From the Department of Medicine (Division of
Infectious Diseases) and the § Department of Microbiology
and Immunology, the University of British Columbia Faculties of
Medicine and Science and The Research Institute of the Vancouver
Hospital and Health Sciences Center,
Vancouver, British Columbia, Canada V57 3J5, and the
¶ Department of Epidemiology, School of Public Health, the
University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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Lipoarabinomannan (LAM) is a putative virulence
factor of Mycobacterium tuberculosis that inhibits monocyte
functions, and this may involve antagonism of cell signaling pathways.
The effects of LAM on protein tyrosine phosphorylation in cells of the
human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein
kinase. To examine whether these effects of LAM could be related to
activation of a phosphatase, fractions from LAM-treated cells were
analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of
cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells,
using phosphorylated mitogen-activated protein kinase as substrate,
indicated that LAM promoted increased activity of SHP-1 in
vivo. LAM also activated SHP-1 directly in vitro.
Exposure of cells to LAM also attenuated the expression of tumor
necrosis factor-, interleukin-12, and major histocompatibility class
II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.
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INTRODUCTION |
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It has been estimated that there are approximately eight million new cases and three million deaths annually, worldwide, from tuberculosis (1). It is also believed that the causative agent of this disease, Mycobacterium tuberculosis, infects one-third of the world's population (2). M. tuberculosis infects and resides exclusively within mononuclear phagocytes, and its ability to evade being killed within phagocytic cells has likely contributed to its longevity as a highly successful pathogen. Although many other microbes are killed when ingested by phagocytes, M. tuberculosis prevents its destruction by impairing critical macrophage functional responses (3-7)1 using mechanisms that are not well understood.
Following ingestion by macrophages, M. tuberculosis modifies
the phagosome such that it does not fuse with lysosomes (3), an
avoidance tactic that allows it to evade proteolytic destruction. Evidence also suggests that the phagosome membrane is disrupted in a
manner that may allow entry of the organism or its products into the
host cell cytosol (6, 9, 10). Within the host cell, M. tuberculosis is able to replicate (11) and induce a state of
diminished responsiveness to further stimulation (3, 4, 6, 7,
12).1 For example, infected macrophages are poorly
responsive to interferon- (IFN-
)2 as assessed by
both the expression of major histocompatibility complex (MHC) class II
molecules1 and intracellular killing (13). Of critical
interest is the identity of the virulence factors responsible for
macrophage deactivation. One potential candidate is the mycobacterial
cell wall glycolipid, lipoarabinomannan (LAM).
LAM is complex molecule consisting of a phosphatidylinositol
moiety that anchors a large mannose core to the mycobacterial cell wall
(14, 15). The mannose core contains multiple branched, arabinofuranosyl
side chains. Comparative analyses of LAMs from different species of
mycobacteria have shown that the non-reducing termini of the
arabinofuranosyl side chains are differentially modified. For example,
M. tuberculosis and Mycobacterium leprae modify
the termini with mannose residues thereby yielding "man-LAM," whereas rapidly growing mycobacterial species use inositolphosphates thereby giving rise to "ara-LAM" (16). It is thought that these contrasting modifications are responsible for the marked differences in
the biological activities of man-LAM and ara-LAM (16-18). Studies have
shown that, in comparison to man-LAM, ara-LAM is a potent inducer of
tumor necrosis factor (TNF)- and interleukins-1, -6, and -10 (17,
19-22). However, both LAMs appear to be equipotent with respect to
induction of transforming growth factor (TGF)-
(22). In addition,
the early response genes, c-fos, KC,
iNOS, and JE are induced by ara-LAM but not by
man-LAM (18, 23). These findings suggest that M. tuberculosis has evolved mechanisms to "mask" man-LAM such
that it has a diminished capacity to induce cytokines and other
monocyte gene products that may be detrimental to the organism's
survival.
