Neutrophil direction sensing and superoxideproduction linked by the GTPase-activatingprotein GIT2

Yuichi Mazaki1, Shigeru Hashimoto1, Tohru Tsujimura2, Masaki Morishige1,3, Ari Hashimoto1,Kosuke Aritake4, Atsuko Yamada1, Jin-Min Nam1, Hiroshi Kiyonari5, Kazuki Nakao5 & Hisataka Sabe1,6

In neutrophils, superoxide anion production generally accompanies chemotaxis and functions in killing invading pathogens. The

GIT2 GTPase-activating protein binds to the guanine nucleotide–exchange factor aPIX. Here we show that GIT2 was necessary

for directional chemotaxis and for the suppression of superoxide production in G protein–coupled receptor–stimulated

neutrophils. GIT2 was also necessary for the orientation of superoxide production toward chemoattractant sources. GIT2

suppressed the activity of ADP ribosylation factor 1 and was a component of the Gbc subunit–mediated direction-sensing

machinery ‘downstream’ of G protein–coupled receptor signaling. This study establishes a function for GIT2 in linking

chemotaxis and superoxide production in neutrophils and shows that loss of GIT2 in vivo leads to an immunodeficient state.

Neutrophils are important in innate immunity and in the initiation ofan acute response to infection. During such a response, neutrophilsare activated, move toward the site of inflammation and activelyproduce antimicrobial agents, including many reactive oxygen species(ROS). These ROS are first produced as superoxide anions by NADPHoxidase and are essential for the killing of invaders after neutrophilphagocytosis1. Neutrophil production of superoxide anions is tightlycoupled to the activation state of neutrophils. Strict suppression ofsuperoxide production in resting neutrophils is essential for theprevention of nonspecific injury to blood vessel endothelial cells,tissues and organs. Moreover, the direction of superoxide productionis regulated to coincide with the direction of chemotaxis, which allowsmore efficient recognition and killing of targets.

Most chemoattractants, including bacterial products, complementfragments and chemokines, bind to cell surface receptors linked to theGai family of heterotrimeric G proteins (G protein–coupled receptors(GPCRs))2. Gbg subunits, which are released after activation ofheterotrimeric G proteins by GPCR stimulation, bind to p21-activating protein kinase 1 (PAK1), which simultaneously binds toaPIX, a Dbl family Rac (small GTPase) and Cdc42 (GTP-binding Rhofamily protein) guanine nucleotide–exchange factor; thus, a linearcomplex of Gbg-PAK1-aPIX is formed3. This Gbg-PAK1-aPIXcomplex is pivotal in direction sensing and the persistence ofGPCR-stimulated neutrophils3. PAK1, Rac and Cdc42 also influenceactin cytoskeletal organization4.

NADPH oxidase consists of five unique subunits: two (p22phox andgp91phox) are membrane bound and three (p40phox, p47phox andp67phox) are located in the cytosol1. The assembly of several otherproteins, such as Rac2, is necessary for its oxidase activity1. AfterGPCR stimulation, all five subunits localize to the plasma membranefor their final assembly and activation. In addition to Rac2, the activityof other small GTPases, such as ADP ribosylation factors (Arfs), hasbeen linked to oxidase activation in neutrophils, although their precisefunctions remain unclear5,6.

GIT proteins are GTPase-activating proteins that regulate Arf familyGTPases. GIT1 and GIT2 are structurally conserved7,8. In additionto negatively regulating Arf activity, GIT proteins are thought to beinvolved in linking the regulation of Arf proteins with other intracel-lular signaling events9. Of note, the Spa2 homology domains of GITproteins bind to the GIT-binding domain of aPIX7,8,10.

Although the molecular mechanisms underlying both chemotaxisand superoxide anion production in neutrophils have been studiedextensively, the molecules and mechanisms that connect those twoprocesses remain mostly unknown. Here we demonstrate that mouseneutrophils express GIT2 but not GIT1. We generated GIT2-deficientmice and found that GIT2 is essential for neutrophil function in vivo.Loss of GIT2 in neutrophils resulted in impaired GPCR-inducedchemotactic direction sensing and superoxide ‘hyperproduction’.Moreover, the direction of superoxide production in GIT2-deficientneutrophils was not oriented toward the chemoattractant source.

