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2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase1
Department of Medical Microbiology, Linköping University, Linköping, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 (PLC2). Several tyrosine-phosphorylated proteins
were associated with the PLC2 precipitates from Mtb-stimulated
neutrophils, of which pp46 was characterized as the Shc adapter
protein. A role for PLC2-Shc association in the generation of ROI is
supported by the observations that stimulation with Mtb causes the
activation of p38 mitogen-activated protein kinase (MAPK), a downstream
target of the Shc/Ras signaling cascade, and that the effect of
genistein on ROI production coincided with its ability to inhibit both
PLC2-Shc association and p38 MAPK activation. Moreover, pretreatment
of neutrophils with a PLC inhibitor markedly suppresses the
Mtb-stimulated ROI production as well as p38 MAPK activation in these
cells. Taken together, these results indicate that stimulation of
neutrophils with Mtb triggers the tyrosine phosphorylation of PLC2
and its association with Shc, and that such association is critical for
the Mtb-stimulated ROI production through activating p38
MAPK. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The host response to infection entails a carefully choreographed series of inflammatory events with macrophages and neutrophils playing a critical role in the acute phase before the development of acquired immunity specifically directed against the pathogen. Although tuberculosis is characterized by the predominant migration of monocytes/macrophages to the site of infection, the earliest response to the invasion of tissue by mycobacteria is primarily an influx of neutrophils (3, 4). Emerging evidence indicates that neutrophils play a significant protective role in the acute phase of tuberculosis (5, 6, 7). When exposed to mycobacteria, neutrophils exhibit the typical early bactericidal responses, such as phagocytosis (8, 9), generation of reactive oxygen intermediates (ROI)3 (10), exocytosis of specific granules (11), and ultimately killing of mycobacteria (9, 12). In contrast to macrophages, which utilize reactive nitrogen intermediates as the major bactericidal molecules, the production of ROI in neutrophils appears to be essential for killing the ingested microbes. Several lines of evidence suggest the potential role of ROI in the antimycobacterial activities of neutrophils: 1) phagocytosis of live or heat-killed M. tuberculosis is associated with an increased production of ROI in human neutrophils (10); 2) ROI generated by peroxidase or catalase H2O2-halide can directly kill M. tuberculosis (13) and growth of M. tuberculosis is markedly inhibited under high oxygen tension (14); and 3) an increased susceptibility to infection with M. tuberculosis was found both in patients with chronic granulomatous disease, an inherited defect in phagocyte ROI production, and in the knock-out mouse model of chronic granulomatous disease (15, 16).
The major source of ROI in neutrophils is the NADPH oxidase, a
multicomponent enzyme that catalyzes the transfer of electrons from
NADPH to molecular oxygen, resulting in the production of superoxide
and hydrogen peroxide. It is generally believed that activation of
protein kinase C (PKC) and activation of protein tyrosine kinases (PTK)
are the two critical events in regulating neutrophil functions in
response to a variety of extracellular stimuli (17). Such
activation leads to the phosphorylation of regulatory proteins and the
activation of different phospholipases and kinases, e.g., Shc protein,
phospholipase C2 (PLC2), PtdIns 3-kinase, and mitogen-activated
protein kinases (MAPK) (18, 19, 20). The involvement of these
intracellular signals in neutrophil activation is supported by the
observation that pharmacological agents which regulate these protein
phosphorylation and kinase activities are also potent modulators of
neutrophil microbicidal responsiveness (20, 21).
MAPK have been demonstrated to represent an important signaling pathway in neutrophils (22, 23, 24, 25, 26). MAPK can be grouped into three families: c-Jun N-terminal kinase, p38, and p44/42 MAPK, each with apparently unique signaling pathways. Studies in neutrophils in response to a variety of stimuli indicate that the production of ROI is primarily regulated by p38 MAPK and to a lesser extent by p44/42 MAPK (22, 23, 24). Although the precise mechanisms by which MAPK is activated are not clear, it has been shown that stimulation of neutrophils with FMLP activates tyrosine kinase Lyn and subsequent phosphorylation of Shc, an adapter protein which activates Ras/Raf/MAPK pathways via Shc-GRB2-Sos complexes (27). Apart from Shc and Ras, recent studies with inhibitors indicate a potential role of PLC in the FMLP-stimulated MAPK activation (28).