Other studies have revealed that LAM, derived from M. tuberculosis, actively inhibits properties of both macrophages and
T cells. In macrophages for example, LAM is able to block many actions of IFN-, including tumor cell killing (24, 25), intracellular killing of toxoplasma (21, 24, 25), and increased expression of several
IFN-
-inducible genes (26). It has been suggested that these actions
of LAM may be related to inhibition of protein kinase C (PKC) to
scavenging of cytotoxic oxygen free radicals, or to both (26). In T
cells, LAM has been shown to suppress antigen-driven proliferation of a
CD4+ T cell clone (27). It has also been shown to inhibit
the accumulation of mRNA for interleukins-2 and -3, granulocyte-macrophage colony-stimulating factor, and the interleukin-2
receptor
-chain in Jurkat T cells stimulated with phytohemagglutinin
and phorbol esters (28).
This study examined the hypothesis that the inhibitory effects of LAM on mononuclear phagocytes may be related to altered cell signaling. The results show that LAM promotes tyrosine dephosphorylation of multiple proteins including MAPK. In the latter instance, this leads to impaired activation of the enzyme. These effects of LAM may be explained by the action of the Src homology 2-containing tyrosine phosphatase-1 (SHP-1), a tyrosine phosphatase known to be important for attenuating activation signals (29, 30). The results presented show that SHP-1 is activated by LAM both in vivo and in vitro.
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MATERIALS AND METHODS |
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Reagents--
Phorbol 12-myristate 13-acetate (PMA) was
purchased from Sigma. Protein G-Sepharose was from Pharmacia Biotech
Inc. Horseradish peroxidase-conjugated goat anti-rabbit antibodies,
protein A-agarose, and electrophoresis reagents and supplies were
purchased from Bio-Rad. The THP-1 cell line was obtained from the
American Type Culture Collection (Rockville, MD). THP-1 cells, derived
from a patient with acute monocytic leukemia, are phagocytic and
possess other characteristics of monocytes including expression of Fc and C3b receptors (31). THP-1wt cell line (THP-1 cells stably expressing the glycosylphosphatidylinositol-linked CD14) was kindly provided by Dr. Richard Ulevitch (The Scripps Research Institute, La
Jolla, CA). RPMI 1640 and Hank's balanced salt solution were from Stem
Cell Technologies (Vancouver, British Columbia). Enhanced chemiluminescence reagents and ECL film were from Amersham
International (Oakville, Ontario). Lipopolysaccharide
(Escherichia coli O1227:B8) was purchased from Difco. Human
AB+ serum was provided by the Canadian Red Cross
(Vancouver, British Columbia). Anti-phosphotyrosine monoclonal antibody
4G10, anti-MAPK-ct, and MAPK-glutathione S-transferase (GST)
were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-SHP-1 was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Recombinant, human IFN- was from Genentech Inc. (South San
Francisco, CA). Riboprobe systems buffers, RQ1 RNase-free DNase, T7,
T3, and SP6 RNA polymerases, and proteinase K were from Promega Corp.
(Madison, WI). RNase T1, Trizol, and tRNA were from Life Technologies,
Inc. Unless stated otherwise, all reagents were the highest quality
available.
Mycobacterial Lipids-- Endotoxin-free LAM, lipomannan, and phosphatidylinositolmannoside were generously provided by Dr. P. J. Brennan (Colorado State University, Ft. Collins, CO, through National Institutes of Health Contract NO1-A1-25147). In all experiments, unless otherwise indicated, the LAM (mannose capped) used was derived from the virulent, erdman strain of M. tuberculosis (32, 33). Lipomannan is similar to LAM except that it does not contain the arabinofuranosyl side chains (34). Phosphatidylinositolmannoside is similar to lipomannan except that the mannan core contains fewer mannose residues (34). Both of the latter were derived from the virulent H37rv strain of M. tuberculosis.
Isolation of Monocytes and Cell Culture--
Fractions of
peripheral blood enriched in white blood cells were obtained from the
Cell Separator Unit (Vancouver Hospital and Health Sciences Center).
