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The Herman B Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology) and Medical and Molecular Genetics, The James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, IN 46202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-primed BM neutrophils and in peritoneal exudate neutrophils. We
investigated receptor-mediated activation of the NADPH oxidase in the
current study, finding that superoxide production in
rac2-/- BM and peritoneal exudate
neutrophils was normal in response to opsonized zymosan, reduced to
22% of wild type in response to IgG-coated SRBC, and almost absent in
response to fMLP. In wild-type murine BM neutrophils, phosphorylation
of extracellular signal-regulated kinase 1/2 (ERK1/2), p38
mitogen-activated protein kinase, c-Jun N-terminal kinase, and Akt was
induced by PMA or fMLP, which was decreased in
rac2-/- neutrophils for ERK1/2 and p38.
Activation of p38 by either opsonized zymosan or IgG-coated SRBC was
similar in wild-type and rac2-/- cells.
Inhibition of ERK1/2 or p38 activation using either PD98059 or
SB203580, respectively, had only a modest effect on fMLP-elicited
superoxide production and no effect on the PMA-induced response. These
data provide genetic evidence supporting an important role for Rac2 in
regulating neutrophil NADPH oxidase activation downstream of
chemoattractant and Fc
receptors. The effect of Rac2 deficiency on
superoxide production is probably exerted through multiple pathways,
including those independent of mitogen-activated protein kinase
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Rho family GTPases regulate a wide spectrum of cellular functions, including cytoskeletal organization, transcription, cell growth, development, and superoxide production (8, 9, 10, 11, 12, 13). Alternating between GDP- and GTP-bound states, Rho GTPases function as intermediary switches, mediating the transfer of signals from receptors and their associated kinases to downstream effectors (12). Three Rho GTPase subclasses based on homology to the three prototypic members, RhoA, Rac1, and Cdc42Hs, are currently recognized. These encompass seven distinct proteins, including Rho (A, B, and C isoforms), Rac (1, 2, and 3 isoforms), Cdc42 (Cdc42Hs and G25K isoforms), RhoD, RhoG, RhoE, and Tc10. Rac1 and Rac2 share 92% identity and differ primarily in the C-terminal 10 residues, where Rac1, but not Rac2, contains a polybasic sequence. Rac1 and Rac3 share 77% identity, and Rac2 and Rac3 share 83% identity (14, 15, 16, 17, 18). Murine Rac1 has 100% amino acid identity and 92% nucleotide homology with human Rac1, and murine Rac2 differs by only two amino acids from human Rac2 (14, 15). Rac1 and Rac3 are expressed in a wide variety of tissues, whereas Rac2 expression is highly restricted to hematopoietic cells (14, 15, 16, 17, 18).
One of the first cellular functions ascribed to Rac was the activation of the phagocyte NADPH oxidase (6, 7). The GTP-bound form of Rac binds to both p67phox and probably also to flavocytochrome b in the assembled oxidase complex (4, 5, 19, 20, 21, 22). Superoxide generation in cell-free assays is substantially increased by the addition of Rac (6, 7), and studies using either Rac2 transgenes or antisense oligonucleotides to manipulate Rac levels suggest that Rac regulates NADPH oxidase activity in intact cells (23, 24). Neutrophil activation with either fMLP or phorbol ester stimulates the formation of Rac-GTP (25, 26). Rac2 is the predominant form in human neutrophils (22, 27). In the yeast two-hybrid system, p67phox has higher affinity for Rac2 than for Rac1 (19). Recombinant isoprenylated Rac1 and Rac2 were equipotent in a cell-free NADPH oxidase assay using purified flavocytochrome b and recombinant cytosolic phox proteins, but Rac2 was more active than Rac1 when neutrophil cytosol was added (28).
To examine whether Rac2 has a specialized function in the NADPH oxidase and other neutrophil functions, mice with a targeted disruption of the hematopoietic-specific rac2 gene were generated (11). Rac2-null mice exhibited decreased exudate formation in vivo and an increased susceptibility in invasive Aspergillosis. Phorbol ester-activated NADPH oxidase activity in bone marrow (BM)3 rac2-/- neutrophils was reduced by about 4-fold. However, the requirement for Rac2 was not absolute, in that deficient superoxide production by rac2-/- BM neutrophils could be partially overcome by first priming with TNF, and phorbol ester-elicited superoxide production in rac2-/- exudate neutrophils was almost normal. Other functional defects in rac2-/- neutrophils include diminished L-selection-mediated adhesion to glycam-1, impaired actin polymerization, and chemotaxis in response to fMLP and other chemoattractants signaling through G protein-coupled receptors, and decreased fMLP-induced activation of p42/p44 and p38 mitogen-activated protein (MAP) kinase (11). Taken together, these findings suggested that Rac2 regulates multiple cellular functions in neutrophils. Additional studies in mast cells (29) and T cells (30) have demonstrated a variety of functional abnormalities associated with intracellular signaling defects. These include reduced chemotaxis and growth factor-induced survival in mast cells, with impaired activation of Akt (29).