Received 5 December 2005; accepted 24 April 2006; published online 21 May 2006; corrected online 2 June 2006 (details online); doi:10.1038/ni1349

1Department of Molecular Biology, Osaka Bioscience Institute, Suita 565-0874, Japan. 2Department of Pathology, Hyogo College of Medicine, Nishinomiya 663-8501,Japan. 3Department of Neurosurgery, School of Medicine, Oita University, Oita 879-5593, Japan. 4Department of Molecular Behavioral Biology, Osaka BioscienceInstitute, Suita 565-0874, Japan. 5Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN Kobe, Kobe 650-0047, Japan.6Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. Correspondence should be addressed to H.S. (sabe@obi.or.jp).

724 VOLUME 7 NUMBER 7 JULY 2006 NATURE IMMUNOLOGY

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

In GIT2-deficient neutrophils, Arf1 was hyperactivated and Rac1 washypoactivated. Production of phosphatidylinositol 3,4,5-triphosphate(PIP3) and activation of PAK1 and the serine-threonine kinase Aktwere defective, but F-actin assembly was not impaired. We also foundthat GIT2 acted as a component of the Gbg-mediated direction-sensing machinery and was necessary for polarized recruitment andaccumulation of this direction-sensing machinery to the leading edgeduring chemotaxis. These data establish GIT2 as a key participantregulating neutrophil function and, hence, innate immunity.

RESULTS

Immunodeficiency of GIT2-deficient mice

We disrupted Git2 in mice by homologous recombination in embryo-nic stem cells (Supplementary Fig. 1 online). We used Southern blotanalysis of SphI-digested genomic DNA (probe, SupplementaryFig. 1) to ensure proper targeting (Supplementary Fig. 1). Git2–/–

mice were born at the expected mendelian ratio and were fertile, butoften developed splenomegaly (Fig. 1a). Histochemical analysis sug-gested the occurrence of extramedullary hematopoiesis, generatingmainly granulocytes in the red pulp of abnormally enlarged spleensand increasing granulocyte production in the bone marrow of Git2–/–

mice with splenomegaly (Fig. 1b). The lungs of Git2–/– mice withsplenomegaly were firm and stiff and were infiltrated by manylymphocytes, similar to that noted with pneumonia. However, fewneutrophils infiltrated the lungs of Git2–/– mice (Fig. 1b) and therewere no substantial differences in the numbers of neutrophils andother hematopoietic cells in peripheral blood of apparently healthyGit2–/– mice without splenomegaly and their Git2+/– littermates(Supplementary Table 1 online).

Those results suggested that Git2–/– mice might be highly suscep-tible to infection, which may occur sporadically with some pathogenseven in specific pathogen–free housing conditions. Neutrophils arevery important for the immunological defense against fungal infec-tion11. We therefore infected mice by intranasal injection of Aspergillusfumigatus conidia and found more fungi in the lungs of Git2–/– micethan Git2+/– mice (Fig. 1c). Histological analysis showed the presenceof ‘hyperinflammation’ in infected lungs of Git2–/– mice and thepresence of more fungal elements and infiltration of more lympho-cytes and macrophages, but not neutrophils, in infected lungs of

Git2–/– mice than Git2+/– mice (Fig. 1d). Therefore, although granu-locytes are actively produced in Git2–/– mice, their neutrophil progenydo not efficiently reach peripheral inflammatory regions, and it islikely that such defective neutrophil function in Git2–/– mice con-tributes to their high susceptibility to infection.