Monocytes were enriched (85-95% pure) by adherence as described
previously (35). Monolayers of adherent cells were treated with either
LAM, LPS, or both, rinsed with ice-cold PBS, snap-frozen using liquid
nitrogen, and stored at 70 °C prior to analysis. Cell lysates were
prepared by lysing cells on ice (20 min) in lysis buffer (20 mM Tris, pH 8.0, 1% Triton X-100, 137 mM NaCl,
10% glycerol, 2 mM EDTA, 1 mM
Na3VO4, 5 mM NaF, 100 nM microcystin, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml
aprotinin). Protein concentrations were determined using Bio-Rad
ProteinDC and bovine serum albumin as standard. Monocyte
cell lines were maintained in RPMI supplemented with 10%
heat-inactivated fetal calf serum (FCS). For acute studies, cells were
rendered quiescent by culture in RPMI without FCS for 12-16 h at a
concentration of 5 × 105 cells/ml. For chronic
studies, log phase cells were washed in Hanks' solution and
resuspended in RPMI without FCS, followed by the addition of
mycobacterial cell wall components. Following treatments, cells were
lysed immediately, and detergent-soluble material was frozen at
70 °C until further analysis.
Cellular Fractionation and Translocation Assay-- Following incubation of THP-1wt cells with either LAM or LPS, cells were fractionated essentially as described previously (36). In brief, monocytes were scraped into hypotonic fractionation buffer (10 mM Tris, pH 7.4, 4.5 mM EGTA, 2.5 mM EDTA, 1.0 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 100 nM microcystin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin) and lysed for 20 min at 4 °C while rotating. Lysates were then centrifuged at 100,000 × g for 30 min to separate cytosolic from particulate fractions. The resulting pellets were extracted in fractionation buffer containing 1% Triton X-100 for 20 min and centrifuged (16,000 × g, 20 min, 4 °C) to separate detergent-insoluble and -soluble material. The resulting supernatant was taken to represent a membrane fraction. Twenty micrograms of cytosolic and membrane fractions were then subjected to SDS-PAGE and immunoblotting using anti-SHP-1 or anti-MAPK-ct antibodies.
Analysis of HLA-DR Cell Surface Expression--
THP-1 cells were
seeded at a density of 105 per cm2 and allowed
to adhere and differentiate in the presence of PMA (20 ng/ml) at
37 °C in a humidified atmosphere of 5% CO2 for 24 h. Cells were then washed three times with Hanks' balanced salt
solution and adherent monolayers were replenished with culture medium. LAM or LPS was added to the cells for an additional 24 h followed by treatment with IFN- for 24 h more. To measure cell surface expression of HLA-DR, control and treated cells were incubated with
anti-HLA-DR monoclonal antibody (clone HL38, Catlag Laboratories, San
Francisco, CA) for 30 min and then washed twice and labeled with
fluorescein isothiocyanate-conjugated F(ab)
2 sheep
anti-mouse IgG (Sigma) for 30 min. All staining and washing procedures
were performed at 4 °C in Hanks' balanced salt solution containing 0.1% NaN3 and 1% FCS. To control for cell viability,
cells were incubated with propidium iodide (0.5 µg/ml in staining
buffer) for 10 min. Cells were then washed twice and fixed in 2%
paraformaldehyde in staining buffer. Fluorescence was analyzed using a
Coulter Elite flow cytometer (Hialeah, FL). Viable cells were
identified by exclusion of propidium iodide. Relative fluorescence
intensities of 5000 cells were recorded as single-parameter histograms
(log scale, 1024 channels, 4 log decades), and the mean fluorescence intensity (MFI) was calculated for each histogram. Results are expressed as MFI index which corresponds to the ratio: MFI of cells
incubated with specific antibody/MFI of cells incubated with irrelevant
isotype-matched IgG.
Western Blotting, Immunoprecipitation, and Densitometry-- Whole cell lysates, prepared in lysis buffer, were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine monoclonal antibody (4G10), anti-SHP-1, or anti-MAPK-ct by previously described methods (37). SHP-1 was immunoprecipitated by incubating 0.5-1.5 mg of lysate with anti-SHP-1 for 2 h at 4 °C, followed by the addition of 40 µl of protein G-Sepharose for an additional 2 h. To assess the amount of individual proteins immunoprecipitated in each sample, after detection of bound 4G10 Ab, membranes were stripped and reprobed with anti-SHP-1 or anti-MAPK-ct and developed by enhanced chemiluminescence as described (37). Densitometry was performed using a Howtek Scanner and Quantity One software (PDI Bioscience, Aurora, Ontario, Canada).
