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Originally published online as doi:10.1189/jlb.1102525 on July 1, 2003

Published online before print July 1, 2003
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(Journal of Leukocyte Biology. 2003;74:611-619.)
© 2003 by Society for Leukocyte Biology

Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils

Maria Forsberg*, Pia Druid{dagger}, Limin Zheng*, Olle Stendahl* and Eva Särndahl{dagger},1

Departments of
{dagger} Cell Biology and
* Medical Microbiology, Faculty of Health Sciences, Linköping University, Sweden

1Correspondence: Department of Cell Biology, Faculty of Health Sciences, Linköping University, SE- 581 85 Linköping, Sweden. E-mail: eva.sarndahl{at}ibk.liu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (Fc{gamma}R)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and Fc{gamma}R and CR3- and Fc{gamma}R-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or Fc{gamma}Rs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.

Key Words: Rho–GTPases • phagocytosis • NADPH oxidase • ß2 integrin • leukocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocytes play an important role in host defense against invading pathogens and infection by performing effector functions such as chemotaxis, phagocytosis, and respiratory burst. Phagocytosis in neutrophils and macrophages mainly occurs by two different mechanisms: one where particles coated with C3b/C3bi fragments of the complement system are recognized by complement receptors (CR)1 and CR3 (ß2integrin) and the other where particles coated with immunoglobulin G (IgG) are recognized by Fc receptors (FcRs) [1 ]. Upon activation, these receptors transduce intracellular signals that trigger rearrangement of the cytoskeleton and particle ingestion and the killing of microorganisms by means of reactive oxygen intermediates (ROIs) and release of bactericidal proteins and peptides [2 3 4 5 6 ].

ROIs are formed by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multicomponent electron transport system. Proper functioning of this enzyme system is critical for host defense. This becomes evident in patients with a defective enzyme system, such as chronic granulomatous disease, and in patients defective in any component of the enzyme complex, all of whom suffer from reoccurring infections [6 7 8 ]. Rac2, a small guanosine 5'-triphosphate (GTP)-binding protein that forms one of the components of the NADPH-oxidase enzyme system, has been shown to be crucial for NADPH-oxidase activity in cell-free systems [9 ] and in patient with Rac2 dysfunctions [10 , 11 ]. In resting cells, Rac2 remains guanosine 5'-diphosphate-bound in the cytoplasm. Once activated, Rac2 becomes GTP-bound, and a small but vital fraction moves to the NADPH-oxidase enzyme complex in the plasma or phagosome membrane [12 , 13 ].

Rac and Cdc42 are members of the Rho family of GTPases, which are involved in regulating the actin cytoskeleton in various cellular functions [14 15 16 ]. To date, most of the knowledge on their function has been acquired from studies of nonhematopoietic cells such as fibroblasts. The role that Rac and Cdc42 play in inflammatory cells is, conversely, less clear at this point. As these cells differ in several aspects regarding the nature of the cytoskeletal rearrangements involving different Rho–GTPases, e.g., the formation of focal adhesions and stress fibers, the knowledge acquired from one cell type is not necessarily applicable to another. Discrepancies are also evident when comparing the different signaling strategies among different types of phagocytes. For example, the requirement for calcium during phagocytosis is different between macrophages and neutrophils [17 , 18 ]. Macrophages produce only a small amount of ROIs compared with neutrophils [19 ]. In macrophages, Cdc42 and Rac are necessary for FcR for IgG (Fc{gamma}R)-mediated phagocytosis, whereas CR3-mediated phagocytosis, which does not trigger the NADPH oxidase in these cells, is mediated by Rho [20 ]. In contrast, the NADPH oxidase is activated in neutrophils also upon CR3-mediated stimulation [21 ], suggesting that Rac2 activation is involved. Until now, Rac2 and Cdc42 have only been shown to be activated in response to formyl-Met-Leu-Phe (fMLP) and platelet-activating factor in neutrophils [22 , 23 ]. Evidence of Rho–GTPase activation following phagocytosis is however lacking in these cells.

The p21-activated kinases (PAKs) are downstream targets of Rac and Cdc42. PAKs are serine/threonine kinases known to exist in four different isoforms, PAK1–4. They have been implicated in mitogen-activated protein kinase signaling pathways, apoptosis, and cytoskeletal regulation [24 ]. In a recombinant system, PAK is able to phosphorylate the cytosolic component of the NADPH oxidase p47phox, indicating an important role for PAK in regulation of the oxidase [25 ].

