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Prepublished online as a Blood First Edition Paper on January 2, 2003; DOI 10.1182/blood-2001-12-0180.
PHAGOCYTES
From the Immunology Program, H. Lee Moffitt Cancer
Center & Research Institute, University of South Florida College of
Medicine, Department of Interdisciplinary Oncology, Tampa; and Los
Alamos National Laboratories, NM.
Elevated levels of mitogen-activated protein kinase/extracellular
regulatory kinase (MAPK/ERK) activity are frequently found in
some cancer cells. In efforts to reduce tumor growth, attempts have
been made to develop cancer therapeutic agents targeting the MAPK.
Here, by use of biologic, biochemical, and gene manipulation methods in
human polymorphonuclear neutrophils (PMNs), we have identified a key
pathway important in normal cell function involving MAPK/ERK in PMNs
for growth inhibition of Candida albicans. Contact with
C albicans triggered MAPK/ERK activation in PMNs within 5 minutes, and blocking of MAPK/ERK activation, either by the
pharmacologic reagent PD098059 or by dominant-negative MAPK
kinase (MEK) expression via vaccinia viral delivery, suppressed
antimicrobial activity. Rac and Cdc42, but not Ras or Rho, were
responsible for this MAPK/ERK activation. Expression of
dominant-negative Rac (N17Rac) or Cdc42 (N17Cdc42) eliminated not
only C albicans- mediated ERK phosphorylation but
also phagocytosis and granule migration toward the ingested microbes,
whereas dominant-negative Ras (N17Ras) and Rho (N19Rho) did not.
PAK1 (p21-activated kinase 1) activation is induced by C
albicans, suggesting that PAK1 may also be involved in the
Rac1 activation of MAPK/ERK. We conclude from these data that
Rac/Cdc42-dependent activation of MAPK/ERK is a critical event in
the immediate phagocytic response of PMNs to microbial challenge.
Therefore, use of MAPK pharmacologic inhibitors for the treatment of
cancer may result in the interruption of normal neutrophil function. A
balance between therapeutic outcome and undesirable side effects must
be attained to achieve successful and safe anticancer therapy.
(Blood. 2003;101:3240-3248) Mitogen-activated protein kinases (MAPK),
also referred to as extracellular regulatory kinases (ERK1/2), are
crucial constituents of signaling pathways that are important in
oncogenic transformation.1-4 There has been a dramatic
increase in interest in the role of the MAPK pathway in governing
neoplastic cell behavior in a variety of human tumors.5-10
Much of this interest has focused on pharmacologic inhibition of cell
growth by disruption of the MAPK pathway with either small molecules or
by overexpression of proteins with a dominant-negative phenotype. Using
a highly potent and selective inhibitor of MAPK kinase (MEK), which
lies directly upstream of MAPK, inhibited tumor growth and enhanced
tumor apoptosis in both mouse and human tumors.11,12
However, because MAPK regulates multiple biologic activities in normal
cells, including cell proliferation, differentiation, cell cycle
traverse, and survival, it is important to assess potential undesirable
effects of MEK inhibitors on normal physiological processes.
Human polymorphonuclear leukocytes or neutrophils (PMNs) constitute the
body's foremost defense against invading
microorganisms,13,14 and dysregulation of neutrophil
antimicrobial activity has severe clinical consequences.15
This is best exemplified in the infection rate observed in neutropenic
cancer patients receiving cytotoxic chemotherapeutic agents or in
patients with genetic defects in neutrophil function as seen in chronic
granulomatous disease.16,17 The ability of PMNs to combat
microbial pathogens is due to a number of specific activities,
including (1) adherence of neutrophils to endothelium, (2) migration or
chemotaxis to an inflammatory site, (3) ingestion or phagocytosis of
pathogens into phagosomes, and (4) degranulation and killing of
invading microorganisms.14 All of these components must
act in concert for effective elimination of microbes. However, it is
unclear how these events are achieved and what signals control them.