RNase Protection Assay--
THP-1wt cells were preincubated with
either LAM or LPS for 16-20 h, followed by the addition of 1 µg/ml
LPS for 2 h. RNA was extracted from cells using Trizol according
to the manufacturer's protocol. Equal amounts of RNA were subjected to
an RNase protection assay, essentially as described previously (38).
Two cytokine-specific riboprobe template sets were tested, HL-14 and
HL-21. These were assembled from EcoRI-linearized and
purified subclones. The HL-14 template set synthesized riboprobes
specific for interleukins-1, -1
, -6, -10, TNF-
, TNF
,
granulocyte-macrophage colony-stimulating factor, TGF-
1, and rpL32.
The HL-21 template set synthesized riboprobes specific for
interleukins-2, -4, -5, -12 (p40 and p35), and -13, CD4, CD8, and
rpL32. The respective nucleotide sequences and GenBank Accession
numbers of the individual clones were previously described.3
Phosphatase Assays--
Assays for protein tyrosine phosphatase
activity were carried out using either para-nitrophenol
phosphate (pNPP) or phosphorylated MAPK-1-GST as substrates. SHP-1 was
immunoprecipitated from equal amounts of cell lysate protein as
described above. For MAPK-1-GST dephosphorylation, the
immunoprecipitates were further washed three times in phosphatase assay
buffer (25 mM imidazole, pH 7.0, 1 mM EDTA) and
resuspended in phosphatase assay buffer supplemented with 1 mM dithiothreitol (DTT). MAPK-1-GST coupled to
glutathione-agarose was autophosphorylated in buffer containing 25 mM -glycerophosphate, 20 mM MOPS, pH 7.2, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 mM ATP, and 1 mM DTT for 30 min
at 30 °C. The agarose beads were washed two times in
autophosphorylation buffer without ATP and two times in phosphatase
buffer without DTT. After resuspension in phosphatase assay buffer
containing 1 mM DTT, the MAPK-1-GST beads were incubated
with the SHP-1 immunoprecipitates for 1 h at 30 °C. The
reactions were stopped by the addition of an equal volume of 2 × Laemmli buffer followed by immunoblotting with anti-phosphotyrosine antibodies. Blots were then stripped and reprobed with either anti-MAPK-1-GST or anti-SHP-1 to confirm equal loading of substrates and phosphatase enzyme. When pNPP was used as a substrate, the SHP-1
immunoprecipitates were washed further in a buffer containing 50 mM Hepes, pH 7.0, 5 mM EDTA and, 10 mM DTT for 1 h at 37 °C. Immunoprecipitates were
ultimately resuspended in the same buffer containing 2 mM
pNPP followed by an incubation for 4 h at 30 °C. Absorbances of
the samples were read at 405 nM as described previously (39). Activity is expressed as a percent of control. Phosphatase activity in cytosolic and membrane fractions was measured by incubating 25-50 µg of each fraction from control or treated cells in a buffer containing 50 mM imidazole Cl, pH 7.2, 1.0 mM
EDTA, and 16 mM pNPP, for 30 min at 30 °C. Absorbances
of the samples were measured as described above and expressed as
percent of control optical density.