GIT2 in neutrophil chemotaxis

In contrast to other tissues and cell types, which constitutively expressboth GIT1 and GIT2, neutrophils expressed exclusively GIT2 (Fig. 2a).Git2–/– neutrophils did not express GIT2 protein (Fig. 2b). Toinvestigate the function of GIT2 in inflammatory responses, wemeasured agonist-induced neutrophil infiltration into subcutaneousair pouches. The accumulation of neutrophils in air pouches duringthe 4-hour interval after the injection of interleukin 8 (IL-8) into theair pouches was less in Git2–/– mice than in Git2+/– mice (Fig. 3a). Wethen prepared freshly isolated bone marrow neutrophils and investi-gated their chemotactic properties in vitro. We first used transwellchemotaxis assays to assess direction sensing12. In this assay, twochambers were separated by a filter with a pore size of 3 mm; itsunderside was soaked in chemoattractant medium. We placed neu-trophils in the top chamber and allowed them to migrate to the bottomchamber. Similar to wild-type neutrophils13,14, Git2+/– neutrophilsshowed a 2.5- to 3.5-fold increase in migratory activity in responseto various chemoattractants, such as a formyl peptide (N-formyl-Met-Leu-Phe (fMLP)), C5a and IL-8 (Fig. 3b). However, such responseswere considerably impaired in Git2–/– neutrophils: there was augmen-tation of only about 1.6-fold in response to these chemoattractants(Fig. 3b). Notably, neutrophils deficient in phosphatidylinositol-3-OHkinase-g (PI(3)K-g) show similarly impaired but not completelyblocked transwell chemotactic activity13–15. We next investigated two-dimensional chemotaxis using Zigmond chambers, in which cells wereplaced in a chemoattractant gradient16 (Fig. 3c). Like wild-typeneutrophils3, Git2+/– neutrophils moved up the fMLP gradient sothat about 83% of the cells ended up within a 1201 arc facing thefMLP source. However, only about 43% of Git2–/– neutrophilsmigrated to positions within the 1201 arc. Tracing the migration trackssuggested that the reduction was not due to changes in migration ratesbut instead was mainly due to a reduction in directional persistence(Fig. 3c). However, complete loss of GIT2 did not cause a complete

a b

c d

250

200

150

100

Wei

ght o

f spl

een

(mg)

50

0

0

5

10

15

CF

U (

× 10

3 )

Git2+/–

+/– –/–

Git2–/–

Git2+/–

Git2–/–

Git2

–/–

Git2

+/–

Git2

–/–

Git2

+/–

Spleen Bone marrow LungFigure 1 Immune effects of Git2 deficiency.

(a) Splenomegaly in Git2–/– mice. Top, weights

of the spleens of 10- to 13-week-old mice

(n ¼ 15 for each genotype (horizontal axis); small

horizontal bars indicate median values). Bottom,

representative photographs of spleens. Scale bar,

1 cm. (b) Staining of tissue sections from 12-

week-old mice (genotypes, left margin) withhematoxylin and eosin (spleen, left column, and

bone marrow and lung) or with a granulocyte-

specific antibody (anti-Gr-1; spleen, right

column). (c,d) Susceptibility to fungal infection.

Mice were injected intranasally with aspergillus

conidia for 3 consecutive days and fungal growth

was assessed 3 d after the last infection.

(c) Quantification of fungal growth in the lungs

(n ¼ 10 for each genotype). CFU, colony-forming

units. Error bars, s.e.m. (d) Staining of infected

lungs with hematoxylin and eosin (left) and

Gomori methanamine silver stain (right). Scale

bars (b,d), 50 mm. Images (b,d) represent more

than ten mice of each genotype.

NATURE IMMUNOLOGY VOLUME 7 NUMBER 7 JULY 2006 725

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

loss of directionality, as completely randomly migrating cells wouldshow a value of 33% in the 1201 arc. In contrast, adhesion to fibrinogenwas not altered in Git2–/– neutrophils and could be augmented byfMLP (Fig. 3d). Therefore, GIT2 is very important but not absolutelyessential for direction sensing and persistency, similar to PI(3)K-g,during GPCR-mediated chemotaxis of neutrophils. GIT2 may bedispensable for neutrophil adhesion and migration.

Gbc-PAK1-aPIX-GIT2 complex

GIT2 binds strongly to aPIX7. We next investigated whether GIT2 actsas a component of the Gbg-PAK1-aPIX direction-sensing machinery3.We expressed a glutathione S-transferase (GST) fusion form of theGIT2 protein in Cos-7 cells, together with Gb1, Gg2, PAK1 and aPIX.We first confirmed that Gb1g2, PAK1 and aPIX form a linear complexin this order (data not shown and Fig. 4a), as reported before3. Wefound that Gb1 (and hence also Gg2) precipitated together with GST-GIT2 only in the presence of both PAK1 and aPIX (Fig. 4a). There-fore, GIT2 is a component of the Gbg-signal-ing complex and forms a linear complex ofGbg-PAK1-aPIX-GIT2. This complex seemsto be analogous to the PAK1-aPIX-GIT1(PKL) complex formed ‘downstream’ ofT cell receptor engagement, which is neces-sary for immunological synapse function17.