In the present study, we used rac2-/-
mice as a genetic approach to investigate the role of Rac2 in
receptor-mediated activation of the neutrophil NADPH oxidase. The
results support the concept that Rac2 plays an important, but
stimulus-specific, role in regulating NADPH oxidase activation and
other functional responses of neutrophils, particularly those
downstream of chemoattractant receptors and Fc receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rac2 and p47phox Abs were gifts from G. Bokoch and U. Knaus (The Scripps Research Institute, La Jolla, CA) and D. Lambeth and D. Uhlinger (Emory University, Atlanta, GA), respectively. Rabbit polyclonal Abs against p42/44, Thr202/Tyr204-phosphorylated p42/44, Thr183/Tyr185-phosphorylated c-Jun N-terminal kinase (JNK)/SARK, p38 MAP kinase, Thr180/Tyr182-phosphorylated p38 MAP kinase, Akt, and Ser473-phosphorylated Akt were purchased from New England Biolabs (Beverly, MA). Mouse mAbs against Rac1 and phosphotyrosine (4G10) were purchased from Upstate Biotechnology (Lake Placid, NY), and those for JNK1/2 and CD16/CD32 were purchased from PharMingen (San Diego, CA). Highly purified recombinant human Rac1 and Rac2 were provided by D. Lambeth. Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. PBS (pH 7.2), HBSS (without Ca2+, Mg2+, and phenol red), and HBSS with 1.26 mM Ca2+ and 0.4 mM Mg2+ were purchased from Life Technologies (Grand Island, NY). Other buffers used in this study include HBSS with 0.1% BSA and 1% glucose (pH 7.25–7.4) (HBSS-BG); PBS with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose (PBSG); and lysis buffer (20 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 2 mM Na3VO4, 0.01 mM phenylarsine oxide, 20 µg/ml chymostatin, 2 mM PMSF, 10 µM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride).
Animals
Rac2 knockout (rac2-/-) mice had
previously been generated by targeted disruption of the rac2
gene (11). Rac2 knockout mice used in this study had been
backcrossed into C57BL/6J mice for 
11 generations. C57BL/6J mice were
used for wild-type controls. Mice were housed in microisolator cages
under specific pathogen-free conditions and were fed autoclaved food
and acidified water ad libitum. Mice used in these experiments were
8–16 wk of age.
Isolation of neutrophils
Murine neutrophils were obtained from the BM and peritoneal cavity as previously described (11). BM neutrophils were isolated from femurs and tibias flushed with 3 ml of HBSS-BG using a 22-gauge needle (Becton Dickinson, Franklin Lakes, NJ). The cells were pelleted by centrifugation at 600 x g for 10 min at 4°C and suspended in 3 ml of 45% Percoll (Pharmacia, Uppsala, Sweden). Two milliliters each of the 62, 55, and 50% Percoll solutions was layered successively onto 3 ml of the 81% solution. One hundred percent Percoll stock solution was made by mixing 9 vol of Percoll with 1 vol of 10x HBSS. Finally, cells in 45% Percoll were layered on top of the gradient. After centrifugation at 1600 x g for 30 min at 10°C, the cell band between the 81 and 62% layer was collected and washed twice in 10 ml of HBSS-BG and suspended in 3 ml of HBSS-BG. These cells were layered over 3 ml of Histopaque 1119 and centrifuged at 1600 x g for 30 min at 10°C to remove contaminating RBC. The cells between the Histopaque and HBSS-BG layers were harvested and washed twice in 10 ml of HBSS-BG. The resulting cell population was 73–78% neutrophils by assessment of nuclear morphology of Diff-Quick (Dade, Miami, FL)-stained cells, with the remainder being mononuclear cells. The final cell preparation was kept on ice in PBS (without Ca2+ and Mg2+) until further processing. For some studies BM neutrophils were cultured for 24 h at 37°C in DMEM containing 20% FCS and 1 ng/ml IL-3 before measuring NADPH oxidase activity. To elicit peritoneal exudate neutrophils, mice were injected with 1 ml of 3% thioglycolate. Peritoneal cells were harvested after 18 h as previously described (31). All reagents used were endotoxin tested (<0.1 ng endotoxin/ml by the Limulus lysate assay) to minimize inadvertent priming during the isolation procedure.
Human neutrophils were separated from peripheral venous blood obtained from healthy volunteers as approved by the institutional review board of the Indiana University School of Medicine. Heparinized whole blood was sedimented using 6% dextran 70 (McGaw, Irvine, CA), and the granulocytes were purified from the leukocyte-rich serum by Ficoll-Hypaque. Remaining erythrocytes were removed by hypotonic lysis.
FcRII/III expression
Flow cytometry was used to quantitate the relative amount of
cell surface FcRII/III expression per neutrophils. BM neutrophils
(2 x 105/ml) in PBS containing 0.1% BSA
were incubated with 2 µg/ml FITC-conjugated anti-mouse CD16/CD32
or FITC-conjugated anti-mouse IgG2b isotype (PharMingen) for 30 min
on ice. After washing cells twice with PBS containing 0.1% BSA, flow
cytometric analysis was performed using a FACScan (Becton Dickinson). A
minimum of 10,000 neutrophil events was recorded and analyzed using
CellQuest (Becton Dickinson).