A precise understanding of the signaling mechanisms by which
neutrophils mediate phagocytosis and microbicidal activity, however, has not been an easy task to pursue, primarily due to the fragility of
freshly isolated neutrophils and the inability to develop long-term culture of neutrophils, which are terminally differentiated cells. Difficulty in gene delivery via plasmid transfection is another obstacle in the study of PMNs, preventing molecular analysis of the
signaling pathways related to PMN function.18 Most of the data have been gathered from experiments where phagocytosis-related neutrophil receptors, such as FcR, CR3, and
N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor, are
transfected into either fibroblasts or macrophage tumor cell
lines.19-21 By such manipulations, Syk activation has been
shown to be required for immunoglobulin G (IgG)-mediated phagocytosis through the FcR.21 Furthermore, it has been
reported that 2 distinct mechanisms of opsonized phagocytosis exist.
Opsonized phagocytosis mediated by the FcR involves Rac and Cdc42,
whereas the complement receptor signaling involves Rho.19
In other cases, the use of pharmacologic agents in neutrophils has
helped to identify the need for MAPK/ERK and Syk in microbicidal
function.18,22-24 Studies of knock-out mice have
also provided another means to establish phosphatidylinositol-3
kinase (PI-3K) as a critical signal molecule in neutrophil
migration and function.25 How these molecules are
integrated to control neutrophil function, however, has not been established.
In the present study, we show that human neutrophils are very receptive
to gene transfer by recombinant vaccinia viruses, providing us with a
potent tool to probe which pathways are triggered by microbes and what
function they control in PMNs. Using Candida albicans as a
model pathogen, we have identified a specific Rac/Cdc42-dependent but
Ras/Rho-independent MAPK/ERK pathway that controls PMN function against
C albicans. Furthermore, we show that this control is at the
level of lytic granule movement and phagocytosis within PMNs.
Preparation of PMNs
Reagents and cell culture
Vaccinia viral delivery of dominant-negative proteins The advantages of vaccinia virus as a gene delivery vehicle are based on the fact that it has a high infection rate and infects a wide range of cell types including leukocytes. Additionally, the DNA replication cycle takes place in the cytoplasm of the host cells and uses the virus's own transcription system. Therefore, a short time is required for a significant amount of protein expression (2 to 4 hours), which is independent of the cellular biosynthetic capacity.31,32 Recombinant vaccinia viruses encoding dominant-negative MEK1, Rac (N17Rac), Cdc42 (N17Cdc42), and Rho (N19Rho) were constructed using the vector pSP11 in recombination with the WR strain of vaccinia.33 Vaccinia virus containing kinase-deficient PAK1 (KD) was kindly provided by Elizabeth Hong-Geller (Los Alamos National Laboratory, NM). Vaccinia virus expressing CD56, a large granular lymphocyte-specific surface marker, was used as a control. The procedure of vaccinia viral generation and infection has been described previously.34-37 Briefly, 5 × 106 PMN cells per treatment group were incubated with the vaccinia virus constructs for 2 hours at 37°C serum-free medium at a multiplicity of infection (MOI) of 4 to 6. The cells were washed and then further incubated in serum-containing medium for 2 hours at 37°C prior to the growth inhibition of C albicans assay, Western blotting, and in vitro kinase assays. Cell viability was evaluated by flow cytometric analysis after annexin V-phycoerythrin (PE) staining in some experiments.Western blotting analysis Freshly purified PMNs were treated with either serum-free RPMI 1640 (5 × 106 cells per treatment), dimethyl sulfoxide (DMSO), or PD098059 for 2 hours at 37°C and then washed and mixed with paraformaldehyde-fixed C albicans at a ratio of 1:10 (PMNs/C albicans). The cells were pelleted rapidly at 1000 rpm in a microcentrifuge at 4°C followed by incubation for 0 to 5 minutes at 37°C. Then, the cells were solubilized by incubation at 4°C for 30 minutes in 1% Nonidet P-40 (NP-40), 10 mM Tris (tris(hydroxymethyl)aminomethane), 140 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM iodoacetamide, 50 mM NaF, 1 mM EDTA (ethylenediaminetetraacetic acid), 0.4 mM sodium orthovanadate, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 10 µg/mL aprotinin. Cell lysates were centrifuged at 12000g for 15 minutes to remove nuclei and cell debris. The protein concentration of the soluble extracts was determined by using the Bio-Rad (Bradford, Hercules, CA) protein assay. For Western blots, 50 µg of the protein per lane was separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to Immobilon membranes, and reacted with the desired antibody. The proteins were detected by the enhanced chemiluminescence detection system (ECL, Amersham, Piscataway, NJ).In vitro kinase assay Cell lysates from 5 × 106 PMNs were immunoprecipitated with 5 µg anti-PAK1 polyclonal antibody. The immunoprecipitated proteins were suspended in 30 µL kinase reaction buffer containing 10 mM Tris (pH 7.2), 10 mM MnCl2, 10 mM MgCl2, 10 µCi (0.37 MBq) -32P
adenosine triphosphate (ATP) (Amersham), and 1 µM unlabeled ATP and
then incubated at room temperature (RT) for 20 minutes with 4 µg myelin basic protein (MBP) (Upstate Biotechnology, Lake Placid,
NY) as an exogenous substrate. The reaction was terminated by addition
of sample buffer and quick centrifugation, and equal volume aliquots
from each sample were analyzed by electrophoresis using 10%
SDS-polyacrylamide gels. After transfer, phosphoproteins were detected
by autoradiography.
