimulated
macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DMEM, penicillin/streptomycin, Dulbecco’s PBS (DPBS), and
Trizol reagent were purchased from Life Technologies (Grand Island,
NY). FBS, characterized for low level of endotoxin, was obtained from
HyClone (Logan, UT). Recombinant murine IFN- was purchased from
Amgen (Thousand Oaks, CA). Nonidet P-40 and PMSF were purchased from
United States Biochemicals (Cleveland, OH). Sodium deoxycholate, sodium
orthovanadate, sodium fluoride, Tween 80, BSA, aprotinin, leupeptin,
p-nitrophenyl guanidinobenzoate, and rabbit anti-mouse
IgG polyclonal HRP-conjugated Ab were purchased from Sigma (St. Louis,
MO). Anti-murine p91 mouse mAb (C-111), anti-murine p84/p91 rabbit
polyclonal Ab (M-22), anti-phosphotyrosine mouse mAb (PY-20),
protein A/G PLUS-Agarose, and goat anti-human/murine CD64 (Fc
receptor type I) Ab (N-19) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Ab against murine Fc receptor type II and III
(FcBlock), FITC anti mouse I-Ab (used to analyze
the Ia protein expression of cells derived from 129/J mice), and FITC
anti mouse I-Ak Ab (used to analyze the surface
Ia expression of cells derived from B10A mice) were obtained from
PharMingen (Mississauga, ON, Canada).
[-32P]dCTP and ECL chemiluminescent reagent
were purchased from Amersham (Amersham, U.K.). Middelebrook 7H9
broth was purchased from Difco Laboratories (Detroit, MI). BBL
Middelebrook OADC Enrichment was purchased from Becton Dickinson
(Mississauga, ON, Canada).
Bacteria
M. bovis BCG substrain Montreal was cultivated using constant rotation at 37°C for 2 wk in Middelebrook 7H9 broth supplemented with 10% Middelebrook OADC Enrichment and containing 0.05% Tween 80. After culture reached concentration of 0.6–1.0 OD600, the cells were collected and briefly sonicated to break down bacterial clumps and filtered through a 5-µm syringe filter to eliminate remaining clumps. After estimation of cell concentration, the culture was aliquoted and frozen in 15% glycerol solution.
Cell lines
Macrophage cell lines were derived from the bone marrow of
B10A.Bcgr and 129/J mice expressing the
wild type of the Nramp1 gene (B10R cell line and 129.M
cell line, respectively), from B10.A mice carrying a mutated
Nramp1 gene (point mutation at D169; B10S macrophage cell
line), and from 129/Nramp1 gene knockout mice with
genetically disrupted Nramp-1 (51) gene in the
129/J embryonic stem cells (129/Nramp1-KO cell line), as
previously described (52). Cell lines were cultured in
DMEM supplemented with 10% heat inactivated FBS and
penicillin/streptomycin antibiotic mixture. The subconfluent cell
cultures were used for all of the experiments.
FACS analysis of cell surface Ia expression
Macrophage cell lines were plated at a concentration of
0.5–1 x 106/ml and treated with IFN-
and/or M. bovis BCG for 48 h. Cells were removed from
the flasks, washed in DPBS, and resuspended in DPBS containing 5% BSA
and 0.1% sodium azide. FcR were blocked for 15 min at 4°C using Ab
against FcR type II/III (FcBlock; PharMingen) and anti-murine/human
CD64 Abs against FcR type I (N-19; Santa Cruz Biotechnology). Cells
were washed three times and incubated for 15 min at 4°C with
strain-specific anti I-Ak (B10R and B10S) and
I-Ab (129 M) Abs directly labeled with FITC
(PharMingen). After washing, cells were fixed in 1% solution of
paraformaldehyde in PBS for 30 min at room temperature. Stained cells
were analyzed using a dual laser FACStarPlus flow
cytometer (Becton Dickinson). A green fluorescence histogram of 1000
channel resolution was collected from 10,000 cells counted for each
sample analyzed.