Opsonization of zymosan and preparation of IgG-sensitized SRBC
Zymosan particles were prepared as described previously (32), opsonized with normal human serum for 1 h at 37°C, washed with PBS, and then diluted in PBS. Ab-coated SRBC (IgG-SRBC) were freshly prepared in magnesium- and calcium-free PBS by incubating 109/ml SRBC (Cappel; ICN Biomedicals, Costa Mesa, CA) with an equal volume of 2 µg/ml IgG rabbit-anti-SRBC Ab (Cappel) as previously described (33).
Immunoblot analysis of Rac1 and Rac2 expression
Lysates of mouse BM neutrophils and human peripheral blood neutrophils were prepared, and protein was measured with the bicinchoninic acid protein kit from Pierce (Rockford, IL) using BSA as a standard. Lysates were subjected to SDS-PAGE and immunoblotting as previously described (11, 34). Blots were probed with either mouse mAb for Rac1 or a rabbit polyclonal Ab for Rac2 and developed with the ECL method (Amersham, Arlington Heights, IL) as previously described (35). The Ab specificity of the Rac1 and Rac2 Abs was confirmed by immunoblotting Escherichia coli-produced human recombinant Rac1 and Rac2, supplied by D. Lambeth (Emory University). Integrated densitometry was employed to measure the relative intensity of signals using an Eagle Eye II Still Video System and associated software (Stratagene, La Jolla, CA).
Measurement of NADPH oxidase activity
Superoxide production was measured in a quantitative kinetic
assay based on the reduction of cytochrome c. The assay was
performed at 37°C using a Thermomax microplate reader and associated
SOFTMAX version 2.02 software (Molecular Devices, Menlo Park, CA) as
reported previously (34, 35). Briefly, for the soluble
stimuli, cells were suspended (2.5 x 105
cells/well) in 250 µl of PBSG containing 75 µM ferricytochrome
c and activated by the addition of 200 ng/ml PMA or 10 µM
fMLP. For opsonized particle stimulation, cells were preincubated for
30 min at 37°C with 100 µg/ml opsonized zymosan (OpZ) or a 10-fold
excess of IgG-SRBC (2.5 x 106 IgG-SRBC in
0.25 ml for 2.5 x 105 BM neutrophils)
before adding PBSG containing ferricytochrome c.
FcR-activated oxidant production in BM neutrophils was also measured
using the Fc OxyBurst reagent (Molecular Probes, Eugene, OR), according
to the manufacturer’s instructions. Briefly, BM neutrophils were
suspended at 2 x 106/ml in PBSG and then
stimulated with Fc OxybBurst at a final concentration of 140 µg/ml.
Flow cytometric analysis was performed using a FACScan (Becton
Dickinson), and data were analyzed using CellQuest. For measurement of
nitroblue tetrazolium (NBT) reduction, neutrophils suspended in IMDM
were placed a glass chamber slide (Nunc, Naperville, IL) for 1 h
before activation with different stimuli. After an additional
incubation for 20 min at 37°C, slides were fixed and counterstained
with safranin, and the percentage of NBT-positive neutrophils and the
intensity of staining were determined by evaluating 100 randomly
selected cells (34).
MAP kinase activation
To examine the phosphorylation of MAP kinases, paired samples of wild-type and rac2-/- BM neutrophils were suspended in HBSS (1 x 107 cells/ml) and stimulated for varying periods of time with 200 ng/ml PMA, 10 µM fMLP, 100 µg/ml OpZ, or a 10-fold excess of IgG-SRBC at 37°C. The activation was terminated by addition of cold PBS and placement on ice. Thereafter, cells (1 x 107 cells/ml) were pelleted and lysed in 200 µl of lysis buffer at 4°C for 30 min. Cell lysates were clarified by centrifuging for 2 min at 18,000 x g at 4°C. Samples then were heated at 95°C for 5 min with 0.2 vol of 5x Laemmli sample buffer and 10 µg of cell lysate (10 µl for IgG-SRBC samples) resolved on 10% SDS-PAGE. Immunoblot analysis was conducted as described above. Expression of total p42/44, p38, JNK, and Akt was detected using rabbit polyclonal Abs against p42/44, p38, and Akt and a mouse mAb against JNK1/2. Activation of p42/44, p38, JNK1/2 MAP kinases, and Akt and tyrosine phosphorylation were determined using phospho-specific p42/p44, p38, JNK, Akt, and tyrosine Abs. Integrated densitometry was employed to measure the relative intensity of the protein signal using an Eagle Eye II Still Video System.
MAP kinase inhibitors
To investigate whether MAP kinase inhibitors affect superoxide production in murine BM neutrophils, the p38 MAP kinase inhibitor, SB203580 (Calbiochem, La Jolla, CA) and the MAP kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, PD98059 (Calbiochem), were used. BM neutrophils were incubated with SB203580 (1, 5, and 10 µM) or PD98059 (5, 25, and 50 µM) for 30 min at 37°C. Following incubation, cells were analyzed for cytochrome c superoxide production using the reduction assay and for MAP kinase activation in response to various stimuli as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has previously been shown that Rac2 is the predominant Rac
isoform in human neutrophils (22). We compared the
expression of Rac1 and Rac2 in mouse BM neutrophils to those in human
neutrophils by immunoblotting with specific Abs for Rac1 and Rac2. At
least three to five times more Rac1 was expressed in mouse neutrophils
on a milligram protein basis compared with human neutrophils, whereas
similar levels of Rac2 were detected in mouse and human neutrophils
(Fig. 1
). As previously reported
(11), the expression of Rac1 in murine
rac2-/- neutrophils was similar to that
in wild-type neutrophils.