Immunostaining PMNs, untreated or pretreated with 50 µM PD098059 for 2 hours at 37°C, were added to C albicans prestained red by using the Sigma PKH26 red fluorescent cell linker kit at a 1:10 PMN/C albicans ratio in a total volume of 100 µL. The cells were spun rapidly at 1000 rpm for 1 minute in a cold microcentrifuge and then incubated for 0 to 5 minutes at 37°C. DMSO-treated PMNs were included as a control. The cells were then centrifuged onto microscope slides and fixed at 20°C with
methanol-acetone (3:1) for 20 minutes. The slides were air-dried and
rehydrated for 2 hours in several changes of PBS. All procedures were
performed at room temperature. Monoclonal antigranzyme B was used to
detect granule mobilization38 toward ingested C
albicans in PMNs. The slides were washed several times with PBS
and covered with coverslips in mounting media with antifade.
Immunofluorescence was observed with a Leitz Orthoplan 2 microscope
(Photometrics Ltd, Tucson, AZ), and images were captured by a CCD
camera with the Smart Capture Program (Vysis, Downers Grove, IL).
Growth inhibition of C albicans PMN function against C albicans was assessed by 3H-glucose incorporation into residual C albicans after incubation with PMNs treated or untreated with PD098059 or infected with vaccinia viral constructs. PMNs were first serially diluted 2-fold from 6 × 105/mL to 7.5 × 104/mL in RPMI 1640 containing 2% FCS, and 50 µL of each cell suspension was added to triplicate wells of a 96-well flat-bottomed microtiter plate; 500 C albicans in 25 µL of medium was added to each well to achieve the effector-target (E/T) ratios of 60:1, 30:1, 15:1, and 7.5:1. C albicans was also added to empty wells to serve as controls. After incubation for 18 hours at 37°C, 50 µL of sterile water containing 10 µCi/mL of 3H-glucose (New England Nuclear Research Products, Boston, MA) was added for 3 hours at 37°C. The proliferating fungi that incorporated 3H-glucose were collected by adding 50 µL sodium hypochloride (bleach) immediately before processing through a harvester with distilled water onto glass fiber filters and counted on a scintillation counter.39 The mean ± SE of the triplicate cultures
was determined, and the percent growth inhibiton of C
albicans was calculated as follows: % growth inhibition = cpm C albicans alone cpm effector/C albicans × 100 cpm C albicans alone.
Each experiment was performed at least 3 times with different healthy donors, and only the 60:1 E-T ratios are reported in the figures for clarity.
Rapid stimulation of MAPK phosphorylation by C albicans Our first attempt to investigate the signaling pathways required for microbial killing in PMNs was to examine if C albicans could activate MAPK. Exposure of human PMNs to C albicans rapidly induced a transient activation of MAPK as detected by Western blotting with antiactive MAPK that recognized the phosphorylated TEY motif. MAPK activation peaked at 2 minutes and declined by 5 minutes (Figure 1A). This is in sharp contrast to MAPK activation by granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 U/mL), a survival cytokine that consistently induced a long-lasting MAPK/ERK activity (Figure 1B), indicating that C albicans-induced MAPK/ERK activation may be associated with immediate PMN antifungal activity.