DNA probes
The pGEM-Aß containing a 500-bp PstI fragment of the I-Aßd gene was kindly provided by Dr R. Germain (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). The GAPDH probe was generated by PCR amplification of a 456-bp cDNA fragment using the following oligonucleotide primers: sense primer, 5'-CCC TTC ATT GAC CTC AAC TAC ATG G-3'; antisense primer, 5'-AGT CTT CTG GGT GGC AGT GAT GG-3'. The PCR product was subcloned in pBluescript KS+ and sequenced. The CIITA probe was obtained by PCR amplification of the 341-bp fragment of macrophage cDNA using the following oligonucleotide primers: sense primer, 5'-CTT CTG GCT TCA CCT TCA CG-3' and anti-sense primer, 5'-ATT AAG GAC TCA GGG CTC CC-3'. The products of PCR amplification were subcloned into pGEM-T-Easy vector (Promega, Madison, WI).
Northern blot analysis
To isolate total cellular RNA, 
10 million cells, treated with
IFN- and/or M. bovis BCG, were lysed using either
guanidinium isothiocyanate solution or Trizol reagent, and RNA was
isolated as previously described (53). A total of 15–20
µg of total cellular RNA extracted from B10R and B10S macrophages was
always separated on the same agarose gel (1.2%) containing 2.2 M
formaldehyde. The membranes were then hybridized for 18 h at
42°C with labeled probe (106 cpm/ml).
Subsequently, the membranes were washed three times with 2x SSC, 0.1%
SDS (10 min, 43°C), and then three times with 0.1x SSC, 0.1% SDS
(10 min, 55°C) before autoradiography. The densitometry analysis data
was obtained using PhosphoImager (Storm 860; Molecular Dynamics,
Sunnyvale, CA) and analyzed using ImageQuant image analysis
software (Molecular Dynamics). The presented results represent the
averages of the relative values from the three independent experiments,
standardized against GAPDH mRNA levels.
Immunoprecipitation of STAT1 protein
Analysis of STAT1 phosphorylation was performed according to
the immunoprecipitation protocol provided by Santa Cruz Biotechnology
technical services. Briefly, IFN--stimulated cells were lysed in
RIPA buffer (PBS (pH 7.5) containing 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 25 mM sodium
fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µg/ml PMSF,
and 0.025 mM p-nitrophenyl guanidinobenzoate), mixed with
rabbit polyclonal anti-p84/p91 Abs and incubated overnight at
4°C. The immunocomplexes were precipitated for 2 h at 4°C
using protein A/G agarose beads. After washing the agarose beads with
RIPA buffer, STAT1 proteins were eluted from immunocomplexes by heating
at 95°C for 5 min in SDS-PAGE sample buffer. Proteins were subjected
to SDS-PAGE using 8 or 10% running gel, according to standard
protocols, and transferred to the polyvinylidene difluoride membranes.
Nonspecific binding was blocked overnight at 4°C using 10% FBS in
PBS containing 0.05% Tween 20. To assess the level of STAT1
phosphoprotein, the membranes were incubated in a solution of mouse
mAbs against phosphotyrosine (at a 1:1000 dilution in blocking buffer)
at room temperature for 1 h, followed by incubation with a
solution of anti-mouse IgG HRP-conjugated Abs (1:10,000 dilution in
blocking buffer, 1 h at room temperature). The signal was
visualized using ECL reagent. Subsequently, the
anti-phosphotyrosine Abs were removed from membranes by a 30-min
incubation at 55°C in buffer containing 62.5 mM Tris-HCl (pH 6.7),
2% SDS, and 70 mM 2-ME, and the total amount of the immunoprecipitated
STAT1 was assessed using mouse mAbs against STAT1 protein,
following the same protocol as used for anti-phosphotyrosine Abs.
The densities of the bands corresponding to phosphorylated STAT1
protein levels were determined and normalized against the total amount
of immunoprecipitated STAT1 protein. The exposure times for loading
controls used in normalization were always within the linear range of
the film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that the induction of the
I-Aß gene occurs12–18 h post IFN-
stimulation and then continues to rise for at least another 36 h
(33). Therefore, in the experiments described below, we
tested I-Aß mRNA expression 24 h after
IFN- stimulation of macrophage cell lines derived from the bone
marrow of B10A.Bcgr (B10R) and from B10.A
mice carrying the susceptibility associated mutation of
Nramp1 gene (point mutation at D169; B10S). As shown in Fig. 1
, following 24 h of treatment with
10 U/ml of IFN-, B10R macrophages expressed a high level of
I-Aß mRNA, whereas B10S macrophages expressed
I-Aß mRNA at a very low level. The results of
the densitometric analysis of four independent experiments are
presented in Fig. 1B. Infection with M. bovis BCG
decreased the ability of macrophages to express
I-Aß mRNA in response to IFN- by 75% in
B10R cells when a 10:1 bacteria to macrophage ratio was used (Fig. 1).