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To examine the activation of the NADPH oxidase by physiological
agonists, three different receptor-mediated stimuli were tested.
Superoxide production in response to fMLP was 
10-fold higher in
freshly isolated wild-type BM neutrophils (2.5 ± 2.3
nmol/min/107 cells) than in those isolated from
rac2-/- mice (0.2 ± 0.7
nmol/min/107 cells; Fig. 2A). BM neutrophils were also
cultured with 1 ng/ml IL-3 for 24 h at 37°C to enhance
responsiveness to fMLP stimulation (36, 37) (Fig. 2B). fMLP-elicited superoxide production by IL-3-treated
wild-type BM neutrophils increased 2-fold compared with that by
freshly isolated BM neutrophils (p < 0.05),
but was still virtually undetectable in
rac2-/- cells. Consistent with previous
observations (11), PMA-elicited NADPH oxidase activity was
reduced by about 4-fold in freshly isolated
rac2-/- BM neutrophils (3.6 ± 1.2
nmol/min/107 cells) compared with wild-type
neutrophils (17.3 ± 3.8 nmol/min/107 cells)
(Fig. 2A). Culture in IL-3 resulted in a modest increase in
PMA-elicited superoxide production by BM neutrophils (22.5 ± 6.5
nmol/min/107 cells), although this was still
significantly reduced in cells isolated from
rac2-/- mice (9.6 ± 3.2
nmol/min/107 cells).
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). Superoxide
production elicited by IgG-SRBC was reduced by 5-fold in
rac2-/- BM neutrophils compared with
wild-type neutrophils (wild-type, 6.9 ± 2.5
nmol/min/107 cells;
rac2-/-, 1.5 ± 2.4
nmol/min/107 cells). The expression of FcR, as
measured by flow cytometry using an Ab against Fc III/II receptor
(CD16/CD32), was similar for both wild-type and
rac2-/- genotypes (data not shown). This
suggests that the significant impairment in activation of the NADPH
oxidase by IgG-SRBC in rac2-/- BM
neutrophils does not reflect reduced expression of Fc receptors. In
contrast, activation of the respiratory burst by serum OpZ, which can
also interact with the 
2 integrin CR3 receptor
for activated complement protein C3bi, was preserved in
rac2-/- BM neutrophils. There was no
difference in OpZ-induced superoxide release between genotypes
(wild-type, 7.3 ± 3.0 nmol/min/107 cells;
rac2-/-, 6.0
± 2.2 nmol/min/107 cells; Fig. 3).
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). Stimulation with fMLP did not induce
sufficient superoxide production that could be detected in the
cytochrome c assay in either wild-type or
rac2-/- peritoneal exudate cells
(data not shown). In contrast to our initial observation that
PMA-activated superoxide production was not significantly reduced in
rac2-/- peritoneal exudate neutrophils
(11), we found an 2-fold reduction in
rac2-/- peritoneal exudate neutrophils
compared with wild-type (wild type, 14.9 ± 2.4
nmol/min/107 cells;
rac2-/-, 7.7 ± 2.8
nmol/min/107 cells; p < 0.01).
This may reflect differences in the mouse genetic background. In the
original study, we used C57BL/6J x 129Sv
F2, F3, and
F4 littermate mice, whereas in the current study,
mice were in the C57BL/6J background.
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). However, the
relative numbers of NBT-positive cells detected upon activation with
either fMLP or IgG-SRBC were significantly decreased in
rac2-/- cells (Table I). Activation with
either PMA or OpZ produced intense cell-associated formazan deposits in
both wild-type and rac2-/- neutrophils,
which were concentrated in the phagosome in the case of OpZ-activated
neutrophils, whereas fMLP or IgG-SRBC stimulation produced less intense
formazan staining of cells (data not shown). Phagocytosis of
serum-opsonized zymosan particles was equivalent in wild-type and
rac2-/- neutrophils. The number of
particles ingested per 100 neutrophils was 131 ± 19 for wild-type
and 137 ± 26 for rac2-/-
(n = 5 for both genotypes). Ingestion of IgG-SRBC was
rarely seen for either wild-type or rac2-/-neutrophils, perhaps because the rabbit IgG used for
opsonization is a poor stimulus inducing phagocytosis in murine
neutrophils under our conditions.
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The decreased fraction of NBT-positive neutrophils in
rac2-/- mice in response to fMLP or
IgG-SRBC also indicates that reduced superoxide release measured in the
cytochrome c assay could reflect either fewer numbers of
activated rac2-/- neutrophils and/or
decreased neutrophil NADPH oxidase activity. To examine this question
directly for FcR-elicited NADPH oxidase activity, we used the Fc
OxyBurst reagent to monitor oxidant production by flow cytometry. As
shown in Fig. 3
B, incubation with Fc Oxyburst produced a
shift in fluorescence, corresponding to oxidant production, in a
subpopulation of BM neutrophils. The shift in fluorescence was
significantly decreased in rac2-/-
neutrophils compared with wild-type cells (666 ± 76 vs 1753
± 501 U after 30 min; n = 3–5; p =
0.011; Fig. 3C). The relative number of responding
neutrophils was also decreased in rac2-/-
mice, with 27 ± 9% of rac2-/-
cells exhibiting a shift in fluorescence by 30 min compared with
48 ± 25% for wild-type cells, although this difference was not
statistically significant. These results suggest that decreased
superoxide production by FcR-activated
rac2-/- neutrophils is due to both a
decrease in cellular oxidant production as well as a smaller proportion
of responding cells.