GIT2 in Gbc polarization

Gbg-signaling molecules are recruited to theleading edge of neutrophils during chemo-taxis, where polarized F-actin assemblyoccurs3. We noted such polarized recruitmentof endogenous Gb, PAK1, aPIX and GIT2 inGit2+/– but not Git2–/– neutrophils placed inan fMLP gradient (Fig. 4b). Therefore, GIT2is a component of the Gbg-signaling machin-ery and is necessary for the polarized recruit-ment of the Gbg-signaling machinery to theF-actin assembled leading edge in GPCR-stimulated neutrophils.

GIT2 in polarization of F-actin assembly

In the experiments reported above, we notedthat well polarized F-actin assembly stilloccurred in Git2–/– neutrophils stimulatedby fMLP (Fig. 4b). Measurement of thetotal amount of cellular F-actin assembled

after fMLP stimulation also showed no notable differences in Git2–/–

versus Git2+/– neutrophils (Fig. 4c). We then examined the peripheralsites of F-actin assembly, with regard to the direction of the chemoat-tractant source, in neutrophils placed in a chemoattractant gradient.We found that although about 80% of Git2+/– neutrophils showedperipheral F-actin assembly facing the fMLP source, only about 51%of Git2–/– neutrophils showed such F-actin assembly facing the source(Fig. 4b). Therefore, although Git2–/– neutrophils retain the ability topolymerize F-actin and form the leading edge, orientation of leadingedges toward the direction of the chemoattractant source seems to bemostly impaired in the absence of GIT2. Neutrophils generally migratetoward the direction of F-actin assembled leading edges. These resultsare therefore consistent with our results reported above showingimpaired direction sensing and persistence of Git2–/– neutrophils.

Defective PAK1 activation in Git2–/– neutrophils

PAK1 is activated by GPCR stimulation in neutrophils3,4. In areconstitution experiment using Cos-7 cells, PAK1 was activatedwhen Gbg, aPIX and Cdc42 were overexpressed3. We next measuredPAK1 activation in Git2–/– neutrophils. Phosphorylation of PAK1stimulated by fMLP was mostly impaired, although not completelyblocked, in Git2–/– neutrophils, in contrast to normal phosphorylationof PAK1 in Git2+/– neutrophils (Fig. 4d). Therefore, GIT2 is requiredfor the efficient activation of PAK1 in neutrophils. As shown above,GIT2 was necessary for the polarized recruitment of Gbg-signalingmolecules to the leading edge. It is thus conceivable that such anaccumulation of Gbg-signaling molecules, including PAK1 itself, byGIT2 is necessary for the efficient activation of PAK1 in GPCR-stimulated neutrophils.

GIT2 in PIP3 production and Akt activation

PIP3, a product of PI(3)K, is essential for the chemotactic direction-ality of GPCR-stimulated neutrophils18–20. PIP3 is produced and

a bEmbryo Adult tissue

Git1

Git2

Gapdh

Peripheralleukocyte

9.5

dpc

11.5

dpc

14.5

dpc

Brain

Live

rM

uscle

Splee

nKid

ney

Heart

Test

isLu

ng +/– –/–

Anti-GIT2

Thym

usLy

mph

ocyt

eNeu

troph

ilM

acro

phag

e

Figure 2 Expression of Git1 and Git2. (a) RT-PCR analysis of Git1,

Git2 and Gapdh (glyceraldehyde phosphate dehydrogenase) expression

in various tissues (above lanes) of C57BL/6 mice. dpc, days after coitus.

(b) GIT2 protein expression in mouse bone marrow neutrophils; genotype,

above lanes: +/–, Git2+/–; –/–, Git2–/–. Data are representative of

three experiments.

a

c

b dGit2+/–

Git2–/–

Git2+/– Git2–/–

Git2+/–

Git2–/–Git2+/–

Git2–/–

Git2+/–

Git2–/–Git2+/–

Git2–/–

8

Neu

trop

hils

/pou

ch (

× 10

6 )

Che

mot

axis

(%

of c

ontr

ol)

Adh

eren

t cel

ls (

%)

Tim

e in

terv

al (

s)

Spe

ed (

µm/m

in)