In this paper, we investigated the activation of Rac2 and Cdc42 after CR3 and Fc{gamma}R ligation and phagocytosis using a glutathione S-transferase (GST) pulldown assay [22 ]. To verify these results, we also measured the phosphorylation of their common downstream target, PAK, and studied the translocation of Rac2. CR3 and Fc{gamma}R engagement and CR3- and Fc{gamma}R-mediated phagocytosis activated Rac2 and Cdc42. Furthermore, PAK became phosphorylated, and a translocation of Rac2 to the membrane fraction was detected following receptor engagement of CR3 and Fc{gamma}Rs. Our data indicate that in neutrophils, Rac2 and Cdc42 are important in FcR- and CR3-induced activation of the cell and for proper signal transduction leading to the generation of oxygen radicals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Merthiolate (sodium ethylmercurithiosalicylate), dimethyl sulfoxide, fMLP, dimethylpimpelimedate, luminol (5-amino-2,3-dihydro-1,4-phthalazindione), EDTA, EGTA, Triton X-100, and fluorescein isothiocyanate (FITC) were purchased from Sigma Chemical Co. (St. Louis, MO). Horseradish peroxidase (HRP), pepstatin, phenylmethanesulfonyl fluoride (PMSF), leupeptin, aprotinin, guanosine 5'-0-(thio)triphosphate; GTP{gamma}S), Nonidet P-40 (NP-40), Pefablock SC [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)], and bovine serum albumin were from Roche Diagnostics Corporation (Indianapolis, IN). Ficoll-Paque, glutathione-Sepharose beads, and enhanced chemiluminescence (ECL) reagents were from Pharmacia-Amersham (Uppsala, Sweden). PolymorphprepTM and LymphoprepTM were from Nycomed Pharmacia (Oslo, Norway); ethanol amine was from BDH Laboratory Suppliers (Poole, UK); N-acetyl-S-farnesyl-L-cysteine (AFC), N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), N-acetyl-S-geranyl-L-cysteine (AGC), and Pansorbins® were from Calbiochem-Behring (La Jolla, CA). All the chemicals used were of an analytical grade.

Antibodies
Anti-CD18 monoclonal antibody (mAb) IB4 (mIgG2b) was purchased from Ancell Corporation (Bayport, MN); mAb directed against Fc{gamma}RII (IV3, mIgG2b) was from Medarex Inc. (Annandale, NJ); anti-Fc{gamma}RIII mAb (VD2, mIgG2a) was from CLB (Amsterdam, The Netherlands); anti-PAK1/2/3 and antiphospho-PAK1/PAK2 antibodies were from Cell Signaling Technology (Beverly, MA); anti-Cdc42 mAb was from BD Transduction Laboratories (San Diego, CA); anti-Rac2 antibody (rabbit polyclonal, C-11) was from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Rac mAb was from Upstate Biotechnology (Waltham, MA); and HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, and mouse IgG (i.e., negative control) were from Dakopatts AB (Glostrup, Denmark).

Preparation of human neutrophils
Peripheral human blood was drawn from healthy volunteers and collected in heparin-containing vacutainer tubes, and neutrophils were isolated as described previously [26 27 28 ]. Briefly, whole blood was layered over a gradient consisting of LymphoprepTM layered over PolymorphprepTM and was centrifuged for 40 min at 480 g at room temperature. The band rich in polymorphonuclear cells was collected, and contaminating erythrocytes were removed by hypotonic lysis. The obtained cell suspension, consisting of >95% neutrophils, was washed twice, resuspended in Krebs-Ringer phosphate buffer [120 mM NaCl, 1.2 mM MgSO4x7 H2O, 1.7 mM KH2PO4, 8.3 mM Na2HPO4x2 H2O, 1 mM CaCl2, 4.9 mM KCl, and 10 mM glucose (KRG), pH 7.3]. The cells were kept on ice until used.

Preparation of antibody-coated Pansorbins®
Pansorbins® (10% w/v, specially hardened and heat-killed Staphylococcus aureus; Cowan I strain) were incubated with 0.3 mg mAb/ml for 1 h under continuous shaking and were then washed twice in 0.2 M boric acid. The coupling step was initiated by resuspending the Pansorbins® in 0.2 M boric acid supplemented with 20 mM dimethylpimpelimidate for 30 min. The termination was achieved by washing and incubating the Pansorbins® in ethanolamine (0.2 M, pH 8) for 2 h. The antibody-coated Pansorbins® were finally resuspended in phosphate-buffered saline (PBS) containing merthiolate (0.01% w/v).