Activation of intracellular kinases in activated murine neutrophils
We previously found that phosphorylation of ERK1/2 and p38 MAP
kinases induced by fMLP was diminished in
rac2-/- BM neutrophils (11).
To expand on these initial observations, we compared
chemoattractant-activated MAP kinase activation in mouse BM neutrophils
to stimulation by PMA, OpZ, or IgG-SRBC, using Abs to detect the
corresponding phosphorylated form of the protein (Fig. 5). Preliminary studies (see also Fig. 6A) determined that
PMA-induced phosphorylation of ERK1/2 and p38 was initiated within 2
min, which increased further by 10 min. fMLP-induced
phosphorylation of ERK1/2 and p38 peaked at 2 min, with
phosphorylated ERK1/2 declining by 5 min (data not shown).
Phosphorylated p38 was detected 5 min after the addition of IgG-SRBC
and persisted for at least 60 min; we did not detect ERK1/2
phosphorylation in response to IgG-SRBC over this same period (data not
shown). In subsequent studies, we examined agonist-induced MAP kinase
protein phosphorylation at 2 min for fMLP, 10 min for PMA, and 30 min
for opsonized particles. As shown in the representative blots in Fig. 5, phosphorylation of ERK1/2 induced by either fMLP or PMA was markedly
reduced in rac2-/- BM neutrophils
compared with wild-type neutrophils. Only a small decrease in p38
phosphorylation was typically seen for fMLP or PMA-activated
rac2-/- BM neutrophils compared with
wild-type cells, although this difference was more prominent in some
experiments (compare Figs. 5 and 6A). Activation of p38 by
either OpZ or IgG-SRBC was similar in wild-type and
rac2-/- cells. Phosphorylation of JNK was
also detectable after stimulation with either PMA or fMLP, which was
consistently decreased in fMLP-activated
rac2-/- cells. Previous studies have
suggested that Rac is downstream of phosphoinositide-3 kinase (PI3K)
activation induced by chemoattractants and other signals (39, 40). In rac2-/- mast cells, PI3K
activation by stem cell factor is normal, but phosphorylation of
PI3-dependent kinase Akt (41) is decreased
(29). Phosphorylation of Akt was not decreased in
rac2-/- BM neutrophils stimulated with
either PMA or fMLP, and actually appeared to be increased in
fMLP-treated rac2-/- cells compared
with wild-type cells (Fig. 5), confirming our initial observations of
fMLP-activated neutrophils (11). Under the conditions
used, Akt phosphorylation was not observed in response to OpZ or
IgG-SRBC.
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We next investigated whether the decreased phosphorylation of
ERK1/2 or p38 observed in rac2-/- BM
neutrophils activated by fMLP or PMA was functionally linked to NADPH
oxidase activation. Studies in human neutrophils have found variable
results as to whether NADPH oxidase activity can be suppressed by
inhibiting phosphorylation of either ERK1/2 using the MEK-1 inhibitor
PD098059 or of p38 using SB203580 (42, 43, 44, 45, 46, 47). In initial
studies we established that phosphorylation of ERK1/2 or p38 could be
effectively inhibited by the corresponding inhibitor (Fig. 6A). However, PD098059 or SB203580 did not significantly
inhibit PMA-elicited superoxide production in either wild-type or
rac2-/- BM neutrophils, either when given
alone (Fig. 6B) or in combination (data not shown).
fMLP-induced superoxide production was diminished by about 30% in
wild-type BM neutrophils treated with either inhibitor, although this
reached statistical significance only for PD098059 (Fig. 6C). The residual superoxide-generating activity in
fMLP-activated rac2-/- BM neutrophils was
also partially sensitive to inhibition by PD098059 or SB203580. These
results suggest that the effect of Rac2 deficiency on activation of the
NADPH oxidase in murine neutrophils may be mediated largely through
non-MAP kinase-dependent pathways.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and, probably, Rac3 (18). In neutrophils,
the absence of Rac2 was associated with significant defects in
chemotaxis, L-selectin-mediated rolling, F-actin generation, and
phorbol ester-induced superoxide production. These defects were
reflected in vivo by baseline neutrophilia, reduced inflammatory
peritoneal exudate formation, and increased mortality infected with
Asperigillus fumigatus (11). The importance of
Rac2 in regulating neutrophil function has also been highlighted by the
recent discovery of a new genetic immunodeficiency syndrome in humans
due to a dominant negative mutation of Rac2 (48, 49).
Neutrophils isolated from the affected patient exhibited
functional abnormalities similar to those seen in
rac2-/- neutrophils, with defects in
chemotaxis and NADPH oxidase activation in response to some, but not
all, agonists along with diminished L-selectin-mediated adhesion.