MAPK is associated with PMN killing of C albicans To examine if MAPK/ERK controls function in PMNs, an inhibitor of MEK, PD098059, was added to PMNs for 2 hours prior to the addition of C albicans. While control PMNs cultured in medium alone or medium containing the inhibitor solvent DMSO significantly suppressed the growth of C albicans, increasing doses of PD098059 prevented PMN-mediated growth inhibition of C albicans (Figure 2A). As a control, whole-cell lysates prepared from the same pools of PMNs were evaluated for active MAPK/ERK, and the doses of PD098059 that inhibited PMN antimicrobial function also correspondingly inactivated MAPK/ERK phosphorylation (Figure 2B). Although phagocytosis mediated by fMLP has been demonstrated to involve p38 MAPK, addition of a p38 MAPK-selective inhibitor, SB203580, failed to reverse PMN-mediated growth suppression of C albicans (data not shown). These results suggest that MAPK/ERK activation and not p38 MAPK is required for the direct antimicrobial killing function of PMNs.
Vaccinia viral expression of dominant-negative MEK1 but not Ras prevented PMN antimicrobial activity To confirm the involvement of MAPK/ERK in antimicrobial activity, PMNs were either mock infected or infected with vaccinia virus encoding dominant-negative MEK1 (dnMEK1). Infection was carried out for 4 hours, which provided sufficient time for ample production of proteins from viral delivery without loss of cell viability. CD56, which is not endogenously expressed in PMNs, was also introduced into a separate PMN pool as a nonspecific infection control. MAPK phosphorylation was induced by C albicans after 5 minutes in mock-infected PMNs. However, overexpression of dnMEK1 ablated MAPK/ERK phosphorylation in response to C albicans (Figure 3A). Expression of the irrelevant gene, CD56, had no effect on MAPK/ERK activation. Because the Ras/Raf/MAPK pathway is thought to be the predominant signaling event linked to MAPK activation,40 we next examined whether Ras was involved in MAPK activation of PMNs and antifungal activity. Dominant-negative Ras (N17Ras) expression in PMNs, unlike dnMEK1, had no suppressive effects (Figure 3A).
Aliquots of cells from the same experiment were then examined for antimicrobial activity. Medium-cultured PMNs, mock-infected PMNs, and CD56-infected PMNs were all capable of suppressing the growth of C albicans in vitro. Expression of dnMEK1, but not N17Ras, significantly down-regulated PMN antimicrobial activity (Figure 3B). Because PMNs rapidly undergo spontaneous cell death in vitro, we examined the PMN viability after mock infection and infection with CD56 vaccinia virus after 12 and 24 hours. Staining with annexin V-PE was used as an early indicator of apoptosis. A viability dye (7-aminoactinomycin D [7-AAD]) was also added to discriminate live and dead cells, and cells were analyzed by flow cytometry. We found that CD56 vaccinia virus infection did not affect the amount or rate of spontaneous cell death in this assay (Figure 3C). These results confirm that the reduced MAPK activity and antifungal activity was not caused by reduced viability due to the infection. Taken together, the findings with PD098059 and dnMEK1 reveal the necessity of MAPK activation in direct PMN antimicrobial function. However, this pathway is Ras independent. To support our observation with N17Ras, we next used a specific
farnesyl-transferase inhibitor, FTI277, which blocks Ras
function.41 FTI277 treatment of PMNs had no effect on MAPK
activation induced by C albicans (Figure
4A) and resulted in no inhibition of
antifungal activity (Figure 4B). Importantly, FTI277 did not decrease
PMN viability in these experiments as determined by annexin V-PE
staining (data not shown). To ensure that FTI277 and N17Ras worked
appropriately to block Ras activation in PMNs, we included GM-CSF as a
control. GM-CSF (100 IU/mL) was added for 5 minutes in the presence of N17Ras and FTI277 (Figure 5). Both FTI277
(15 µM) and N17Ras were able to block GM-CSF-induced but not C
albicans-induced MAPK activation. These results indicate that
within the same PMNs, GM-CSF triggers a Ras-dependent MAPK pathway,
whereas microbe-triggered MAPK is Ras independent.