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RESULTS |
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Effects of LAM on PMA-induced Tyrosine Phosphorylation and Activation of MAPK-- To examine whether LAM modulates activation of MAPK, THP-1wt cells were preincubated with LAM for 16 h followed by the addition of PMA (100 nM) for 15 min. PMA reproducibly induced the tyrosine phosphorylation of a 42-kDa protein which is positioned exactly with that of p42MAPK2 (Fig. 1A). Phosphorylation of this band was not evident in cells treated with either 0.1% serum (lane 1) or LAM alone (lane 3). On the other hand, tyrosine phosphorylation of p42MAPK2, in response to PMA, was significantly attenuated in cells preincubated with LAM. Two other parameters of MAPK activation were also assessed. In response to PMA and secondary to the activation of MAPK kinase, MAPK becomes tyrosine-, serine-, and threonine-phosphorylated leading to its activation (40-42). These phosphorylations are associated with retarded mobility (bandshifting) of the kinases as assessed by SDS-PAGE and immunoblotting. As can be seen in Fig. 1B, bandshifting in response to PMA was clearly observed for both the p42MAPK2 and p44MAPK1 isoforms. Of particular interest was the finding that, although LAM markedly attenuated tyrosine phosphorylation of p42MAPK2, this did not impair the bandshifting otherwise expected following treatment with PMA. Based on this finding, it would appear that bandshifting is a function of serine/threonine phosphorylations and that LAM selectively affects tyrosine phosphorylation of MAPK. Of interest, while bandshifting was observed for p44MAPK1, PMA was unable to induce tyrosine phosphorylation of this isoform, consistent with a previous report of the actions of PMA on p44MAPK1 and p42MAPK2 (43).
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Effects of LAM on LPS-induced Tyrosine Phosphorylation of Monocyte
Proteins--
To determine whether other tyrosine phosphorylation
events are regulated by LAM, the effects of LAM on LPS-induced tyrosine phosphorylation of proteins was assessed in normal human monocytes (Fig. 2). As expected, LPS treatment
induced increased tyrosine phosphorylation of multiple monocyte
proteins. Of interest, pretreatment of cells with LAM (2 µg/ml) for
16 h abrogated the effects of LPS in a manner similar to that
observed with tyrosine phosphorylation of p42MAPK2 induced by
PMA. The autoradiogram shown in Fig. 2A was analyzed by
densitometry to identify bands that underwent increased phosphotyrosine labeling in response to LPS and to quantitate the effects of LAM on
these events. The results of this analysis (Fig. 2B) show
that LPS induced marked increases in phosphotyrosine content in at least eight proteins. Pretreatment of cells with LAM virtually eliminated all of these increases. In contrast to the marked effects of
LPS in phosphotyrosine labeling of certain bands (Fig. 2, A and B), proteins in the 50-60-kDa range were less affected
by LPS. Although the overall results of LAM were to promote tyrosine dephosphorylation, occasionally slight, likely insignificant, increases
in tyrosine phosphorylation were observed in response to LAM alone.
These changes were not consistently reproducible. In parallel
experiments, it was also observed that LAM abrogated tyrosine
phosphorylation of monocyte proteins induced by IFN- (data not
shown).
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Effects of LAM Precursors on the Tyrosine Phosphorylation of Cytosolic and Membrane Proteins-- When precursors of LAM were examined for their abilities to affect levels of tyrosine phosphorylation of cytosolic proteins, apparent structure-dependent effects were observed (Fig. 3). In comparison to control cells, LAM (2 µg/ml) markedly reduced levels of tyrosine phosphorylation of multiple cytosolic proteins. Lipomannan (LM, 2 µg/ml) similarly led to diminished tyrosine phosphorylation of proteins in the cytosol, but not to the same extent as LAM. Diminished levels of tyrosine phosphorylation in response to phosphatidylinositolmannoside (PIM, 2 µg/ml) were also evident, but these changes were less pronounced than those observed with either LAM or LM. Although reductions in protein tyrosine phosphorylation were also seen with LAM and LM in the membrane fraction, it was notable that PIM induced increased tyrosine phosphorylation of proteins in the 50-70-kDa range and the 30-40-kDa range. The effects of PIM in both the cytosolic and membrane fractions were similar to those seen with LPS which was used at a concentration reflecting the maximum potential level of LPS contamination in any of the preparations. In other experiments, ara-LAM, derived from Mycobacterium smegmatis, an avirulent environmental species of mycobacterium, was examined for its acute effects on tyrosine phosphorylation. Unlike LAM from M. tuberculosis, the arabinofuranosyl side chains of ara-LAM are not capped with mannose residues. Rather, they are capped with inositolphosphates imparting to ara-LAM agonist properties with respect to monocyte activation. In contrast to the results depicted in Fig. 3 for LAM, ara-LAM was observed to increase levels of tyrosine phosphorylation of multiple proteins (data not shown) consistent with its activating properties.
