Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase Cγ2 (PLCγ2). Several tyrosine-phosphorylated proteins were associated with the PLCγ2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLCγ2-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 PLCγ2-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 PLCγ2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.
It has been estimated that Mycobacterium tuberculosis infects one-third of the world’s population (1), causing eight million new cases and three million deaths annually (2). Tuberculosis is caused by infection with M. tuberculosis, a facultative intracellular pathogen that survives and/or replicates within the phagosome of macrophages. The ability of M. tuberculosis to infect and cause disease depends on its capacity to evade killing by phagocytic cells.
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 Cγ2 (PLCγ2), 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.
Materials and Methods
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-PLCγ2 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 × 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 × 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.
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 × 106) in 0.9 ml KRG were stimulated with 0.1 ml Mtb (4 × 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 × 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 × 107/sample) were lysed with 1 ml RIPA buffer at 4°C for 20 min and then centrifuged at 14,000 × g for 10 min. The supernatant was precleared once with protein A-agarose; incubated with 1 μg anti-PLCγ2 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 PLCγ2 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.
The role of protein kinases and calcium in the generation of ROI induced in neutrophils during phagocytosis of Mtb
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.
Because PKC and PTK are the two major activators of respiratory burst in neutrophils, we investigated which of these signals was involved in the currently observed Mtb-stimulated ROI production. Neutrophils were pretreated with 25 μM genistein, a PTK inhibitor (34), or 100 nM staurosporine, a potent PKC inhibitor (35), for 5 min at 37°C and then stimulated with Mtb in the presence of the same inhibitor. Genistein almost completely blocked the Mtb-stimulated ROI production in neutrophils, whereas staurosporine, which inhibits >90% of PMA-stimulated ROI production, had only marginal inhibitory effects (Fig. 1⇑). 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. 2⇓A). 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).
Tyrosine phosphorylation of PLCγ2 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. PLCγ, one of the three types of PLC in human cells, is activated by tyrosine phosphorylation, and neutrophils primarily express PLCγ2 isoform (37). To determine whether PLCγ2 is activated in neutrophils after Mtb stimulation, the whole cell lysates were precipitated with an anti-PLCγ2 Ab and blotted for tyrosine phosphorylation. The results showed that PLCγ2 is phosphorylated on tyrosine in Mtb-stimulated cells (Fig. 2⇑B). Pretreatment of neutrophils with genistein completely abolished the tyrosine phosphorylation of PLCγ2, whereas staurosporine had no significant effect (Fig. 2⇑C). Reprobing of the same blots with an anti-PLCγ2 Ab revealed that each lane of the gel received similar amounts of PLCγ2 (Fig. 2⇑, B and C).
Several tyrosine-phosphorylated proteins were coprecipitated with PLCγ2 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. 2⇑B).
Association of Shc with PLCγ2 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 PLCγ2 in Mtb-stimulated neutrophils, lysates from unstimulated and stimulated cells were precipitated with an anti-PLCγ2 Ab, blotted with anti-Shc Ab, and vice versa. As shown in Fig. 3⇓A, an increased amount of tyrosine-phosphorylated p46 Shc was found in PLCγ2 immunoprecipitates obtained from Mtb-stimulated neutrophils. When the cell lysates were precipitated with anti-Shc Ab and analyzed for PLCγ2 and mAb 4G10, an increased amount of tyrosine-phosphorylated PLCγ2 was observed in anti-Shc precipitates from Mtb-stimulated neutrophils (Fig. 3⇓B).
Activation of p38 MAPK in Mtb-stimulated neutrophils
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 PLCγ2-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. 4⇓A). 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. 4⇓B). Comparison of the kinetics for the tyrosine phosphorylation of PLCγ2 and MAPK in Mtb-stimulated neutrophils revealed that activation of PLCγ2 preceded that of p38 MAPK (Fig. 5⇓).
To investigate the role of p38 MAPK in Mtb-induced ROI production, neutrophils were pretreated with SB203580, a specific p38 MAPK inhibitor, before stimulation. This inhibitor suppressed the Mtb-stimulated ROI production in neutrophils in a dose-dependent manner (Fig. 6⇓). 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).
Effect of PLC inhibitor on the Mtb-stimulated ROI production and p38 MAPK activation
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 × 107 compared with 15.7 ± 7 × 107 (n = 3) in control cells, whereas its inactive structural analogue U73343 has no effect (13.6 ± 3 × 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⇓).
Although emerging evidence indicates that human neutrophils play an important role during mycobacterial infections (5, 6, 7), the molecular mechanisms underlying the bactericidal responses of neutrophils toward mycobacteria are largely unknown. The present study shows that the ROI production in neutrophils during phagocytosis of Mtb is strictly dependent on tyrosine kinase activation because stimulation with Mtb induces a rapid increase in tyrosine phosphorylation of cellular proteins, and pretreatment with genistein blocks both tyrosine phosphorylation and ROI production in these cells. One of the major tyrosine-phosphorylated proteins is PLCγ2, which is associated with the adapter protein Shc in the Mtb-stimulated neutrophils. Furthermore, our results indicate that the PLCγ2-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 PLCγ2, a lipid-metabolizing enzyme which has been shown to regulate many functional activities including respiratory burst in neutrophils (40, 41). Tyrosine phosphorylation of PLCγ2 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 PLCγ2 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 PLCγ2 and Ras in a tyrosine kinase-dependent manner (42, 43). Nonopsonized Mtb did not induce any detectable tyrosine phosphorylation of PLCγ2 in neutrophils (data not shown). Thus the involvement of a mycobacterial cell wall component in the activation of PLCγ2 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 PLCγ2 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 PLCγ2 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 PLCγ1 was necessary and sufficient to trigger the Ras-dependent signaling cascade (46).
Recent studies in neutrophils stimulated with FMLP and TNF-α 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 PLCγ2 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 PLCγ2-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-γ-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).
↵1 This work was supported by the Swedish Medical Research Council, the County of Östergötland, Reumatiker Förbundet, and the Swedish Foundation for International Cooperation in Research and Higher Education.
↵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; PLCγ2, phospholipase Cγ2; 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 May 3, 1999.
- Accepted November 2, 1999.
- Copyright © 2000 by The American Association of Immunologists