Although neutrophils play a critical role in the acute phase of mycobacterial infection (5, 6, 7), little is known about the molecular mechanisms underlying the bactericidal responses of neutrophils toward mycobacteria. In this paper, we establish an in vitro infection model of human neutrophils with an attenuated strain of M. tuberculosis H37Ra (Mtb) to investigate the signaling pathways involved and how they are coordinated in the generation of ROI in Mtb-stimulated neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ab and chemicals used and their sources are as follows:
anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology Lake Placid,
NY); anti-p38 MAPK and anti-phospho-p38 MAPK (New England
Biolabs, Beverly, MA); anti-PLC2 Ab, anti-Shc mAb, and
protein A-agarose (it has been observed by others (29) and
confirmed by us that this anti-Shc mAb bound only to 46- and 52-kDa
isoformes in lysate of human neutrophils by Western blot) (Santa Cruz
Biotechnology, Santa Cruz, CA); electrophoresis reagents (Pharmacia
Biotech, Uppsala, Sweden); enhanced chemiluminescence (CL) (Amersham,
Bucks, U.K.); U73122, an inhibitor of PLC (30, 31), and
its inactive structural analogue U73343 (Biomol, Plymouth Meeting, PA);
1,2-bis-5-methyl-amino-phenoxylethane-N,N,N'-tetra-acetoxymethyl
acetate (MAPT/AM), EGTA, and SB203580 (Calbiochem-Behring, La Jolla,
CA); staurosporine, catalase, superoxide dismutase (SOD), HRP, BSA,
leupeptin, pepstatin, and aprotinin (Boehringer Mannheim, Mannheim,
Germany); genistein (Research Biochemicals, Natick, MA); polymorphprep
and lymphoprep (Nycomed, Oslo, Norway); and all other reagents were
from Sigma (St. Louis, MO) unless otherwise indicated in the
text.
Preparation of mycobacteria
Attenuated H37Ra strain of Mtb (ATCC 25177) was obtained from the American Type Culture Collection (Manassas, VA), cultured, and prepared for use in experiments. C3b/bi-opsonized mycobacteria were provided by using 5% normal human serum, as previously described (9). Along with this study, C3b/bi-opsonized Mtb was referred to Mtb only.
Single mycobacterial suspension was prepared by using a syringe connected to a 27-gauge 3/4 0.4 x 19 needle. After several passages through the needle, the bacterial suspension was filtered using a sterile pasture pipette equipped with cotton wool. A significant portion (>95%) of the bacteria was in nonaggregated form as determined microscopically. The viability of mycobacteria in each step was assessed by comparing bacterial counts determined by microscopy together with CFU assay.
Preparation and treatment of human neutrophils
Human neutrophils were isolated from peripheral blood of healthy
volunteers. Density-gradient separation on polymorphprep
(24) was performed at 340 x g for 45 min
at room temperature. The pale-red granulocyte layer was washed and the
contaminated erythrocytes were lysed by a brief hypotonic lysis.
Neutrophils of 
96% purity were resuspended either in
Krebs-Ringer-glucose buffer containing 1 mM Ca2+
and Mg2+ (KRG) or without
Ca2+ (KRG-Ca2+). Where
indicated, depletion of intracellular calcium in neutrophils was
conducted by using 25 µM MAPT/AM and 1 mM EGTA, which reduces
intracellular calcium concentration to <20 nM (32).
Simultaneously, control cells were split into two parts: one was
resuspended in KRG and the other in
KRG-Ca2+.
To inhibit p38 MAPK, neutrophils were preincubated with the indicated concentrations of SB203580, an inhibitor of p38 MAPK (33), for 15 min at 4°C and then for an additional 15 min at 37°C. To inhibit PTK or PKC activity, cells were pretreated with 25 µM genistein (34) or 100 nM staurosporine (35), respectively, for 5 min at 37°C and then stimulated with Mtb in the presence of the same inhibitors. To inhibit PLC activity, cells were pretreated with 2 µM U73122 for 10 min at 37°C. The cell viability in each step was >95% as examined with trypan blue exclusion.