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Activation of SHP-1--
Attenuation by LAM of tyrosine
phosphorylation in monocytes could be explained by inhibition of
tyrosine kinases, activation of phosphotyrosine phosphatases, or both.
To address whether a phosphatase is involved, initial experiments were
done in which cells were exposed to either LAM (2 µg/ml), LPS (36 pg/ml, the maximum potential contaminating concentration in LAM
preparations), IFN- (200 units/ml), or control medium for 30 min.
Cytosolic and membrane fractions were prepared and examined for
evidence of phosphatase activation. As shown in Fig.
4, in response to LAM, a
membrane-localized increase in pNPPase activity of approximately 1.7-fold was observed. In contrast, no change in cytosolic pNPPase activity was observed. In cells exposed to IFN-
or LPS, pNPPase activity was similar to control levels in both cytosolic and membrane fractions.
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Attenuation of LPS-induced Up-regulation of TNF- and IL-12
p40 mRNA by LAM--
To address whether the effects of LAM on
monocyte tyrosine phosphorylation correlate with diminished functional
responses to LPS, mRNA levels were measured for TGF-
, TNF-
,
CD4, and IL-12 p40 by RNase protection assay. As shown in Fig.
7, treatment of THP-1wt cells with LPS (1 µg/mL) up-regulated the expression of both TNF
and IL-12 p40
mRNAs within 2 h. In contrast, levels of neither TGF-
1 nor
CD4 changed in response to LPS. When cells were preincubated with LAM
(2 µg/ml) for 16-20 h, the LPS-induced increases in both TNF-
and
IL-12 p40 mRNAs were significantly attenuated. In contrast, LAM had
no effects on the steady state levels of TGF-
1 or CD4. Cells were
also incubated in 36 pg/ml LPS (cLPS) overnight prior to stimulation
with LPS. This preincubation in cLPS represents the apparent level of
LPS-like activity contaminating the LAM preparations as assessed by
Limulus amebocyte assay. Preincubation of cells with this
concentration of LPS had negligible effects on the subsequent
enhancement of TNF
and IL-12 p40 mRNA levels in response to a
second incubation with LPS.
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Attenuation of Interferon--induced Up-regulation of Cell Surface
MHC Class II Expression by LAM--
To examine whether the effects of
LAM on IFN-
-induced protein tyrosine phosphorylation in THP-1 cells
(data not shown) correlates with diminished functional responses to
this cytokine, cell surface expression of MHC class II expression was
analyzed. Differentiated THP-1 cells were treated with various
concentrations of LAM or control vehicle for 20 h. Interferon-
was then added for an additional 24 h. Cell surface MHC class II
levels were measured by fluorescence-activated cell sorter analysis. As
shown in Fig. 8, treatment of cells with interferon-
(200 units/ml) produced a 4-fold increase in the level
of cell surface MHC class II. Pretreatment of cells with LAM led to a
nearly complete abrogation of interferon-
-induced MHC class II
expression. Similar results were obtained with normal human monocytes
(data not shown).
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DISCUSSION |
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To maintain itself stably in its human reservoir, M. tuberculosis has evolved strategies to undermine both innate host resistance and acquired immune responses. The mechanisms involved most likely include the elaboration of immunosuppressive molecules, one of which is LAM. A great deal of evidence supports the notion that LAM is a potential immunosuppressive molecule (21, 24-26); however, its precise mode of action is still not known. The present study examined the possibility that LAM may affect cell signaling pathways in macrophages and focused on the critical phenomena of protein tyrosine phosphorylation and tyrosine dephosphorylation.