Chemoattractant-induced superoxide production, chemotaxis, and
degranulation were markedly deficient (48, 49) as was
fMLP-primed phagocytosis of IgG-SRBC (48). However, only a
small decrease was observed in the phorbol ester-activated respiratory
burst (48, 49). In the current study, we examined receptor-mediated activation of the NADPH oxidase in rac2-/- neutrophils to further characterize the role of Rac2 in the regulation of neutrophil superoxide production. NADPH oxidase activity was normal in opsonized zymosan-activated rac2-/- neutrophils, but was deficient in response to fMLP or to IgG-opsonized particles despite the expression of Rac1 at substantially higher levels compared with human neutrophils. The agonist-specific defects observed in activation of the respiratory burst in murine rac2-/- neutrophils are consistent with those reported for human neutrophils expressing a dominant negative Rac2 mutant. We had previously observed that the impaired phorbol ester-induced activation of superoxide production in rac2-/- neutrophils could be partly surmounted by in vivo or in vitro priming with inflammatory cytokines (11). Taken together, these data show that the requirement for Rac2 in superoxide production is not absolute and suggest that specific signaling pathways leading to activation of the neutrophil respiratory burst are selectively regulated by Rac2.
There are at least two mechanisms by which the absence of Rac2 could lead to defects in neutrophil superoxide production. Rac is a component of the enzymatically active NADPH oxidase complex, and either Rac1 or Rac2 is required for enzyme activity in recombinant cell-free oxidase assays. The GTP-bound form of Rac can bind directly to p67phox and to at least one other site in the NADPH oxidase (5, 19, 50). Decreased respiratory burst activity in rac2-/- neutrophils might reflect a preferred role for Rac2 due either to increased affinity to one or more other subunits of the NADPH oxidase and subsequent enhanced enzymatic activity or to differences in activation or subcellular localization of Rac2 compared with Rac1. Alternatively, Rac2 deficiency may lead to signaling abnormalities that affect phosphorylation of p47phox and subsequent translocation of p47phox/p67phox/p40phox and/or recruitment of Rac-GTP to the enzyme complex. At the current time it is unknown whether the predominant effect of Rac2 deficiency on the respiratory burst reflects its function in the NADPH oxidase complex itself or in the preceding steps leading to enzyme assembly. However, since multiple functional abnormalities in other BM-derived cells have been identified in rac2-/- mice (11, 29, 30), we speculate that defects in intracellular signaling pathways account for at least some of the observed decrease in superoxide production in response to specific agonists.
Why Rac1 is unable to substitute for Rac2 in superoxide production, chemotaxis, and other functional responses that are impaired in Rac2-deficient blood cells also remains to be defined. These two isoforms may be selectively activated by specific exchange factors or interact differentially with downstream effectors. For example, the Rac-GEF Tiam-1 (51) is a strong inducer of p21-activated kinase 1 activation, but not of JNK (52), two kinases that are both strongly activated by Rac-GTP. This selectivity could arise either through differences in subcellular localization or in relative affinities for different Rac isoforms with interacting proteins. For example, Rac1 has been shown to bind to and stimulate p21-activated kinase 1 activity more efficiently that Rac2 (53). In addition, studies using the yeast two-hybrid system have suggested that the affinity of Rac2 for p67phox is higher than that for Rac1 (19), and Rac2 is more active than Rac1 in stimulating NADPH oxidase activity in the presence of neutrophil cytosol (28). Another potential mechanism for functional defects in rac2-/- cells is that the overall level of Rac-GTP might be a rate-limiting step in the activation of downstream functions, and signaling cascades triggered by different neutrophil agonists may vary in the amount of activated Rac generated.
Rac has been implicated as a participant in many of the multiple signaling pathways that are triggered upon neutrophil activation by soluble or particulate agonists (1, 39, 54, 55). How these complex and often redundant signals are ultimately linked to specific functional responses, such as activation of the otherwise dormant NADPH oxidase, remains incompletely understood. However, from studies using biochemical, pharmacologic, or genetic approaches, it is clear that different agonists activate both overlapping and distinct signaling cascades (1, 39, 54, 56, 57, 58, 59) that could potentially lead to selective activation of Rac2 and the respiratory burst.
Signaling defects downstream of heterotrimeric G protein-coupled
receptors for chemoattractants are a prominent feature of Rac2
deficiency. We previously reported that that chemotaxis and F-actin
polymerization in response to fMLP or leukotriene B4 was substantially
diminished in rac2-/- neutrophils
(11) and now show that fMLP-induced activation of
superoxide production also significantly impaired. These features of
the rac2-/- phenotype have some overlap
with mice deficient in the p110 catalytic subunit of PI3K
(57, 58, 59). PI3K-deficient leukocytes are unable to
generate phosphatidyl-inositol 3,4,5-trisphosphate upon G
protein-coupled receptor stimulation, in association with deficient
chemoattractant-induced chemotaxis and NADPH oxidase activity.
fMLP-induced Rac activation was reported to be unaffected in
PI3K-deficient mice (58). This suggests that
PI3K-dependent signaling and a parallel pathway(s) linked to Rac2
activation are both required for inducing a respiratory burst by fMLP
and related agonists.