Phagocytosis
Neutrophils were incubated with Mtb (final volume, 1 ml) at the indicated ratio of cell/Mtb for different times at 37°C under occasional agitation. In some experiments, to investigate whether the reduced ROI production in inhibitor-treated neutrophils is due to the impairment of phagocytosis, we used FITC-conjugated mycobacteria. The bacteria were conjugated with FITC, opsonized as previously described (9), and then incubated with neutrophils (1:20 cell/Mtb) under the same conditions as described above. The numbers of ingested Mtb per 100 cells were analyzed with trypan blue exclusion (9).
Oxidative burst assay
The production of ROI in neutrophils was measured as luminol-enhanced CL in a six-channel Biolumat model LB 9505 (Berthold, Wildblad, Germany). Neutrophils (2 x 106) in 0.9 ml KRG were stimulated with 0.1 ml Mtb (4 x 108/ml) or 10-7 M PMA, and light emission was recorded continuously in the presence of HRP (4 U). Calcium-depleted neutrophils incubated in KRG-Ca+2 were used to investigate the role of calcium in Mtb-stimulated ROI production in neutrophils. Intracellular production of ROI was measured in the presence of 50 U SOD and 2000 U catalase (the cell-impermeable scavengers for O2- and H2O2, respectively).
Protein tyrosine phosphorylation and p38 MAPK activation
Neutrophils were incubated with opsonized Mtb at the ratio of 1:30 (cell:Mtb) as described above. Neutrophils alone that had been kept at 4°C served as a control. Reactions were terminated by adding 0.5 ml ice-cold PBS with 1 mM Na3VO4 and by using rapid centrifugation at 4°C. The pellets were resuspended in 0.4 ml of lysis buffer A containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 50 mM Tris (pH 7.4), 10 mM NaF, 10 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, and 10 µg/ml of aprotinin, leupeptin, and pepstatin and were kept on ice for 20 min. After centrifugation at 8000 x g for 10 min, the supernatants were regarded as Triton-soluble fractions and the pellets were regarded as Triton-insoluble fractions. These cellular fractions were dissolved in Laemmli sample buffer (36) and heated at 90°C for 5 min. Equal amounts of proteins (20 µg/lane) were separated on 7.5% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with BSA, and the presence of tyrosine-phosphorylated proteins on the blots was detected with anti-phosphotyrosine mAb 4G10 and a commercially enhanced CL kit.
To detect the activation of p38 MAPK, the cells were lysed in RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, and protease inhibitors as in lysis buffer A). Equal amounts of cellular proteins were separated on 10% SDS-PAGE and analyzed by Western blot with Ab specifically against (Thr180/Tyr182) phosphorylated p38 MAPK (active-p38). To confirm that each line received the same amount of proteins, the blots were stripped and reprobed with anti-p38 MAPK Ab (total-p38), which recognized both phosphorylated and nonphosphorylated p38 MAPK.
Immunoprecipitation and Western blot
After stimulation, neutrophils (2 x
107/sample) were lysed with 1 ml RIPA buffer at
4°C for 20 min and then centrifuged at 14,000 x g
for 10 min. The supernatant was precleared once with protein A-agarose;
incubated with 1 µg anti-PLC2 Ab, 1 µg anti-Shc Ab, or
0.2 µg of anti-total p38 MAPK Ab for 1 h; and then incubated
with 30 µl 50% (v/v) protein A-agarose for an additional 1 h at
4°C. The beads were washed three times with RIPA buffer, resuspended
in sample buffer, and boiled. The proteins eluted from beads were
separated by SDS-PAGE and immunoblotted as described above. In some
experiments, the membranes were stripped and reprobed with Ab as
specified in the figure legends.
To compare the extent of tyrosine phosphorylation of PLC2 and p38
MAPK and, thereby, to uncover the time relationship-activation of these
proteins during phagocytic process, the intensity of
tyrosine-phosphorylated proteins was measured by densitometric assay
using a Howtek scanner and Quantity One Software (Advanced American
Biotechnology, Fullerton, CA).