6

4

2

0

200 200

fMLP fMLP

100 200 200100 100100

100 100

100100

None fMLP None fMLPC5a IL-80

82.5 ± 4.8% 43.4 ± 3.8%

50

0

0 0

2

4

6

8

10

100

200

300

400

20

40

60

80

100

150

200

250

300

350

400

Figure 3 Git2 in neutrophil chemotaxis. (a) Air pouch assay. Neutrophil numbers in exudates isolated

4 h after injection of IL-8 into subcutaneous dorsal air pouches (n ¼ 5 mice). (b) Transwell chemotaxis

assay. Neutrophil migration in response to fMLP, C5a or IL-8, measured for 90 min in transwell cham-

bers (n ¼ 11 wells for each stimulus). Basal activity of Git2+/– neutrophils is considered 100%. (c) Zig-

mond chamber assay. Left (scatterplots), positions of cells relative to their original positions after 20 minof migration in an fMLP gradient (single experiment). Below, percentage of cells that ended up within a

1201 arc facing the fMLP source. Right, directional persistency, calculated as time interval between

direction changes (second from right); and migration rates (far right). More than 25 cells were assigned

scores in four independent experiments. (d) Cell adhesion assay. Neutrophils were placed onto fibrinogen-

coated dishes for 60 min with (fMLP) or without (None) fMLP and the percentage of adherent cells

was determined (n ¼ 6 wells). Error bars, s.e.m. Data in a,b,d are representative of three experiments.

726 VOLUME 7 NUMBER 7 JULY 2006 NATURE IMMUNOLOGY

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

accumulates at the leading edge in GPCR-stimulated neutrophils. Wefound that PIP3 production and accumulation at the leading edgewere mostly lost in Git2–/– neutrophils, in contrast to the normal PIP3

production and accumulation in Git2+/– neutrophils (Fig. 4b,e). Akt isknown to be activated and hence autophosphorylated through thebinding of PIP3 to its pleckstrin homology domain21. We found thatAkt autophosphorylation and recruitment of activated Akt to theleading edge were also substantially lost in fMLP-stimulated Git2–/–

neutrophils (Fig. 4b,d). These results indicate that GIT2 is necessaryfor the efficient production of PIP3 and activation of Akt in GPCR-stimulated neutrophils. Gbg can activate the p110g catalytic subunitof PI(3)K by direct interaction22,23. Therefore, our results also suggest

that Gbg itself is not enough for the efficient activation of PI(3)K inneutrophils and that GIT2 may also be necessary.

Superoxide anion hyperproduction in Git2–/– neutrophils

We next examined superoxide anion production in bone marrowneutrophils (Fig. 5a). In contrast to the reduction in chemotacticactivity, we found that GPCR-mediated superoxide production wasenhanced in Git2–/– neutrophils compared with that in Git2+/–

neutrophils. There was more superoxide production in Git2–/– neu-trophils, even in the absence of any stimuli (Fig. 5a). Brefeldin A(BFA) inhibits the activities of Arf proteins24 and has been shown tosuppress fMLP-induced superoxide production6. BFA also suppressed

Gβ

αPIXPAK1 Precipitation

Total

–fMLP +fMLP

0None fMLP

50

F-a

ctin

form

atio

n (%

)

100

0 10 40Time (s)150 Git2

+/–

Git2–/–

Git2+/–

Actin Merged Actin Merged Actin Merged Actin

Gra

dien

t

Merged

Git2–/– Git2+/– Git2–/–

Git2+/–

Git2–/–

Git2GST-GIT2:

Gβγ:

αPIX:PAK1:

Gβ

Gβ

αPIXPAK1

PAK1

GIT2

αPIX

PIP3

p-Akt

p22

GIT2

p-PAK1

PAK1

p-Akt

Akt Rel

ativ

e P

IP3

(%

)

0

100

200

300

400

500

None fMLP

–– –– – –

– –++

+ ++ ++ + +

– ++

+/– –/– +/– –/––/–+/–

a

b

c d e

Figure 4 Impaired GPCR-coupled direction sensing in Git2–/– neutrophils. (a) Precipitation and immunoblot analysis of complex formation. Cos-7 cells were

transfected with histidine-tagged GST-GIT2, Gbg, aPIX and PAK1; precipitation together with GST-GIT2 was analyzed by immunoblot with antibody to the

Express tag. Bottom, protein expression in total cell lysates. Representative of two experiments. (b) Microscopy of neutrophils left unstimulated or placed

in an fMLP gradient in Zigmond chambers for 15 min, after which they were fixed and stained with antibodies specific for various proteins (left margin).