FITC labeling and opsonization of Pansorbins®
Pansorbins® were resuspended in a carbonate buffer (0.2 M, pH 10) supplemented with (0.5 mg/ml) FITC for 30 min at 37°C as described by Hed [29 ]. After washing, the Pansorbins® were resuspended in PBS supplemented with merthiolate. Opsonization with complement was obtained by incubating FITC-labeled Pansorbins® with 20% normal human serum (NHS) at 37°C for 30 min. To obtain the opsonization with IgG, the Pansorbins® were incubated with 20% heat-inactivated (37°C, 30 min) serum isolated from rabbits immunized with Pansorbins®. After washing three times, the Pansorbins® were diluted in KRG.

Phagocytosis
A fluorescence-quenching method [29 ], which distinguishes between extracellulary and intracellularly located Pansorbins®, was used to determine phagocytosis. Neutrophils and FITC-labeled Pansorbins® were incubated at a ratio of 1:100 at 37°C for 5, 10, or 20 min. After incubation, the mixtures were kept on ice and examined by microscope. One drop of the neutrophil–Pansorbin® mixture and one drop of trypan blue (2 mg/ml in 25 mM citrate-phosphate buffer and 25 mM NaCl, pH 7.4) were placed on a microscopic slide. Particles bound extracellulary were quenched by trypan blue, whereas the ingested bacteria remained fluorescent when the slide was examined in an incident-light fluorescence microscope. At least 50 cells were counted for each sample. The fraction of cells containing fluorescent bacteria was used as a measure of phagocytosis (=phagocytic index).

The neutrophil respiratory burst activity
The respiratory burst in neutrophils was measured using a luminol ECL system [chemiluminescence (CL)]. The CL activity was measured using a six-channel Bioluminat LB9505 (Berthold Co., Wibald, Germany) using disposable 4-ml polypropene tubes. Neutrophils (1x106) were incubated for 5 min at 37°C with luminol (2x10-5 M), HRP (4 U), with or without inhibitor. After equilibrating at 37°C, stimulus was added at a ratio of 100 Pansorbins® per cell, and the light emission was recorded continuously.

Detection of activated forms of Cdc42 and Rac
To assay the activity of Cdc42 and Rac2, we used a precipitation assay using the p21-binding domain (PBD) of PAK1 expressed as a fusion protein with GST, as described previously [22 ]. Briefly, the cDNA-encoding residues 67–150 of PAK1 were cloned in-frame into the expression vector pGEX-4T3, kindly provided by Dr. Gary M. Bokoch (Scripps Research Institute, La Jolla, CA) and expressed in Escherichia coli. The GST–PBD fusion protein produced was stored at –70°C in a buffer containing 25 mM Tris-HCl, pH 7.5, 0.2 M dithiothreitol (DTT), 1 mM MgCl2, 5% glycerol, 0.5 mM PMSF, 1 mM benzamidine, and 1 µg/ml aprotinin.

Neutrophils (4x106/sample) were preincubated for 5 min at 37°C and thereafter, stimulated for 1–20 min. The reactions were terminated by adding an equal volume of a 2x ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 200 mM NaCl, 2% NP-40, 10% glycerol, 1 mM DTT, 2 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 2.8 µg/ml pepstatin, and 2 mM Na3VO4) and putting the samples on ice. The samples were thereafter incubated under rotation for 30 min at 4°C. Subsequently, the lysates were centrifuged at 10,000 g for 0 min, transferred to new tubes containing glutathione-Sepharose beads precoupled with GST–PBD, and incubated under rotation at 4°C for 30 min. The beads were washed three times with 1x lysis buffer, and the proteins on the beads were eluated with Laemmeli sample buffer [30 ], separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and blotted for Cdc42 and Rac. The presence of protein on the blot was detected using a commercial ECL detection kit (Pharmacia-Amersham), and the bands on the blots were quantified using NIH Image v.1.6.1. To ascertain the specificity of the assay, we added 1 mM EDTA and 100 µM GTP{gamma}S in the cell lysate of fMLP (1 µM)-stimulated cells (15 min, 30°C) as a positive control and added uncoupled GST beads (i.e., beads without the PBD domain of PAK1) as a negative control.