Analysis of data
Differences between experimental groups comprised normally distributed data, which were analyzed for statistical significance using Student’s t test or ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study, we observed that neutrophils are able to
ingest and kill the attenuated strain H37Ra of M.
tuberculosis (9). Because ROI is essential for
killing the microbes ingested by neutrophils, we determined the
production of ROI in neutrophils upon stimulation with Mtb, utilizing
luminol-enhanced CL. The results show that phagocytosis of Mtb is
associated with an increase in ROI production in neutrophils (Fig. 1
). The activation was rapid and occurred
as early as 1 min after stimulation with a peak between 20 and 25 min
before gradually declining to base level at 60 min (data not shown). To
find out whether this Mtb-stimulated ROI is mainly produced
intracellularly, the CL assay was performed in the presence of
extracellular scavengers, SOD and catalase. We found that 90% of
ROI induced during phagocytosis were of an intracellular origin.
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). Depletion of intracellular calcium markedly
suppresses the ROI production, indicating that the intracellular
calcium concentration is required for the maximum production of ROI in
neutrophils stimulated with Mtb (Fig. 1). It should be noted that the
phagocytosis of Mtb is not affected in calcium-depleted or
staurosporine-treated cells and is only slightly inhibited by genistein
(20%). Neutrophils stimulated with Mtb induce protein tyrosine phosphorylation
Because the above-described results indicate that activation of
tyrosine kinases, but not PKC, is a prerequisite for Mtb-induced ROI in
neutrophils, we then investigated which proteins were
tyrosine-phosphorylated during phagocytosis. Stimulation of neutrophils
with Mtb induced tyrosine phosphorylation of multiple cellular proteins
in both Triton-soluble and -insoluble fractions, with an apparent
molecular mass of about 145, 120, 72, 62, 36, and 30 kDa (as shown for
Triton-soluble fraction in Fig. 2A). These
tyrosine-phosphorylated proteins were detectable after stimulation for
1 min with a peak between 10 and 15 min and gradually declined to base
level at 60 min. Pretreatment of neutrophils with genistein resulted in
complete inhibition of this Mtb-induced protein tyrosine
phosphorylation in stimulated cells, whereas staurosporine had no
effect (data not shown).
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2 in neutrophils stimulated with
Mtb
One of the tyrosine-phosphorylated proteins observed in
Mtb-stimulated cells, pp145, was identical in size to PLC, a protein
with multiple structures that could function as both an adapter protein
and a lipid-metabolizing enzyme. PLC, one of the three types of PLC
in human cells, is activated by tyrosine phosphorylation, and
neutrophils primarily express PLC2 isoform (37). To
determine whether PLC2 is activated in neutrophils after Mtb
stimulation, the whole cell lysates were precipitated with an
anti-PLC2 Ab and blotted for tyrosine phosphorylation. The
results showed that PLC2 is phosphorylated on tyrosine in
Mtb-stimulated cells (Fig. 2B). Pretreatment of neutrophils
with genistein completely abolished the tyrosine phosphorylation of
PLC2, whereas staurosporine had no significant effect (Fig. 2C). Reprobing of the same blots with an anti-PLC2 Ab
revealed that each lane of the gel received similar amounts of PLC2
(Fig. 2, B and C).
Several tyrosine-phosphorylated proteins were coprecipitated with
PLC2 in Mtb-stimulated neutrophils. Of the proteins, pp46 was the
most prominent and was detected within 1 min after stimulation with a
peak between 5 and 10 min and a gradual decline thereafter (Fig. 2B).
Association of Shc with PLC2 in Mtb-stimulated neutrophils
The pp46 has a similar electrophoretic mobility to one isoform of
Shc adapter protein. Phosphorylation of Shc appears to activate the
Ras/MAPK pathway (27, 38), a signaling cascade that has
been shown to regulate many functional activities including respiratory
burst in neutrophils (27). To investigate whether p46 Shc
is tyrosine-phosphorylated and associated with PLC2 in
Mtb-stimulated neutrophils, lysates from unstimulated and stimulated
cells were precipitated with an anti-PLC2 Ab, blotted with
anti-Shc Ab, and vice versa. As shown in Fig. 3A, an increased amount of
tyrosine-phosphorylated p46 Shc was found in PLC2 immunoprecipitates
obtained from Mtb-stimulated neutrophils. When the cell lysates were
precipitated with anti-Shc Ab and analyzed for PLC2 and mAb
4G10, an increased amount of tyrosine-phosphorylated PLC2 was
observed in anti-Shc precipitates from Mtb-stimulated neutrophils
(Fig. 3B).