The rac2-/- neutrophils also exhibited a
defect in NADPH oxidase activation upon activation through FcR using
Ig-opsonized particles, but not with zymosan opsonized with serum
opsonins (C3bi, IgG), which can bind to both the
2 integrin receptor CR3 and FcR
(60, 61, 62, 63, 64). This is the first direct evidence that Rac2
plays an important role in regulating the FcR-dependent respiratory
burst in neutrophils, which is likely to occur downstream of the
activation of receptor-associated tyrosine kinases, including Syk and
Hck, that are prominent proximal events upon FcR ligation
(65). Our data also indicate that signals induced either
downstream of the CR3 receptor or through CR3 costimulation of FcRs
(66) are sufficient to activate NADPH oxidase
independently of Rac2 in murine neutrophils. Signaling through Rho
GTPases has previously been shown to play an important role in the
phagocytosis of opsonized particles by macrophages, and different Rho
family members appear to play distinctive roles in this process
(10, 65, 67). In macrophages, Cdc42 and Rac regulate
uptake of Ig-opsonized particles via FcR, whereas Rho plays a
prominent role in CR3-mediated phagocytosis (67). Our
findings that superoxide production in
rac2-/- neutrophils is impaired in
response to FcR ligation but not when CR3 binding is also involved
are consistent with these observations.
Although PMA is not a physiologic stimulus for neutrophil activation,
it is noteworthy that PMA-elicited superoxide production was
substantially decreased in rac2-/-
neutrophils. PMA is a direct activator of protein kinase C (PKC) and is
one the most potent activators of the neutrophil respiratory burst.
Neutrophil activation by most physiologic agonists is accompanied by
activation of PKC, and specific inhibitors of PKC decrease NADPH
oxidase activity (reviewed in Refs. 54 and
55). An early event downstream of agonist binding to
chemoattractant or Fc receptors is the activation of phospholipase
C, which catalyzes the formation of diacylglycerol (DAG) and inositol
1,4,5-trisphosphate by hydrolysis of membrane phospholipids
(54). In turn, DAG directly activates PKC, and inositol
1,4,5-trisphosphate promotes the release of intracellular calcium,
which is important for activation of many PKC isoforms, including the
PKC- and -
species present in neutrophils (54, 55).
DAG can also be generated through a parallel pathway involving
activation of phospholipase D. Direct evidence for PKC- regulation
of NADPH oxidase activity was found in a recent study in which PKC-
activity was ablated either using a specific inhibitor or genetically
using a knockout mouse lacking PKC- (68). In either
case, an 2-fold reduction in neutrophil superoxide production
elicited by IgG-coated particles or PMA was observed. Inhibition of
PKC- expression in HL-60 cells using antisense oligonucleotides also
resulted in decreased NADPH oxidase activity (69). The
deficient respiratory burst in rac2-/-
neutrophils in response to PMA, fMLP, or IgG-opsonized particles may
reflect a common defect in Rac2-dependent events downstream of PKC that
promote oxidase assembly.
We hypothesized that the differential activation of MAP kinase cascades might explain the agonist-dependent effects on neutrophil NADPH oxidase activation in murine rac2-/- neutrophils. MAP kinase activation is a prominent response in human neutrophils activated by either soluble agonists or opsonized particles (54, 70, 71). ERK2/1 (p42/44) MAP kinase is activated via the Ras/Raf pathway, although cross-regulation with Rac-dependent pathways can also occur (72, 73). Many groups have reported that JNK and p38 MAP kinase pathways are downstream of Rac and Cdc42 in various cell types, suggesting an analogous role to that played by Ras in the ERK MAP kinase cascade (12, 13, 17, 74, 75).
We found that both fMLP and PMA induced phosphorylation of ERK1/2 and
p38 in murine BM neutrophils, which was decreased in
rac2-/- cells compared with wild-type
cells, particularly for ERK1/2. However, activation of these MAP
kinases appears to be only in part functionally linked to downstream
activation of superoxide production. Pharmacologic inhibition of either
ERK1/2 or p38 phosphorylation had no significant effect on PMA-induced
superoxide production in wild-type BM neutrophils. Hence, although PMA
activated both ERK1/2 and p38 in murine BM neutrophils in a
Rac2-dependent manner, these events do not play a role in NADPH oxidase
activation and are unlikely to account for the defect in superoxide
generation in PMA-activated rac2-/-
neutrophils. These results are generally consistent with observations
in human neutrophils, where inhibitors of either ERK1/2 or p38 have had
variable results on PMA-induced superoxide production (42, 43, 45, 46, 47). With regard to the fMLP-activated respiratory burst,
inhibition of ERK1/2 has also had inconsistent effects in human
neutrophils (43, 44, 45, 76), whereas at least a partial
decrease in NADPH oxidase activity has been reported in the presence of
p38 inhibitors (42, 43, 47). In fMLP-activated wild-type
murine BM neutrophils, we found that inhibition of either ERK1/2 or p38
produced only an 30% decrease in NADPH oxidase activity. The
residual respiratory burst detected in fMLP-stimulated
rac2-/- BM neutrophils exhibited a
similar partial sensitivity to p38 and ERK1/2 inhibitors. These data
suggest that events downstream of ERK1/2 and p38 regulate the
fMLP-activated respiratory burst in murine BM neutrophils, but that the
absence of Rac2 is likely to affect additional or redundant
fMLP-induced signaling pathways important for NADPH oxidase activation.