C albicans-induced MAPK activation requires Rac and Cdc42 but not Rho Several lines of evidence have suggested that the small guanosine triphosphatases (GTPases), Rac, Cdc42, and Rho, regulate phagocytosis.19,20 These proteins are key regulatory molecules that link surface receptors to cytoskeletal rearrangement.19 Rac has primarily been associated with MAPK-Jun N-terminal kinase (JNK) activation,42,43 but there is growing evidence that Rac and Cdc42 can also activate MAPK/ERK in a pathway that is independent of Ras.44-46 We therefore tested whether the Rho family of small guanosine triphosphate (GTP)-binding proteins might be upstream signals for MAPK activation in PMNs. As shown in Figure 6A, PMNs, mock infected or infected with vaccinia viral constructs expressing CD56, demonstrated marked MAPK activation induced by C albicans. Dominant-negative Rac (N17Rac) and Cdc42 (N17Cdc42), but not Rho (N19Rho), expression in PMNs markedly reduced C albicans-induced MAPK phosphorylation. In addition, N17Rac and N17Cdc42, but not N19Rho, caused a significant decrease in PMN-mediated antifungal activity (Figure 6B). Thus, these results suggest that PMN function against C albicans is controlled by a Ras/Rho-independent but Rac/Cdc42-dependent MAPK/ERK signaling pathway. Importantly, we found that significant amounts of CD56, which is not endogenously expressed in PMNs, are present in these PMNs (Figure 6C). Western blot analysis of whole-cell lysates of PMNs revealed that significant amounts of Rac1 and Cdc42 were expressed in the appropriate samples as evidenced by the slower gel mobility of the recombinant proteins N17Rac and N17Cdc42, which contain myc-tag epitopes, compared with the faster migration of the respective endogenous proteins (Figure 6C). This was further confirmed by vaccinia vector containing green fluorescent protein (GFP) that demonstrated approximately 50% to 60% of cells are positive (data not shown). All these data indicate that the dominant-negative constructs introduced into PMNs are not only highly expressed but also are able to block specific PMN function against C albicans.
Because both N17Rac and N17Cdc42 blocked MAPK and antimicrobial activities, we next speculated that they must belong in the same pathway, particularly based on recent evidence that one could lie downstream of the other.47 We examined if constitutively active V12Cdc42 could rescue PMN function blocked by N17Rac or, alternatively, if V12Rac could rescue PMN function blocked by N17Cdc42. After infection with either N17Rac or N17Cdc42 for 15 minutes, PMNs were infected with constitutively active V12Rac or V12Cdc42. V12Cdc42 had no effect on N17Rac-mediated inhibition of MAPK activation, but V12Rac was able to rescue the blockade effect of N17Cdc42 on MAPK activation (Figure 6D). Virally expressed V12Rac and V12Cdc42 were both equally active in triggering MAPK activation in the presence of an irrelevant CD56 vector. Thus, Rac acts as a downstream effector of Cdc42 in PMNs. C albicans-induced MAPK activation involves PAK1 activation Recent studies have indicated that p21-activated kinase 1 (PAK1) is able to regulate cytoskeletal changes and phagocytic uptake in human neutrophils in response to chemoattractant stimuli.48 PAK1 also serves as a downstream target for both Rac1 and Cdc42.49,50 Many insights into the mechanism of chemotaxis have been made using Dictyostelium cells, which exhibit chemotaxis responses with many similarities to mammalian macrophage and neutrophils. In both mammalian leukocytes and Dictyostelium cells, translocation and phosphorylation of PAK1 is essential for proper cell polarity and chemotaxis.51 Recent data from our laboratory have indicated that PAK1 may act as a specific mediator for target-induced MAPK activation in natural killer (NK) cells.35 Therefore, it is essential to explore the role of PAK1 in C albicans-induced PMN activation. PAK1 protein was immunoprecipitated from the whole-cell lysates of PMNs after C albicans stimulation and analyzed for its kinase activity. PAK1 kinase activity was significantly increased following C albicans stimulation for 5 minutes (Figure 7A). To further examine the role of PAK1 in C albicans-induced killing, we utilized a kinase-defective, dominant-negative PAK1 mutant that was expressed in recombinant vaccinia virus.52 This mutant was used to inhibit PAK1-mediated, Cdc42-dependent responses in PMNs. Expression of kinase-deficient PAK1 markedly suppressed PAK1 kinase activity (Figure 7A). The same PMNs were then tested for antifungal activity. Kinase-deficient PAK1 expression inhibited PMN activity against C albicans growth, whereas PMNs infected with control CD56 viral vector remained highly active against C albicans, equal to that in mock-infected control PMNs (Figure 7B). Examination of whole-cell lysates from the same PMNs demonstrated that kinase-deficient PAK1, unlike control CD56, also markedly inhibited MAPK activation induced by C albicans (Figure 7C). Altogether, these data suggest that PAK1 may act as an upstream effector in C albicans-induced MAPK activation, which drives PMN antifungal activity.