Detection of activated forms of PAK
Human neutrophils (3x106) were preincubated at 37°C for 2 min before stimulated for 5 min. The stimulation was stopped by adding ice-cold PBS supplemented by 1 mM Na3VO4 and simultaneously putting the cells on ice. The chilled cells were rapidly pelleted (10,000 g, 1 min) at 4°C, resuspended in 200 µl lysis buffer containing 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.5, 10 mM EGTA, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1.4 µg/ml pepstatin, and 1 mM PMSF, and incubated under rotation for 30 min at 4°C. After centrifugation at 10,000 g for 10 min, the lysate was suspended in Laemmli sample buffer. The samples were heated for 5 min at 95°C and subjected to SDS-PAGE, electrotransferred to nitrocellulose membrane membranes, and analyzed by immunoblotting with phospho-PAK Ab detecting active PAK. The presence of protein on the blot was detected using a commercial ECL detection kit (Pharmacia-Amersham). To confirm that equal amounts of protein were loaded in each lane, the membranes were stripped and reprobed with an anti-PAK (total PAK) antibody.

Subcellular localization of Rac2
Neutrophils (107) in KRG were preincubated at 37°C for 2 min before stimulated for 5 min. The stimulation was stopped by adding ice-cold KRG supplemented by 1 mM Na3VO4 and putting the cells on ice. The method to isolate subcellular fractions was slightly changed from that described previously [31 , 32 ]. The cells were rapidly pelleted at 4°C, resuspended in 500 µl lysis buffer [2 mM EDTA, 0.5 mM EGTA, 2 mM MgCl2, 20 mM Tris, pH 7.4, and protease inhibitors, i.e., 0.3 mM Pefabloc SC (AEBSF), 1 mM Na3VO4, and 5 µg/ml leupeptin, pepstatin, and aprotinin], and then sonicated. A 200 gcentrifugation and a subsequent 500 g centrifugation for 10 min each at 4°C were used to clarify the samples. The obtained supernatants were further centrifuged twice at 10,000 g for 5 min at 4°C and thereafter, subjected to an ultracentrifugation at 200,000 g for 30 min at 4°C to separate the cytosolic and membrane fractions. The membrane fraction was washed once in lysis buffer and recentrifuged (200,000 g, 15 min). The obtained pellet was dissolved in ice-cold 1% Triton X-100 on ice for 1 h before Laemmli sample buffer was added. Proteins were subjected to SDS-PAGE, electrotransferred to nitrocellulose membranes, and analyzed by immunoblotting with an anti-Rac antibody and a commercial ECL kit.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of Pansorbins® with human neutrophils and subsequent activation of the NADPH oxidase
Previous studies from our laboratory have shown that challenging neutrophils with Pansorbins® coated with antibodies against CR3 or Fc{gamma}R activates the NADPH oxidase and a number of tyrosine kinases [28 , 33 ]. We also found that Pansorbins® coated with anti-CD18 (the ß subunit of CR3) or anti-CD11b antibodies (the {alpha} subunit of CR3) were able to activate the oxidase, whereas anti-CD11a and anti-CD11c did not [21 ]. These antibody-coated Pansorbins® bind readily to the neutrophils but are not ingested, whereas the particles become readily ingested by the neutrophils when opsonized with serum (see Fig. 3 C ). These differential effector functions of antibody-coated Pansorbins® versus opsonized particles could reflect binding to different epitopes and/or different density of the ligand. Notwithstanding, antibody-coated and serum-opsonized particles elicted similar signaling events.



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  		Figure 3. Activation of Rac and Cdc42 in relation to phagocytosis and ROI production in human neutrophils. (A and B) Activation of Rac and Cdc42 was detected using the GST-pulldown assay. The blots were scanned, and the relative intensity of the bands was quantified using NIH Image v.1.6.1. The minimum value, i.e., unstimulated cells, was set at 0, and the values obtained from stimulated cells were accordingly correlated. The maximum value was set at 100% for each individual experiment. Data are given as mean ± SD for n = three to eight separate experiments and are expressed as % of the maximum value. (C) Time kinetics of the phagocytic process of IgG versus serum-opsonized Pansorbins®. Neutrophils were incubated with Pansorbins® at 37°C for various time points as indicated in the figure. Putting the samples on ice stopped the reactions. Phagocytosis was determined by quenching the extracellularly bound particles by trypan blue. Data are shown as mean ± SD of phagocytic index of at least three experiments. (D) Time kinetics showing the ROI production in neutrophils challenged with IgG or serum-opsonized Pansorbis®. The ROI production was measured using CL, and the figure shows one representative experiment out of 10. The insets show an extended version of the ROI response measured under 60 min.