Tyrosine phosphorylation is an important component of all of the signaling pathways examined in this study. PMA is a broad range activator of cells and acts primarily through its ability to activate multiple isoforms of PKC (46). One event that occurs downstream of PKC is activation of the p42MAPK2, and this involves its phosphorylation on tyrosine 183 and threonine 185 N-terminal to kinase subdomain VIII (47). MAPK activation is known to be involved in the regulation of a broad range of cellular processes (48). In the present study, the effects of LAM on PMA-induced tyrosine phosphorylation and activation of the p42MAPK2 was examined (Fig. 1, A-C). As shown, activation of p42MAPK2 was markedly attenuated in LAM-treated cells (Fig. 1C), and this appeared to be related to diminished tyrosine phosphorylation of the kinase (Fig. 1A). The finding that bandshifting of p42MAPK2 was unaffected (Fig. 1B) suggests that other phosphorylation events, involving either threonine residues, serine residues, or both, regulate bandshifting and that these are unaltered in LAM-treated cells. One possibility to explain these findings is that LAM inhibits activation of PKC by PMA. Three lines of evidence, however, argue against this possibility. First, bandshifting was observed in LAM-treated cells indicating that some phosphorylation of MAPK is nevertheless occurring in response to PMA. Second, LAM only weakly inhibits PKC at the concentrations used (26). Third, it was observed that PMA-induced activation of other renaturable kinases, as assessed by "in-gel" assays, were unaltered by LAM pretreatment (data not shown). On the other hand, the finding of diminished tyrosine phosphorylation of p42MAPK2 in the face of apparently normal bandshifting in LAM-treated cells could be explained by selective tyrosine dephosphorylation of MAPK during its activation by PMA. A potential mediator of this effect of LAM is the abundant phosphotyrosine phosphatase SHP-1, which is known to be involved in terminating activation signals. Indeed, it has been reported that MAPK is an in vitro substrate for SHP-1 (45), and the experiments reported here provide direct evidence that SHP-1 is activated by LAM (Figs. 5 and 6).
SHP-1 is thought to be an important negative regulator of a variety
signaling pathways such as those related to the actions of IFN- (29)
and insulin (49), and regulation of its activity is complex. It has
been reported that phosphorylation on tyrosine 538 increases the
activity of SHP-1 (49, 50). In contrast, serine phosphorylation induced
by PKC leads to decreased activity (39). It has also been shown that
SHP-1 is activated by phospholipids suggesting that it may be regulated
by translocation from the cytosol to the membrane (45).
The results of the present study suggest that LAM may regulate SHP-1 by multiple mechanisms. First, it is shown that LAM increases SHP-1 phosphatase activity in vivo as assessed by immunoprecipitation phosphatase assays (Figs. 5B and 6A). The mechanism for this increased activity was investigated by examining SHP-1 tyrosine phosphorylation, direct activation by LAM in vitro, and translocation of the phosphatase to the membrane. Following acute exposure of cells to LAM, it was observed that SHP-1 phosphatase activity is increased (Fig. 5B). However, under these conditions, LAM did not lead to detectable increases in tyrosine phosphorylation of SHP-1 or to its apparent translocation to the membrane (data not shown). Thus, under conditions of short term exposure, the most likely mechanism is related to direct activation of SHP-1 by LAM or possibly to a decrease in the enzyme's serine phosphorylation state. Activation by a direct interaction is supported by the finding that incubation of SHP-1 with LAM in vitro leads to activation of the enzyme (Fig. 5, C and D). This finding is consistent with prior studies in which activation of SHP-1 in vitro by phospholipids has been observed (45).
In contrast to short term incubation, long term exposure of cells to LAM did result in translocation of SHP-1 to the membrane (Fig. 6B) and to an increase in its tyrosine phosphorylation (Fig. 6C). These effects could explain the increase in SHP-1 activity observed during longer term exposure and suggest the possibility that LAM may regulate SHP-1 by multiple mechanisms including (i) direct interactions, (ii) changes in its phosphorylation state, and (iii) subcellular localization.