MAPK activity is required for phagocytosis and intracellular granule mobilization in PMNs In the above experiments, we utilized candidal growth inhibition as an assay for PMN biologic function. However, this assay records both intracellular and extracellular microbicidal effects that is, death by
phagocytosis and by exocytosis of antimicrobial products. For death of
ingested microbes, mobilization of lytic granules into phagosomes is a
critical event.13,14 Both the production of oxygen
intermediates and the release of antimicrobial enzymes contained in
lytic granules determine the intracellular killing capacity of
PMNs.13 It has been reported that granular proteins such
as perforin and granulysin have broad effects against intracellular pathogenic bacteria, fungi, and parasites in vitro.53 But
another key granular protein, granzyme B, has not been reported to be expressed in human PMNs, and its antimicrobial activity has not been
defined. As shown in Figure 8A, granzyme
B is expressed in PMNs, as demonstrated by mRNA expression. Western
blot analysis and immunostaining of PMNs with a monoclonal antibody
specific for granzyme B demonstrated that granzyme B is also present at the protein level (Figure 8A, bottom panel). We then used
granzyme B as a specific marker for lytic granules in PMNs to examine
if the Rac/Cdc42-dependent MAPK pathway controls phagocytosis or granule mobilization. We employed 2-color staining whereby C
albicans was prelabeled with a red flourescent cell linker and the
lytic granules in PMNs were stained with FITC-labeled antigranzyme B. By visual assessment of stained PMNs and C albicans, MAPK
was found to control not only phagocytosis but also granule
polarization toward the ingested microbe (Figure 8B). Phagocytosis was
evaluated by counting the number of PMNs that showed ingested microbes
out of 100 total PMNs on each slide. When the phagocytizing PMNs were located, the position of the granzyme B molecules within the PMNs was
also recorded. Granule movement was then evaluated by counting the
number of PMNs with rings of granzyme B clearly visible around their
ingested microbes out of 100 PMNs containing phagocytized C
albicans. At 0 minutes of exposure to C albicans,
phagosomes were visible in 7.6% ± 1.2% of PMNs, whereas by 5 minutes, 66% ± 2.9% of PMNs had ingested microbes. The solvent
control DMSO had little effect on phagocytosis (67.6% ± 2.4%),
whereas the MEK inhibitor PD098059 greatly reduced the ability of the
treated PMNs to phagocytize microbes (21% ± 1.6%).
We next evaluated whether granule movement toward the ingested microbe was also subject to control by MAPK/ERK. On further visual examination of the PMNs that had phagocytized C albicans, PD098059 also adversely affected granzyme B mobilization toward the ingested microbes. As shown in Figure 8B, only 4% ± 0.5% PD098059-treated PMNs showed granzyme B mobilization as compared with 44.6% ± 4.6% medium- or 41.5% ± 5.0% DMSO-treated control PMNs. Expression of N17Rac, N17Cdc42, and dnMEK1 had similar inhibitory effects on both phagocytosis and granule mobilization (Figure 8C). Two-color staining clearly depicted dispersed FITC-labeled granzyme
B-containing granules (Figure 8Di) in control PMNs and at 0 minutes of
contact with C albicans (Figure 8Diii). After 5 minutes of
C albicans exposure, granzyme B rapidly gathered in PMNs,
surrounding the ingested microbe (Figure 8Div). In PD098059-treated PMNs, granzyme B retained its dispersed intracellular localization (Figure 8Dv). Furthermore, in comparison to CD56 expression (Figure 8Dvi), dnMEK1 in PMNs also suppressed granzyme B intracellular mobilization toward ingested C albicans (Figure 8Dvii).