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The results obtained so far show that stimulation of neutrophils
with Mtb induces a PTK-dependent ROI production and tyrosine
phosphorylation of PLC with subsequent association with Shc. To further
investigate the possible link between Mtb-stimulated ROI production and
PLC2-Shc association, we tested the involvement of p38 MAPK, a
downstream Ras effector that mediates the ROI production in
FMLP-stimulated neutrophils (23, 25). Using the
anti-active p38 MAPK Ab, which recognizes p38 MAPK phosphorylated
on either threonine or tyrosine residues, Western blot analysis showed
that stimulation of neutrophils with Mtb resulted in an increased
amount of phosphorylated p38 MAPK (Fig. 4A). To determine whether p38
MAPK is activated by tyrosine phosphorylation in Mtb-stimulated
neutrophils, the cell lysates were precipitated with an
anti-total-p38 MAPK Ab and blotted with mAb 4G10. The results
showed that stimulation of neutrophils with Mtb resulted in a sustained
increase in tyrosine phosphorylation of p38 MAPK (Fig. 4B).
Comparison of the kinetics for the tyrosine phosphorylation of PLC2
and MAPK in Mtb-stimulated neutrophils revealed that activation of
PLC2 preceded that of p38 MAPK (Fig. 5).
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). It should be noted that
SB203580 at a concentration up to 25 µM did not affect the
phagocytosis of Mtb by neutrophils and had no effect on PMA-induced ROI
production in these cells (data not shown).
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An inhibitor of PLC, U73122, was used to further investigate the
role of PLC in the Mtb-stimulated ROI production and p38 MAPK
activation. The result showed that pretreatment of neutrophils with 2
µM of U73122 completely blocked the Mtb-stimulated ROI production in
these cells, with a value of 0.02 ± 0.01 x
107 compared with 15.7 ± 7 x
107 (n = 3) in control cells,
whereas its inactive structural analogue U73343 has no effect
(13.6 ± 3 x 107, n =
3). Moreover, the activation of p38 MAPK in Mtb-stimulated neutrophils
was markedly attenuated by U73122, but not by U73343 treatment (Fig. 7). SB203580 and staurosporine had no
effect on the Mtb-induced activation of p38 MAPK, whereas genistein
significantly decreased the p38 MAPK activation in Mtb-stimulated
neutrophils (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2, which is
associated with the adapter protein Shc in the Mtb-stimulated
neutrophils. Furthermore, our results indicate that the PLC2-Shc
interaction is critical for triggering the ROI production in
Mtb-stimulated neutrophils by activating p38 MAPK. When human neutrophils are exposed to Mtb, they exhibit the typical early bactericidal responses including phagocytosis and generation of ROI. Our observation that omitting intracellular calcium suppressed the production of ROI without affecting the ingestion of complement-coated mycobacteria suggests that phagocytosis and the generation of ROI induced by mycobacteria have different calcium requirements. This is in contrast to stimulation with IgG-coated erythrocytes or zymosan, with which both phagocytosis and ROI production in neutrophils require a level of intracellular calcium (39). Such a discrepancy indicates that different stimuli might use distinct signaling pathways to regulate neutrophil responses. In agreement with our results, a recent study of neutrophils stimulated with various strains of mycobacteria shows that an enhanced phagocytosis does not increase the exocytosis of specific granule contents (11).