I-Aß mRNA expression in IFN--activated B10S
macrophages infected with M. bovis BCG was also reduced,
compared with uninfected IFN--stimulated B10S macrophages; however,
it is difficult to estimate precisely the level of reduction because
the expression level was already very low.
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To establish whether the observed difference in the level of
I-Aß mRNA expression between macrophages
expressing either the Nramp1r or the
Nramp1s allele of the Nramp1 gene was
similarly reflected at the level of MHC class II surface protein
(Ia) expression, we performed FACS analysis using specific Ab against
Ia molecules. As shown in Fig. 2,
A and B, the stimulation of B10R macrophages with
10 U/ml of IFN- resulted in much higher surface Ia protein
(99.8%) expression, compared with B10S macrophages (15.6%).
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, the infection of macrophages with M. bovis BCG decreased the
ability of both B10R and B10S macrophages to express surface Ia, as
determined by FACS analysis. As shown in Fig. 2, A and
B, infection of macrophages with M. bovis BCG
suppressed the ability of IFN--stimulated macrophages to express
surface Ia protein. The expression of Ia in BCG-infected macrophages
decreased from 99.8% to 75.8% in B10R and from 15.6% to 9.3% in
B10S cells.
Differential tyrosine phosphorylation of STAT1 protein in
resistant and susceptible macrophages
The activation of STAT1 protein requires its phosphorylation at
a specific tyrosine residue. Only phosphorylated STAT1 is
capable of forming homodimers that are subsequently translocated to
the nucleus. Therefore, macrophages carrying the resistant allele of
the Nramp1 gene (B10R and 129.M) and macrophages that do
not express intact Nramp1 protein (B10S), as well as macrophages
derived from Nramp1 knockout mice
(129/Nramp1-KO), were examined for their ability to
phosphorylate STAT1 protein in response to IFN- stimulation.
Macrophages were stimulated with 10 U/ml of IFN- for 5, 10, and 30
min, lysed, and STAT1 protein was immunoprecipitated with
anti-STAT1 Abs and subjected to Western blot analysis using
anti-phosphotyrosine Ab.
As shown in Fig. 3A,
significantly higher levels of the phosphorylated form of STAT1 were
present in B10R, compared with B10S, macrophages. The densitometric
analysis of the level of phosphorylated STAT1 compensated for total
immunoprecipitated STAT1 protein were plotted. In Fig. 3B,
the results show that the level of the phosphorylated STAT1 protein
induced by IFN- in B10R macrophages was at least 4-fold higher,
compared with the B10S macrophages. Similar results were obtained using
129.M macrophages (carrying Nramp1r allele)
and macrophages derived from Nramp1 gene knockout mice on
the same genetic background. As shown in Fig. 4, A and B,
129.M macrophages expressed 2.5–3 times more phosphorylated STAT1,
compared with the 129/Nramp1-KO macrophages.
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and 4 seem to
be more efficient for B10R macrophages than for 129/M (both carrying
the resistant allele of the Nramp1 gene), we decided to
compare the level of I-Aß mRNA and Ia surface
protein expression in the two cell lines following activation with 10
U/ml of IFN-. We have found no significant differences between
IFN--activated B10R and 129/M, either at the level of
I-Aß mRNA expression or at the level of surface
Ia protein (Fig. 5). Therefore, we can
conclude that the apparent difference at the level of STAT1
expression was most likely due to different efficiency of
immunoprecipitation between the two sets of experiments illustrated in
Figs. 3 and 4.
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-induced CIITA mRNA expression
Recently, it has been shown that phosphorylated STAT1 is acting
as a transactivating factor that is able to induce transcription of the
CIITA gene (54). Since we have observed a significant
difference in STAT1 phosphorylation between B10R and B10S
macrophages, it was important to test whether the higher level of
STAT1 phosphorylation observed in B10R cells correlated with higher
level of CIITA mRNA. We analyzed the IFN--induced CIITA mRNA
expression in B10R and B10S macrophages. We found that the expression
of CIITA mRNA was induced as early as 3 h post IFN-
stimulation, reaching highest level at 12–18 h of stimulation both in
B10R and B10S macrophages (data not shown). At each of the time points
analyzed, the expression of CIITA was a few-fold higher in B10R than in
B10S macrophages. Since the expression of I-Aß
mRNA was analyzed 24 h post IFN- stimulation, we also analyzed
the CIITA mRNA expression at this time point following IFN-
stimulation. As shown in Fig. 6,
A and B, and in Fig. 5B, stimulation
with 10 U/ml of IFN- induced CIITA mRNA expression both in B10R and
129/M (both carrying the resistant allele of the Nramp1
gene), as well as in B10S macrophages (carrying the susceptible allele
of the Nramp1 gene). However, the level of induction was
10-fold higher in B10R, compared with B10S, macrophages.