F-actin was visualized by fluorescence-labeled phalloidin. p-Akt, phosphorylated Akt. Images represent more than 20 cells examined in three independent

experiments. Scale bar, 10 mm. Percentage of neutrophils in which most peripheral F-actin assembled within a 1201 arc facing the fMLP source: Git2+/–,

80.2 ± 8.2; Git2–/–, 50.9 ± 5.5. (c) F-actin assembly in neutrophils with (fMLP) or without (None) stimulation for 30 s with fMLP. Data are representative

of three experiments. (d) Immunoblot of Akt and PAK1 phosphorylation (p-) in neutrophils stimulated with fMLP (time, above blots). Representative of three

independent experiments. (e) Flow cytometry measuring PIP3 in neutrophils with (fMLP) or without (None) stimulation for 10 s with fMLP. Error bars,

s.e.m.; data are representative of three experiments. In c,e, values for Git2+/– neutrophils without stimulation are considered 100%.

NATURE IMMUNOLOGY VOLUME 7 NUMBER 7 JULY 2006 727

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

superoxide production in Git2–/– and Git2+/– neutrophils to basalamounts (Fig. 5a). In contrast, phorbol 12-myristate 13-acetate, adirect protein kinase C activator, induces superoxide production inneutrophils independently of GPCR-stimulation13,15. Phorbol 12-myristate 13-acetate induced equivalent superoxide production inGit2–/– and Git2+/– neutrophils (Fig. 5a). We also investigated super-oxide production coupled to phagocytosis. Phagocytosis of immuno-globulin G (IgG)–opsonized zymosan was unaffected in both Git2–/–

and Git2+/– neutrophils, whereas there was hyperproduction of super-oxide anions in Git2–/– neutrophils (Fig. 5a,b). These results indicatethat GIT2 is necessary for the suppressive control or quantitativerestriction of superoxide anion production in GPCR-stimulated neu-trophils. Moreover, GIT2 may also be important for preventing ‘leaky’superoxide anion production in resting (or ‘unstimulated’) neutrophils.

GIT2 in the polarization of superoxide production

The production of superoxide anions and their derivative ROS occursat or near the cell periphery, facing chemoattractants, and is usuallycoupled to the phagocytosis of invading pathogens and production ofsuperoxide anions in the phagosomes1. Such polarized productionof superoxide anions is achieved by the recruitment and assembly ofthe oxidase subunits into plasma membrane areas facing chemoat-tractants1. We next investigated the subcellular localization of NADPHoxidase. Visualization of the p22phox subunit by immunostainingshowed that this subunit was successfully recruited to the F-actinassembled leading edge both in Git2+/– and Git2–/– neutrophils placedin an fMLP gradient (Fig. 4b). We then visualized the location of ROSproduction in these neutrophils in situ by staining cells with nitrobluetetrazolium (Fig. 5c). We found that although about 80% of Git2+/–

neutrophils produced ROS at the cell periphery mostly facing thefMLP source, only about 46% of Git2–/– neutrophils showed such wellorientated ROS production. Therefore, it is likely that althoughNADPH oxidase seems to be successfully recruited to the F-actinassembled leading edge even in Git2–/– neutrophils, this recruitment isoften misoriented in terms of the direction of the chemoattractantsource, as orientation of the F-actin assembled leading edge towardthe chemoattractant source is already considerably impaired inGit2–/– neutrophils.

We obtained further confirmation of the involvement of GIT2 insuperoxide anion production in experiments with HL60 cells (ahuman promyelocytic leukemia cell line). GIT2 expression was‘knocked down’ by duplexes of small interfering RNA and the cellswere allowed to differentiate into neutrophil-like cells. In addition to

wild-type GIT2, a version of GIT2 lacking GTPase-activating proteinactivity (R39K substitution) restored chemotaxis toward fMLP tosome extent but did not mediate suppressive control of superoxideproduction (Supplementary Fig. 2 online), suggesting complexmechanisms of GIT2 function in neutrophils.