 
In the present study, we used Pansorbins® coated with anti-CD18, anti-CD16 (Fc{gamma}RIIIb), and anti-CD32 (Fc{gamma}RIIa) to study early signaling events and serum-opsonized particles to study phagocytosis. Antibody-coated Pansorbins® and Pansorbins® opsonized with serum activated the NADPH oxidase, as measured by CL (Fig. 1 ). The oxidative response elicited by anti-CD18 Pansorbins® was reduced by 80% with Fab–anti-CD18 (data not shown). Moreover, activating the neutrophils with Pansorbins® opsonized with heat-inactivated serum (i.e. complement degraded) induced about 25% of the response obtained with Pansorbins® opsonized with serum (data not shown).



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  		Figure 1. Production of ROI in neutrophils after activation with antibody- and serum-opsonized Pansorbins®. The ROI production was measured using a luminol ECL system. The cells (1x106) were incubated for 5 min at 37°C before adding Pansorbins® coated with antibodies directed against Fc{gamma}RIIa, Fc{gamma}RIIIb, and CR3 or Pansorbins® opsonized with serum (Ops. NHC) or IgG (Ops. IgG; at a ratio of 100 Pansorbins®/cell). The bars represent peak values as percentage of the CR3-mediated ROI production from at least five separate experiments, and data are given as mean ± SEM.

 
Activation of Rac and Cdc42 in human neutrophils
To be able to detect the active, GTP-bound form of Rac and Cdc42, we used an assay in which active forms of Cdc42 and Rac are precipitated from the 10,000 g fraction of lysed cells, using a fusion protein consisting of the PBD of PAK1 and GST (GST–PBD) [22 ]. A rapid activation of Cdc42 and Rac was detected already after 1 min, and this activation was induced after receptor ligation (Fig. 2A ) and phagocytosis (Fig. 2B and 2C) . Densitometric measurement revealed that the activation of Cdc42 and Rac peaked at about 5 min during Fc-mediated phagocytosis and at approximately 10 min during CR3-induced phagocytosis (Fig. 2B and 2C , and Fig. 3A and 3B ). The time kinetics for the activation of Rac and Cdc42 corresponds well with the phagocytic process (Fig. 3C) and the initiation of free oxygen radical production (Fig. 3D) . The fact that phagocytosis reaches a peak after 10 min and the CL response, after 20–30 min indicates that Rac/Cdc42 activation is an early response involved in pseudopod formation but not necessarily in the closure and maturation of the phagosome. The sustained oxidative response shows that the NADPH oxidase is active within the phagosome. GTP{gamma}S, a nonhydrolysable GTP analog that arrests Cdc42 and Rac in their active stage, was added to the cell lysate of fMLP-stimulated cells and was used as a positive control [22 ]. To exclude unspecific binding or activation, beads without the GST–PBD or Pansorbins® coated with mouse IgG were used as negative controls. The results showed a strong activation of Cdc42 and Rac upon stimulation with fMLP + GTP{gamma}S, whereas no activation could be detected upon incubation with mouse IgG-coated Pansorbins® (Fig. 2D) . The noncoated beads did not bind Cdc42 or Rac (Fig. 2D) .



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  		Figure 2. Immunoblot analysis of precipitated Rac and Cdc42 from lysate of human neutrophils using the GST-pulldown assay. Neutrophils (4x106) were preincubated for 5 min at 37°C and then stimulated with Pansorbins® (at a ratio of 100 Pansorbins®/cell) for various time points as indicated in the figure. The activation was stopped, and activated forms of Rac and Cdc42 were precipitated from the lysates using the GST-pulldown assay as described in Materials and Methods. (A) The cells were activated for 1 min with Pansorbins® coated with antibodies directed against Fc{gamma}RIIa, Fc{gamma}RIIIb, and CR3. (B and C) The time kinetics of the activation of Rac and Cdc42 stimulated with IgG or serum-opsonized (NHS) Pansorbins®. (D) Neutrophils stimulated by fMLP + GTP{gamma}S (positive control), cells incubated with Pansorbins® coated with mouse IgG (negative control), or GST-pulldown assay performed with beads lacking the GST–PBD (negative control). Material from 3 x 106 cell equivalents was loaded in each lane, and the electrophoretically separated proteins were detected with Western blot technique using an anti-Rac (1:1000) or anti-Ccd42 antibody (1:250). The upper band on the blot represents the GST fusion protein. Each figure is a representative of at least four separate experiments.