Interestingly, infection of macrophages with M. bovis BCG
suppressed the ability of macrophages to augment CIITA mRNA in response
to IFN- stimulation. In the B10R macrophages infected with M.
bovis BCG, the induction of CIITA mRNA with IFN- was diminished
by 70%, compared with IFN--stimulated noninfected macrophages. The
similar effect of M. bovis BCG infection on the induction of
CIITA by IFN- was observed in B10S macrophages; however, since the
level of CIITA mRNA induction by IFN- was a few-fold lower in B10S
macrophages, compared with B10R macrophages, the level of CIITA in the
BCG-infected B10S macrophages was barely detectable.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Bcg/Lsh/Ity gene was shown to be associated with
regulation of macrophage activation (9, 30, 58). This has
been demonstrated by a wide range of pleiotropic effects, including
regulation of KC chemokine, iNOS and RNI production, respiratory burst,
and IL-1ß and TNF- levels (27, 29, 30, 31, 32). RNI were
shown to play a decisive role in the control of the replication of
intracellular pathogens (21, 22, 23, 24, 25, 26, 27, 28).
A proper analysis of the molecular basis of macrophage activation for effective bactericidal function requires access to homogenous cell populations of defined genetic background. We have generated macrophage cell lines from mouse strains that carry either the Nramp1r or Nramp1s allele (52), and we have made a detailed comparison of these cell lines (27, 33, 59, 60). In addition, we have generated macrophage cell lines from the Nramp1 gene knockout mice on the 129/J genetic background and from their 129/J littermate controls that carry the Nramp1r allele.
Recently, using our macrophage cell lines, we have found that
PKC-specific activity was significantly more increased in the cytosolic
fractions derived from Nramp1r, compared with
Nramp1s, macrophages. Furthermore, during the
course of macrophage activation, particulate fractions from
Nramp1r macrophages contained significantly
greater PKC activity, compared with Nramp1s. The
differences in PKC activity between Nramp1r and
Nramp1s macrophages contributed to altered
responsiveness to IFN- that resulted in more efficient production of
RNI by Nramp1r macrophages.
Nramp1r macrophages also had a superior ability
to phosphorylate endogenous substrate compared with
Nramp1s macrophages (60).
Previously, we found that macrophages carrying the susceptible allele
of Nramp1 expressed much lower levels of MHC class II
surface proteins and I-Aß mRNA when compared
with macrophages with resistant allele (33). We also
demonstrated a significantly reduced amount of produced nitrates, a
decreased production of TNF-, and a decrease in the level of MHC
class II in response to IFN- stimulation in macrophages transfected
with Nramp1 antisense ribozyme (Nramp1-Rb),
compared with the controls transfected with mock vector
(52). Overall, these studies supported the hypothesis that
the Nramp1 gene is involved in the regulation of the early
signaling that occurs in macrophages activated with IFN-.
In this paper, we have focused on the mechanism of IFN--induced
expression of MHC class II Ags by macrophages carrying either the
resistant or susceptible allele of Nramp1 and macrophage
cell lines derived from Nramp1-knockout mice. We also
analyzed the effect of M. bovis BCG infection on the
expression of MHC class II using these cell lines. We found that
infection with M. bovis BCG leads to a very significant
inhibition of IFN--induced I-Aß mRNA and
Ia protein expression in tested macrophage cell lines.
The observed inhibition of I-Aß gene expression
by M. bovis BCG infection may represent a protective
mechanism that allows the pathogen to persist longer in the host. This
phenomenon does not seem to be unique for the infection of macrophages
with M. bovis BCG. It was previously reported that the
protozoan Toxoplasma gondii is able to inhibit MHC class II
expression by infected macrophages. The mechanism of that inhibition is
still unknown, but does not seem to be related to the increased
production of prostaglandin E2, IL-10, TGF-ß, or NO
(61). The infection of macrophages with L.
donovani also leads to the inhibition of IFN--induced MHC
class II gene expression (62, 63, 64). Similarly, it was shown
that murine CMV (MCMV) was able to inhibit MHC class II transcription
(65). Macrophages infected with MCMV were not able to
express Ia in response to IFN- and, consequently, failed to present
Ags and activate CD4+ T lymphocytes. The CMV
virus most likely interferes with the expression of transcription
factors involved in I-Aß gene expression. In
addition, Wadee et al. (66, 67) showed that a 25-kDa
glycoprotein encoded by M. tuberculosis was able to inhibit
MHC class II expression in monocytes.