N17Rac and N17Cdc42, but not N19Rho, had similar effects (Figure
8Dviii-x). These results indicate that phagocytosis and granule
mobilization are simultaneously regulated by the
Cdc42
Most research to date has suggested that signaling by the MAPK/ERK pathway is intimately involved in the ability of growth factors to stimulate proliferation.40,54 The currently accepted view of signaling through the MAPK/ERK cascade is based on studies with established fibroblastoid and transformed cell models that link MAPK/ERK activation with proliferation, whereby the greater the activation, the greater is the proliferative response.55,56 In agreement with increased MAPK/ERK activity correlating with increased transformation and proliferation, elevated levels of MAPK/ERK activity are frequently found in some cancer cells. Indeed, in efforts to reduce tumor growth, attempts have been made to develop cancer therapeutic agents targeting the MAPK/ERK pathway.11 However, emerging evidence indicates that the MAPK/ERK pathway controls normal cell functions, including secretion and reduced nicotinamide adenine dinucleotide phosphate (NADPH) regulation.18 More defined studies from our laboratory have shown that the MAPK pathway is also a key regulator of essential lymphocytic functions in addition to those associated with cell growth and proliferation.38 Depending upon the cell type and stimulus, MAPK activation can, therefore, modulate proliferation/differentiation or particular biologic functions. Here, by use of biologic, biochemical, and gene manipulation methods in human PMNs, we have uncovered yet another function of the MAPK pathway, the ability to control direct microbicidal capacity against C albicans. Our studies demonstrated that C albicans induced rapid MAPK activation in PMNs. Inhibition of MAPK activation, either by the pharmacologic agent PD098059 or by overexpression of dominant-negative MEK1, blocked overall PMN function against C albicans, as monitored by fungal growth inhibition. This antimicrobial activity requires both phagocytosis and granule mobilization toward the ingested microbe, and blockade of MAPK by PD098059 or dnMEK1 sharply reduced not only PMN phagocytic capacity but also the directed targeting of lytic granules. In contrast to the role of MAPK/ERK that we have described here, distinct mechanisms of activation have implicated different signaling pathways to be involved in phagocytosis and degranulation. PD098509 has been reported to affect phagocytosis mediated by fMLP but not degranulation.18,23,24 We believe that the difference in our results and those published using fMLP most likely lies in the mechanism of activation. fMLP induces receptor-mediated signaling through a heterotrimeric G protein that leads to the activation of Src family kinases and p38 MAPK. Of note, we used a unique system of PMN activation directly mediated by C albicans. The exact mechanism for direct microbial stimulation of PMNs has not been elucidated but may involve a unique receptor as well as a unique signaling pathway. Until now, events downstream from Rac and Cdc42 have not been elucidated, probably due to the problems inherent in the previous experimental systems that used receptor-transfected fibroblasts or tumor cell lines, which may not possess the full complement of kinases normally present in neutrophils. Indeed, direct phagocytosis of microbes, not aided by FcR or complement receptors, is not well studied. In the present study, however, using human PMNs we have identified that Rac and Cdc42 critically control phagocytosis and granule movement toward ingested microbes and that this regulation is mediated through MAPK/ERK. Of the small GTPases, N17Rac or N17Cdc42, but not N19Rho, suppressed MAPK/ERK activation triggered by microbe exposure in PMNs and at the same time inactivated phagocytosis and granule movement. On the other hand, neither N17Ras nor the Ras inhibitor FTI277 could interfere with MAPK activation or microbicidal function in PMNs, thus pinpointing a specific Rac/Cdc42-dependent, Ras-independent MAPK pathway for PMN function. It is of significance that biologic function against microbes in PMNs is mediated via a Ras-independent MAPK pathway, similar to that controlling tumor lysis in natural killer cells.34 Whether Ras usage is a key strategy by which cells signal gene expression and cell growth, but not mediate cell function, needs to be further explored. Rac is a required component for NADPH oxidase function in human neutrophils.57 In other cellular systems, Rac has been shown to be