In addition to attenuating PMA-induced tyrosine phosphorylation and
activation of MAPK, LAM was also observed to inhibit both LPS- (Fig. 2)
and IFN- (data not shown)-induced protein tyrosine phosphorylation
in monocytes. Recent evidence indicates that LPS is a potent inducer of
tyrosine phosphorylation (51, 52) and that tyrosine kinase inhibitors
block LPS-induced functional changes such as TNF-
production (51)
and tumoricidal activity (53). Tyrosine phosphorylation is also known
to be a critical element in bringing about functional responses to
IFN-
(54, 55). Indeed, under the conditions of the present study,
attenuation by LAM of tyrosine phosphorylation in response to either
LPS or IFN-
correlated with inhibition of functional responses to
both agonists (Figs. 7 and 8).
The effects of LAM on levels of tyrosine phosphorylation of macrophage proteins appear to be influenced by specific structural features of the molecule. Thus, the results shown in Fig. 3 indicate that the capacity of LAM to diminish tyrosine phosphorylation of macrophage proteins is dependent on the structure of the polysaccharide region (LAM > lipomannan > phosphatidylinositolmannoside). Lipomannan is identical to LAM except for the absence of arabinofuranosyl side chains, and phosphatidylinositolmannoside is identical to lipomannan except for having fewer mannose residues. Thus, these findings suggest that the action of LAM is at least partially mediated through it polysaccharide region.
The ability of LAM to diminish cytokine induction by LPS and functional
responses to IFN- through the effects of SHP-1 has the potential to
modify the course of infection with M. tuberculosis. The
importance of TNF-
in the pathogenesis of the M. tuberculosis infection has become increasingly evident, notably in
granuloma formation. In bacillus Calmette-Guérin infections in
mice, TNF-
deficiency results in poor granuloma formation and
disseminated disease (56). Furthermore, TNF-
induces macrophages to
produce reactive nitrogen oxides that are critical in M. tuberculosis killing (57). Similarly, interleukin-12 (IL-12) plays
a critical role in innate resistance to M. tuberculosis
infections by activating natural killer cells that produce IFN-
,
thereby further activating macrophages (8). Thus, impairing TNF-
and
IL-12 production and responsiveness to IFN-
through effects on SHP-1
may be a major mechanism by which LAM promotes intracellular survival
of M. tuberculosis.
In summary, the data in this study provide support for the hypothesis that LAM is a potential virulence factor that may contribute to the pathogenesis of M. tuberculosis. One mechanism by which LAM may exert its effects is by inhibiting signaling pathways that are necessary for macrophage activation and intracellular killing. This inhibitory effect of LAM may be related to its ability to regulate the activity and subcellular distribution of the protein tyrosine phosphatase, SHP-1.
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ACKNOWLEDGEMENTS |
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We thank Drs. Patrick Brennan and John T. Belisle for providing the mycobacterial derived lipids and Genentech
for providing recombinant human IFN-.
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FOOTNOTES |
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* This work was supported by Medical Research Council of Canada Grant MA-8633 and by a grant from Glaxo Wellcome Action TB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Infectious Diseases, Dept. of Medicine, University of British Columbia, 2733 Heather St., Rm. 452D, Vancouver, British Columbia, Canada V5Z
3J5. E-mail: ethan{at}unixg.ubc.ca.
1 Z. Hmama, R. Gabathuler, W. A. Jeffries, G. de Jong, and N. E. Reiner, submitted for publication.
2
The abbreviations used are: IFN-,
interferon-
; MHC, major histocompatibility complex; LAM,
lipoarabinomannan; TNF-
, tumor necrosis factor-
; PKC, protein
kinase C; MAPK, mitogen-activated protein kinase; PMA, phorbol
12-myristate, 13-acetate; GST, glutathione S-transferase;
FCS, fetal calf serum; MFI, mean fluorescence index; LPS,
lipopolysaccharide; LM, lipomannan; PIM, phosphatidylinositol mannoside; pNPP, para-nitrophenyl phosphate; IL-12,
interleukin-12; TGF-
, transforming growth factor-
; erk,
extracellular-regulated kinase; DTT, dithiothreitol; MOPS,
4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis.
3 Rochford, R., Cannon, M. J., Sabbe, R. E., Adusimilli, K., Picchio, G., Glynn, J. M., Nonnan, D. J., Mosier, D. E., and Hobbs, M., Viral Immunol., in press.
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REFERENCES |
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