PKC and PTK are the two best-known modulators of ROI production in
neutrophils. The present results that the Mtb-stimulated ROI production
is completely blocked by genistein but not by staurosporine indicate
that activation of PTK but not of PKC is essential for this
Mtb-stimulated neutrophil response. In support of this conclusion, the
stimulation of neutrophils with Mtb induces a rapid tyrosine
phosphorylation of multiple cellular proteins including PLC2, a
lipid-metabolizing enzyme which has been shown to regulate many
functional activities including respiratory burst in neutrophils
(40, 41). Tyrosine phosphorylation of PLC2 is
responsible for the generation of second messengers that activate PKC
and mobilize intracellular calcium, both of which are crucial events
for the bactericidal responses of neutrophils (39). The
mechanisms underlying the presently observed activation of PLC2 are
most likely mediated through CD18 because ingestion of serum-opsonized
bacteria is primarily involved by CD11b/CD18 (24) and
ligation of CD18 on neutrophils activates PLC2 and Ras in a tyrosine
kinase-dependent manner (42, 43). Nonopsonized Mtb did not
induce any detectable tyrosine phosphorylation of PLC2 in
neutrophils (data not shown). Thus the involvement of a mycobacterial
cell wall component in the activation of PLC2 can be excluded in our
system.
Apart from its catalytic domain, PLC contains SH2, SH3, and pleckstrin
homology domains (44, 45). The multidomain structure of
PLC raises the possibility that this enzyme carries out additional
signaling pathways during neutrophil activation. This notion is
supported by our finding that several tyrosine-phosphorylated proteins
are associated with PLC2 in Mtb-stimulated neutrophils. One of these
proteins is characterized as Shc, an adapter protein that appears to
activate Ras/MAPK pathway via Shc-GRB2-Sos complexes. Therefore, it is
tempting to suggest that the tyrosine phosphorylation of PLC2 in
Mtb-stimulated neutrophils could be coupled to the Shc-GRB2-Sos pathway
to transduce additional downstream signals, e.g., Ras/MAPK in these
cells. This hypothesis is supported by the observation that U73122, an
inhibitor of PLC, significantly attenuated the p38 MAPK activation in
neutrophils stimulated by FMLP (28) and Mtb (present
study). In agreement with this, it recently has been shown in Jurkat
cells that activation of PLC1 was necessary and sufficient to
trigger the Ras-dependent signaling cascade (46).
Recent studies in neutrophils stimulated with FMLP and TNF-
indicate
that the respiratory burst is primarily mediated by activation of p38
MAPK (23). Such a regulatory mechanism is also involved in
our observation of Mtb-stimulated ROI production because p38 is
activated after stimulation with Mtb, and SB203580, an inhibitor of
p38, suppresses the Mtb-stimulated ROI production. The precise
modulatory mechanisms underlying the activation of p38 MAPK observed in
our study are at present unclear. Stimulation with FMLP induces a rapid
formation of Lyn-Shc-phosphatidylinositol 3-kinase complexes in
neutrophils (27), and wortmannin inhibits the
FMLP-stimulated p38 MAPK activation (28). Therefore, it is
likely that activation of PLC2 and its subsequent association with
Shc are responsible for the activation of p38 MAPK in Mtb-stimulated
cells. It should be noted that additional signaling pathway(s) also
contributes to the activation of respiratory burst because SB203580
partially inhibits the ROI production in Mtb-stimulated neutrophils.
Consistent with this, it recently has been reported that a combination
of inhibitors for p38 and p44/42 MAPK resulted in an additive
inhibitory effect on respiratory burst in neutrophils (24, 47).
Participation of PKC in the activation of p38 MAPK has been shown by
Krump et al. (48). They observed partial inhibition of
FMLP-stimulated tyrosine phosphorylation of p38 MAPK by PKC inhibitor
and a low-level stimulation of p38 MAPK by PMA in neutrophils. However,
neither the PLC2-Shc association nor the p38 MAPK activation in
Mtb-stimulated neutrophils was affected by staurosporine, which argues
against such a regulatory mechanism. This is based on our observations
that staurosporine completely blocked PMA-stimulated ROI production
without affecting Mtb-induced responses and that SB203580 inhibits Mtb-
but not PMA-stimulated ROI production in neutrophils. It has also been
shown that neither PKC depletion nor the PKC inhibitor Calphostin C
affect FMLP-induced activation of p38 MAPK (28).