involved in cytoskeletal alterations and membrane ruffling.42,58 Of note, both Rac and Cdc42 are required for actin reorganization, which is a critical step in PMN phagocytosis of microorganisms.20,59 Indeed, for IgG-mediated phagocytosis, the ingestion of IgG-coated particles is able to activate both Rac and Cdc42.20 Using Swiss 3T3 cells, Cdc42-mediated actin assembly can be blocked by expression of N17Rac, suggesting that Rac is downstream of Cdc42.47 The impact of our work is the discovery that both Rac and Cdc42 not only control phagocytosis but also direct granule movement within PMNs toward the phagosome. Another important finding is that, although Rac has been primarily associated with MAPK-JNK activation in other cell systems,43 Rac preferentially utilizes MAPK/ERK to mediate PMN function. Support for this alternate Rac-MAPK pathway is becoming evident, as reported recently in fibroblasts and epithelial cells.44-46 Involvement of PAK1 in cytoskeletal rearrangement has been reported. Furthermore, PAK1 has been demonstrated to colocalize with actin to form phagocytic cups in activated human PMNs stimulated with fMLP.48 We and others have identified PAK1 as a possible intermediate between Rac and MAPK.44,48,58 Here, we show that PAK-1 is also activated by direct conjugation with C albicans. We have utilized a dominant-negative (kinase-deficient) mutant of PAK1 to further investigate the role of this enzyme in PMN antimicrobial activity. The inhibition of MAPK activation suggests that PAK1 activation is upstream of MAPK. However, the kinase-deficient PAK1 mutant contains an intact CRIB domain for interaction with Rac and Cdc42. Therefore, we cannot completely eliminate the possibility that the kinase-deficient PAK1 may still sequester Cdc42 or Rac1 nonspecifically. Further experiments will be needed to resolve the role of PAK1 in neutrophil-mediated antifungal activity. Recently, several reports have demonstrated that PI-3K plays a
key role in the early stages of neutrophil
chemotaxis.25,60-62 Indeed, fMLP has been shown to
activate a PI-3K-dependent Rac/Cdc42 pathway in neutrophils and
differentiated HL-60 cells.63 We have recently shown that
PI 3-kinase works upstream of Rac in NK cells to control lytic
function.35 Activation of PI 3-kinase by an unknown
C albicans-induced cell surface receptor(s) may lead to the
activation of Rac and Cdc42 in our system. However, there are likely to
be some differences in the signal cascades that control PMN
microbicidal function and NK lytic function, and one distinction we
have already observed is the lack of involvement of Cdc42 in NK
cells. NK cells utilize a PI 3-kinase More importantly, in this study we try to address the question proposed by Duesbery et al.64 They have raised the question of whether MAPK inhibitors, used as a strategy to treat cancer, would potentially lead to undesirable effects on normal physiological processes. Data from our studies indicate that use of MAPK pharmacologic inhibitors for the treatment of cancer may result in the interruption of normal neutrophil function. Because MAPK is a regulator of diverse intracellular signaling pathways involved in normal physiologic processes, a balance between therapeutic outcome and undesirable side effects must be attained to achieve successful and safe anticancer therapy.
We thank the Analytical Microscopy Core, Flow Cytometric Core, and the Molecular Imaging Core facilities of the H. Lee Moffitt Cancer Center.
Submitted December 6, 2001; accepted November 10, 2002.
Prepublished online as Blood First Edition Paper, January 2, 2003; DOI 10.1182/ blood-2001-12-0180.
Supported by American Heart Association grant AHA0256422B and U.S. Public Health Service grant CA83146.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sheng Wei, Immunology Program, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Dr, Tampa, FL 33612; e-mail: wei{at}moffitt.usf.edu.
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Top
Abstract
Introduction
) or granzyme B (
) mobilization by
PD098059. PMNs pretreated with medium, DMSO, or 50 µM PD098059 were
stimulated with C albicans for 0 to 5 minutes at 37°C, and
the number of PMNs showing ingested C albicans of a total of
100 PMNs were counted and expressed as a percent of total
PMNs. The number of PMNs with ingested C albicans
that also contained clearly visible granzyme B surrounding the
phagosome were also counted and expressed as percent of the total PMNs.
(C) Percentages of inhibition of phagocytosis (
Rac
B signalling pathways.
EMBO J.
1998;17:1395-1404