 
Activation of PAK: a downstream target of Cdc42 and Rac
To be able to verify our results on Cdc42 and Rac activation during C3bi and Fc stimulation using the GST-pulldown technique, we detected activation of a downstream target of Cdc42 and Rac, i.e., PAK, which becomes phosphorylated upon binding of active, GTP-bound Cdc42 or Rac [24 ]. By using an antibody specific for the phosphorylated forms of PAK, we were able to detect activated PAK after stimulation with different Pansorbins® (Fig. 4 ).



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  		Figure 4. Immunoblot analysis of the phosphorylation of PAK. Human neutrophils were preincubated at 37°C and stimulated for 5 min by antibody-coated Pansorbins®, i.e., antibodies directed against Fc{gamma}RII (lane 2), Fc{gamma}RIII (lane 3), CR3 (lane 4), or serum-opsonized (ops. NHS) Pansorbins® (lane 5), at a ratio of 100 Pansorbins®/cell. Unstimulated cells, subjected to the same treatment as described above but not stimulated with antibody-coated Pansorbins®, are shown in lane 1. The neutrophils were lysed using a buffer containing 1% Triton X-100. After a 10,000 g centrifugation, the lysate was suspended in Laemmli sample buffer. Material from 2 x 106 neutrophil equivalents was loaded in each lane, and the electrophoretically separated proteins were detected with Western blot technique using an antiphospho-PAK antibody (1:1000). Proteins detected by an anti-PAK antibody (1:1000), i.e., detecting the total amount of PAK, are shown for comparison. The blot shown is a representative of three separate experiments.

 
Neutrophils stimulated via Fc{gamma}RIIa, Fc{gamma}RIIIb, or CR3 induced phosphorylation of PAK (Fig. 4 , lanes 2–4). Phosphorylation of PAK was also induced upon phagocytosis of serum-opsonized Pansorbins® (Fig. 4 , lane 5). These data support our previous finding (Fig. 2) , showing that C3bi and Fc stimulation activates Cdc42 and Rac in human neutrophils.

Role of small GTP-binding proteins during activation of human neutrophils with Pansorbins®
The finding that Cdc42 and Rac become activated following engagement of the phagocytic receptors and upon phagocytosis led us to investigate the potential role of Cdc42 and Rac in the activation of the NADPH oxidase and in the phagocytic process. As neutrophils are difficult to manipulate genetically, we used a pharmaceutical approach to evaluate their role [34 ]. The two methyltranferase inhibitors AFC and AGGC are known to inhibit the prenylcysteine {alpha}-carboxyl methyl esterification of small GTP-binding proteins of the Rho and Ras family [35 , 36 ], including Rac [35 , 37 ] and Cdc42 [38 ], thereby preventing their specific membrane localization [35 ], a key step for the action of Rho–GTPases [39 ].

When neutrophils were pretreated with AFC, AGGC, or their inactive analog AGC, we could detect no significant decrease in phagocytosis of serum-opsonized Pansorbins® in AFC and AGGC-treated cells as compared with control (i.e., AGC-treated cells) or untreated cells (Table 1 ). However, neutrophils treated with AGGC or AFC exhibited a decrease in oxidative activation compared with AGC-treated or untreated cells (Table 2 ). This inhibitory effect on NADPH oxidase activity was more pronounced during phagocytosis (40–65% inhibition) than receptor ligation (20–40% inhibition). Pretreatment with AGGC inhibited the NADPH-oxidase activity more efficiently than did AFC in the same concentration range.