Although neutrophils are the first cells to infiltrate the inflammatory
sites and phagocytose mycobacteria (3, 4), a recent study
in IFN--deficient mice infected with mycobacteria shows that in the
absence of macrophage function, a massive neutrophilia did not protect
those mice from infection (49). These results indicate
that neutrophils play their role mainly at the early stage of
antimycobacterial immunity. In addition to its direct antimycobacterial
responses, e.g., phagocytosis and generation of ROI, neutrophils with
ingested Mtb underwent rapid apoptosis (50), a process
that is vital for the rapid resolution of inflammation. The
phagocytosis of Mtb by neutrophils can inhibit the spread of bacteria
until macrophages accumulate, and ingestion of these apoptotic
neutrophils might directly activate macrophages at sites of
mycobacterial infection. Several studies have shown that after
phagocytosis, neutrophils undergo apoptosis through an
oxygen-dependent pathway (51). Therefore, activation of
respiratory burst in neutrophils by Mtb can also regulate the
inflammatory response by induction of apoptosis. In this context, it
recently has been shown that activation of p38 MAPK is involved in the
apoptosis of neutrophils (52, 53).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nasrin Perskvist, Department of Medical Microbiology, Linköping University, S-581 85, Linköping, Sweden. E-mail address:
3 Abbreviations used in this paper: ROI, reactive oxygen intermediates; PKC, protein kinase C; PTK, protein tyrosine kinase; PLC2, phospholipase C2; MAPK, mitogen-activated protein kinase; Mtb, Mycobacterium tuberculosis H37Ra; KRG, Krebs-Ringer-glucose; CL, chemiluminescence; MAPT/AM, 1,2-bis-5-methyl-amino-phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate.
Received for publication May 3, 1999. Accepted for publication November 2, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 is involved in the activation of phosphoinositide hydrolysis by Fc receptors in human neutrophils. Biochem. Biophys. Res. Commun. 201:1100.[Medline]
2 in HL 60 granulocytes. J. Biol. Chem. 268:3357.Rs in human neutrophils. J. Leukocyte Biol. 53:427.[Abstract]
2 integrin on neutrophils: involment of phospholipase C2 and ins(1, 4, 5)p3. Biochem. J. 317:403.
2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product vav. Proc. Natl. Acad. Sci. USA 93:8431.2 by the M-CSF receptor is necessary for differentiation signaling. EMBO J. 16:5880.[Medline]
and GM-CSF. J. Leukocyte Biol.
64:537.
-deficient mice infected with mycobacteria. Blood 91:2914.This article has been cited by other articles:
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N. Perskvist, M. Long, O. Stendahl, and L. Zheng Mycobacterium tuberculosis Promotes Apoptosis in Human Neutrophils by Activating Caspase-3 and Altering Expression of Bax/Bcl-xL Via an Oxygen-Dependent Pathway J. Immunol., June 15, 2002; 168(12): 6358 - 6365. [Abstract] [Full Text] [PDF]
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S. K. Roach and J. S. Schorey Differential Regulation of the Mitogen-Activated Protein Kinases by Pathogenic and Nonpathogenic Mycobacteria Infect. Immun., June 1, 2002; 70(6): 3040 - 3052. [Abstract] [Full Text] [PDF]
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 G. Cicchetti, P. G. Allen, and M. Glogauer CHEMOTACTIC SIGNALING PATHWAYS IN NEUTROPHILS: FROM RECEPTOR TO ACTIN ASSEMBLY Critical Reviews in Oral Biology & Medicine, May 1, 2002; 13(3): 220 - 228. [Abstract] [Full Text] [PDF]
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 N. Perskvist, K. Roberg, A. Kulyte, and O. Stendahl Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils J. Cell Sci., March 15, 2002; 115(6): 1321 - 1330. [Abstract] [Full Text] [PDF]
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C. Neufert, R. K. Pai, E. H. Noss, M. Berger, W. H. Boom, and C. V. Harding Mycobacterium tuberculosis 19-kDa Lipoprotein Promotes Neutrophil Activation J. Immunol., August 1, 2001; 167(3): 1542 - 1549. [Abstract] [Full Text] [PDF]
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