IFN- has been shown to be a potent inducer of MHC II molecules in a
variety of cell types. The analysis of IFN--knockout mice showed
that they were able to develop normally in the absence of pathogens,
but their macrophages were unable to produce antimicrobial products and
had reduced expression of MHC class II genes. Thus, IFN--deficient
animals were extremely sensitive to infection and died shortly after
administration of the pathogen, i.e., M. bovis
(68). Similarly, the lack of IFN- receptor expression
in knockout mice led to an inability of the animals to control
Mycobacterial as well as other types of infection.
Macrophages derived from IFN- receptor-deficient mice produced much
lower levels of TNF- and NO, making them inefficient in killing
bacteria (69). IFN--induced signal transduction
requires activation of specific receptor coupled to the JAK/STAT signal
transduction system. This primary effect leads to induction of
expression of a number of genes, including many transcription factors
and regulatory proteins. One of them, CIITA, was characterized as a
master regulator of MHC genes, and it was shown to be essential for
both constitutive and inducible expression of MHC class II genes
(43, 70, 71, 72). CIITA belongs to a family of
IFN--inducible genes. Interestingly, it was shown that STAT1
phosphorylation is required for the expression of CIITA gene. The
aberrations in constitutive or infection-induced levels of CIITA
expression may lead to severe immunological disorders or chronic
infections.
Our studies demonstrate that, after IFN- stimulation, CIITA
expression is elevated in macrophages carrying either resistant or
susceptible allele of Nramp1, but the level of expression of
CIITA was higher in macrophages carrying the "r" allele of
Nramp1. Since IFN--inducible expression of CIITA is
STAT1-dependent, we decided to evaluate its phosphorylation in
response to IFN- stimulation. Our results demonstrate that, also at
this level, Nramp1r macrophages are superior to
the Nramp1s macrophages and were able to express
much higher levels of the tyrosine-phosphorylated form of STAT1 in
response to IFN- stimulation. A higher level of phosphorylated
STAT1 protein would lead to a higher level of STAT1 homodimer
being translocated to the nucleus and bound to the GAS element of the
CIITA promoter in Nramp1r, compared with
Nramp1s, macrophages. Consequently the lower
level of CIITA gene expression in susceptible macrophages results in
less efficient transcriptional activation of MHC class II gene
expression. Our data clearly support this hypothesis and explain our
previously published findings, showing a difference between
Nramp1r and Nramp1s
macrophages at the level of transcription activation of
I-Aß (33).
Overall, the presented results indicate that the differential MHC class II expression observed between Nramp1r and Nramp1s macrophages results from differences found at the level of transcription activation of the I-Aß gene controlled by CIITA. Alternative explanations, such as translational control and Ia Ag stability, have also been proposed (73, 74, 75, 76). Recent evidence suggests that one possible function of the Nramp1 protein is the transport of iron out of the bacteria/parasite-containing phagosome (14, 77). Therefore, it is possible that transport of iron by Nramp1 also has an impact on an iron homeostasis in macrophages. Iron and other divalent ions (e.g., Zn2+), were shown to regulate the activity of various transcription factors and other regulatory proteins involved in posttranscriptional regulation of gene expression. The exact link between the regulation of iron metabolism by the Nramp1 gene and transcriptional and posttranscriptional regulation of MHC class II expression remains to be established, and more studies are required to further characterize the molecular mechanism that regulates this important function of macrophages in immunity against bacterial infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Danuta Radzioch, McGill University, Departments of Experimental Medicine and Human Genetics, 1650 Cedar Avenue, L11-218 Montreal, PQ, H3G 1A4, Canada. E-mail address:
3 Abbreviations used in this paper: Nramp1, natural resistance associated macrophage protein 1; CIITA, class II transactivator; BCG, bacillus Calmette-Guérin; PKC, protein kinase C; iNOS, inducible NO synthase; RNI, reactive nitrogen intermediates.
Received for publication February 22, 1999. Accepted for publication June 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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