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  		Table 1. Phagocytosis of NHS- and IgG-Opsonized Pansorbins® by Human Neutrophils

 

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  		Table 2. Effect of AFC, AGGC, and AGC on ROI Production in Human Neutrophils Activated with Pansorbins®

 
Detection of Rac in the membrane fraction of human neutrophils
During activation, a small but essential fraction of Rac translocates to the plasma membrane, where it constitutes an important part of the NADPH oxidase [12 , 13 ]. Using cell fractionation and immunoblot technique, we detected translocation of Rac2 to the membrane fraction in neutrophils stimulated by antibody-coated Pansorbins® (Fig. 5 ).



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  		Figure 5. Immunoblot analysis of the presence of Rac in the membrane fraction of human neutrophils. Cells, preincubated in 37°C, were stimulated by antibody-coated Pansorbins® at a ratio of 100 Pansorbins®/cell for 5 min. The antibodies used were directed against Fc{gamma}RIII (lane 2), CR3 (lane 3), or Fc{gamma}RII (lane 4). Unstimulated cells, subjected to the same treatment as described above but not stimulated with antibody-coated Pansorbins®, are shown in lane 1. The cells were lysed by sonication, and the membrane fractions were obtained performing an ultracentrifugation (200,000 g) on the 10,000 g supernatants. The membrane fraction of 1 x 107 cell equivalents was loaded in each lane, and the electrophoretically separated proteins were detected with Western blot technique using an anti-Rac2 antibody (1:250). Proteins detected in the whole cell lysate (lane 5) are shown for comparison. The blot shown is one representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we report that engagement of and phagocytosis by the phagocytic receptors CR3 and Fc{gamma}R activate Rac as well as Cdc42. This means that a prey does not have to be ingested to activate signal transduction cascades involving these small GTP-binding proteins.

The present data clearly show that Rac and Cdc42 are activated not only by FcR but also after CR3 ligation and that this activation corresponded with phagocytic uptake and ROI production. In transfected COS cells phagocytosing opsonized red blood cells (RBC), only those ingesting IgG–RBC recruited Rac and Cdc42 around the phagosome [20 ]. In neutrophils, conversely, we show that Rac is translocated from the cytosol to the membrane after complement activation as well (Fig. 5) . The detection of Rac in the membrane fraction of cells can be used as an indirect measurement of Rac activation [39 , 40 ] and thereby gives additional support to our observation that Rac is activated upon ligation of Fc{gamma}RIIa, Fc{gamma}RIIIb, and CR3. As these results show a stimulus-induced translocation of Rac2 to a site constituting the NADPH oxidase, they also add support for the involvement of Rac in C3bi- and Fc-induced activation of the NADPH oxidase in human neutrophils.

It is evident that neutrophils and macrophages have different signaling pathways during Fc- and C3bi-mediated phagocytosis. In neutrophils, intracellular calcium is required for Fc-mediated phagocytosis [18 ], whereas ingestion of IgG or C3bi-opsonized particles by macrophages neither triggers a rise of nor uses intracellular calcium [17 , 41 ]. Furthermore, calcium is a sine qua non for degranulation in neutrophils [42 ] but is not necessary for degranulation and phagolysosome fusion in macrophages [41 ]. Whereas Rac and Cdc42 are necessary for FcR-mediated activation and phagocytosis in macrophages, Rho is activated by CR3 ligation and required for complement-mediated ingestion [20 ]. Our finding that not only FcR, but also ligation of CR3, leads to Rac and Cdc42 activation and translocation in neutrophils adds to the diversity between these cells.

To establish if Rac and Cdc42 activation is required for phagocytosis in human neutrophils, the methyltransferase inhibitors AFC and AGGC were used. Normally, the methylation is known to lead to the association of Ras and Rho-family GTPases with membranes and to be important for their interaction with cell membrane components [43 44 45 46 47 ]. By inhibiting the methylation, these inhibitors have been widely used to study the involvement of Ras and Rho–GTPases in several signal-transduction pathways [38 , 48 , 49 ], including in neutrophils [34 , 35 , 37 ]. This treatment had no significant effect on the ingestion of IgG- or C3bi-opsonized bacteria but inhibited the oxidative response, suggesting that Rac and/or Cdc42 are necessary for oxidative activation in human neutrophils. The discrepancy found between the effect of the inhibitors on phagocytosis versus ROI production is intriguing. The requirement for Rac and Cdc42 in phagocytosis is likely to be catalytic in nature. Therefore, a partial decrease in GTPase activity might explain the lack of effect of these inhibitors on phagocytosis. In contrast, Rac2 has been shown to constitute a component of the NADPH oxidase and to physically participate in the generation of ROI [50 ]. Our data imply that Rac and probably Cdc42 might not be completely inhibited by the farnesyl/geranyl transferase inhibitors, reflecting that the turnover of Rac/Cdc42 is too slow for the inhibitors to be completely effective. The difference in the need for Rac in ingestion and oxidative activation, respectively, is however further supported by recent reports on a human neutrophil deficiency syndrome associated with Rac2 mutations [10 , 11 ]. In these patients who lack a functional Rac2, superoxide production, migration, and azurophilic degranulation were severely inhibited. However, phagocytosis of IgG–RBC was not impaired in these neutrophils, with the exception of fMLP-primed cells.