In contrast to studies on human neutrophils (76, 77, 78) or
murine macrophages (79, 80), we were unable to detect
phosphorylated ERK1/2 in murine BM neutrophils stimulated with
opsonized particles. However, p38 MAP kinase was activated with either
OpZ or IgG-SRBC, but to a similar extent in wild-type and
rac2-/- cells. Therefore, although
IgG-SRBC-induced superoxide release was markedly decreased in
rac2-/- neutrophils, this effect appears
to mediated by downstream pathways unrelated to p38.
We also observed activation of JNK in murine BM neutrophils in response to either fMLP or PMA, which was decreased in fMLP-activated rac2-/- neutrophils. Our data agree with previous reports that formyl receptors in neutrophils can couple to the JNK pathway (44, 58) and are also consistent with the important role for Rac/Cdc42 in pathways leading to JNK activation (12, 13). The failure to detect neutrophil JNK activation in response to fMLP in our earlier study (11) may reflect the use of a less sensitive Ab for detecting the phosphorylated form of JNK MAP kinase. The functional consequences of defective JNK activation in rac2-/- neutrophils are currently unknown. However, JNK is believed to play a role in the activation of stress-induced gene expression and apoptosis (12, 13).
In conclusion, the present study provides direct genetic evidence that
Rac2 is a critical regulator of specific signaling pathways that
activate the neutrophil NADPH oxidase, particularly those downstream of
chemoattractant receptors and Fc receptors, despite the concomitant
presence of the highly homologous Rac1 isoform. The effect of Rac2
deficiency on NADPH oxidase activity is probably exerted through
multiple pathways, including those independent of MAP kinase
activation. Ongoing studies are directed at further identifying these
pathways and the specific Rac2 sequences important for regulation of
NADPH oxidase activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary C. Dinauer, Cancer Research Institute R4, Indiana University School of Medicine, 1044 West Walnut Street, Room 402A, Indianapolis, IN 46202.
3 Abbreviations used in this paper: BM, bone marrow; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; HBSS-BG, HBSS with 0.1% BSA and 1% glucose (pH 7.25–7.4); PBSG, PBS with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose; OpZ, opsonized zymosan; IgG-SRBC, Ab-coated SRBC; NBT, nitroblue tetrazolium; MEK-1, MAP kinase kinase-1; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide-3 kinase; DAG, diacylglycerol.
Received for publication August 17, 2000. Accepted for publication October 25, 2000.
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Y. Gu, M. C. Byrne, N. C. Paranavitana, B. Aronow, J. E. Siefring, M. D'Souza, H. F. Horton, L. A. Quilliam, and D. A. Williams Rac2, a Hematopoiesis-Specific Rho GTPase, Specifically Regulates Mast Cell Protease Gene Expression in Bone Marrow-Derived Mast Cells Mol. Cell. Biol., November 1, 2002; 22(21): 7645 - 7657. [Abstract] [Full Text] [PDF]
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S. Li, A. Yamauchi, C. C. Marchal, J. K. Molitoris, L. A. Quilliam, and M. C. Dinauer Chemoattractant-Stimulated Rac Activation in Wild-Type and Rac2-Deficient Murine Neutrophils: Preferential Activation of Rac2 and Rac2 Gene Dosage Effect on Neutrophil Functions J. Immunol., November 1, 2002; 169(9): 5043 - 5051. [Abstract] [Full Text] [PDF]
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M. O. Price, S. J. Atkinson, U. G. Knaus, and M. C. Dinauer Rac Activation Induces NADPH Oxidase Activity in Transgenic COSphox Cells, and the Level of Superoxide Production Is Exchange Factor-dependent J. Biol. Chem., May 17, 2002; 277(21): 19220 - 19228. [Abstract] [Full Text] [PDF]
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M. O. Price, L. C. McPhail, J. D. Lambeth, C.-H. Han, U. G. Knaus, and M. C. Dinauer Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system Blood, April 15, 2002; 99(8): 2653 - 2661. [Abstract] [Full Text] [PDF]
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T. Adachi, R. Vita, S. Sannohe, S. Stafford, R. Alam, H. Kayaba, and J. Chihara The Functional Role of Rho and Rho-Associated Coiled-Coil Forming Protein Kinase in Eotaxin Signaling of Eosinophils J. Immunol., October 15, 2001; 167(8): 4609 - 4615. [Abstract] [Full Text] [PDF]
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,
Wild type; 
, rac2-/-. Data are
expressed as the mean ± SD. *, p < 0.001,
wild type vs rac2-/- (unpaired
t test). A, Superoxide production by
freshly isolated BM neutrophils following stimulation with 200 ng/ml
PMA or 10 µM fMLP was monitored by reduction of ferricytochrome
c (n = 8). B,
Superoxide production by BM neutrophils cultured with 1 ng/ml IL-3 for
24 h at 37°C before stimulation with either PMA or fMLP
(n = 11).