Phosphorylation of a downstream target of Rac and Cdc42, i.e., PAK, has been used in this study to verify the activation of Rac and Cdc42. In human neutrophils, PAK1 relocalizes to the F-actin-rich pseudopodia and phagocytic cups of opsonized zymosan [51 ]. As only a small amount of PAK1 was found around the ingested particles, PAK1 is suggested to be involved in the regulation of the cytoskeletal extensions and initial events required for engulfment of bacteria but not in the subsequent steps of the phagocytic process. Our results indicate a role for Rac and/or Cdc42 in the phagocytosis-induced oxidative activation but not in the ingestion process.

Apart from PAK, we have not investigated the role of other downsteam effector molecules regulating actin polymerization, such as gelsolin, Wiskott-Aldrich syndrome protein (WASP)/neural-WASP, and Arp2/3. Gelsolin-depleted neutrophils and WASP-deficient macrophages from WASP patients show deficient phagocytosis [52 , 53 ]. Furthermore, Arp2/3 localizes to phagosomes and is required for Fc- and C3b-mediated phagocytosis [54 ].

The present report does not allow us to distinguish between the role of Rac and Cdc42. It has been suggested that Cdc42 promotes local formation of actin-rich pseudopods, and Rac has a permissive role in particle entry [54 55 56 ]. Our data show that ligation of CR3 activates Rac and Cdc42 with the same time kinetics. Distinguishing between the roles of Rac and Cdc42 in the different steps of phagocytosis requires genetic manipulation and introduction of specific inhibitors. As these techniques are not readily available for human peripheral blood neutrophils, we are presently developing means to selectively inhibit different signaling pathways. The use of TAT fusion proteins [57 ] to introduce dominant-negative proteins is one way of pursuing these problems. Using this tool, we will also be able to discriminate between the roles of Rac1 and Rac2.

One important reason for elucidating the role of Rho–GTPases and their down-stream targets during phagocytosis is the fact that several microbial toxins and virulence proteins target Rho–GTPases. SptP from Salmonella [58 ], ExoS from Pseudomonas, and YopE from Yersinia [59 ] are all GTPase-activating proteins translocated into the host cells. Furthermore, Rac and/or Cdc42 are required for internalization of Yersinia pseudotuberculosis [60 , 61 ], Listeria [62 ], Salmonella [58 ], and Shigella [63 ].

Another reason is to understand the different functions and roles of neutrophils and macrophages in innate immunity. Whereas integrin and immune receptors in neutrophils trigger an oxidative response and inflammation, these receptors show a more selective role when regulating the multiple functions of macrophages in host defense. As it is evident that neutrophils and macrophages use different signaling pathways during Fc- and C3bi-mediated phagocytosis, respectively, our data add to the diversity in signal transduction between these cells. This suggests a more powerful oxidative response in neutrophils, whereas macrophages exhibit a more nonphlogistic feature benefiting from a weak activation of the NADPH oxidase.


   ACKNOWLEDGEMENTS
 
This work was supported by the King Gustaf V 80-Year Memorial Foundation, the Swedish Association against Rheumatism, the Swedish Association for Medical Research, the County Council of Östergötland, the Swedish Research Council (#5968, #12725, #13103), and the Network for Inflammation Research, funded by the Swedish Foundation for Strategic Research. The authors thank Dr. Gary M. Bokoch (Scripps Research Institute, La Jolla, CA) for generously providing the plasmid-encoding GST–PBD. We thank Ms. Anna Lindström for excellent technical assistance during her final report at the Master Program in Medical Biology and Drs. Jane Wigren and Maria Lerm for helpful discussions.

Received November 4, 2002; revised April 24, 2003; accepted April 25, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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