Negative regulation of Arf1 by GIT2

To further analyze the biochemical basis of GIT2 function in neutro-phils, we investigated the activity of several small GTPases (which areclosely related to the presumed function of GIT2) by quantifying theirGTP-bound substrates in neutrophils using precipitation assays. GIT2acts as a GTPase-activating protein for Arf1, rather than Arf6, in Cos-7cells25, although GIT2 shows similar GTPase-activating protein activityfor Arf1 and Arf6 in vitro26. We found that Arf1 became activated inGit2+/– neutrophils after fMLP treatment, whereas Arf6 seemed toalready have some activity in unstimulated conditions and its activitywas not notably changed after stimulation (Fig. 6a). In contrast,consistent with published data25, Arf1 was hyperactivated in Git2–/–

neutrophils compared with its activity in Git2+/– neutrophils afterfMLP stimulation, whereas we noted no differences in Arf6 activity forGit2–/– versus Git2+/– neutrophils in those conditions (Fig. 6a). Asreported above, BFA suppressed superoxide production even in Git2–/–

neutrophils. BFA is an inhibitor of several Arf guanine nucleotide–exchange factors and hence inhibits the activation of Arf proteins, withthe exception of Arf6 (ref. 24). Therefore, our results here are consistentwith each other as well as with published analyses25 and also with theknown function of BFA. Moreover, Arf6 is also known to be essentialfor IgG phagocytosis27, and it is conceivable that proper Arf6 regulationin Git2–/– neutrophils allows for normal IgG phagocytosis.

Git2 binds to aPIX, the guanine nucleotide–exchange factor for Racand Cdc42. We found that Rac1, Rac2 and Cdc42 were all transiently

0.0None fMLP fMLP

BFAC5a PMA Zymo

0

20

40

60

Pha

gocy

tosi

s (%

) 80

100

0.5

1.0

Sup

erox

ide

form

atio

n (n

mol

/106 c

ells

)

1.5

2.0

2.5

3.0

3.5

4.0Git2+/–

Git2–/–Git2+/–

Git2–/–

Git2+/– Git2–/–

Gra

dien

t

a b

c

Figure 5 Misoriented hyperproduction of superoxide in Git2–/– neutrophils.

(a) Superoxide production over 5 min for neutrophils incubated with various

stimuli (horizontal axis). PMA, phorbol 12-myristate 13-acetate; Zymo,

zymosan. Data are mean values (error bars, s.e.m.) and are representative of

six experiments for each stimulus. (b) Phagocytosis by neutrophils incubated

for 30 min with IgG-zymosan (100 vesicles/cell); data represent percentageof cells containing phagocytosed beads. Data are representative of six

experiments. Error bars, s.e.m. (c) Nitroblue tetrazolium assay in situ of

neutrophils placed in an fMLP gradient in Zigmond chambers in the

presence of nitroblue tetrazolium solution for 15 min before fixation. Images

represent more than 20 cells examined in each of four independent

experiments. Percentage of neutrophils in which most peripheral ROS was

produced within a 1201 arc facing the fMLP source: Git2+/–, 79.0 ± 4.1;

Git2–/–, 45.9 ± 2.6.

+/–Git2

GTP-Arf1

Arf1

GTP-Arf6

Arf6

GTP-Rac1

Rac1

Rac2

GTP-Rac2

GTP-Cdc42

Cdc42

–/– +/– –/–

0Time (s) Time (s)30 0 10 40

+/–Git2 –/– +/– –/– +/– –/–a b

Figure 6 Activity of small GTPases. Neutrophils were stimulated with fMLP

(time, above blots) and the presence of GTP-bound (GTP-, right margin)

Arf1 and Arf6 (a) and Rac1, Rac2 and Cdc42 (b) was determined by

precipitation assay. The lower blot of each pair of blots represents the

total amount of each GTPase in cell lysates. Representative of three

independent experiments.

728 VOLUME 7 NUMBER 7 JULY 2006 NATURE IMMUNOLOGY

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

activated in Git2+/– neutrophils after fMLP stimulation, reachingpeaks at 10 s and then gradually decreasing (Fig. 6b), consistentwith published reports3,28. In contrast, we found that such transientactivation of Rac1 was mostly impaired although not completelyblocked in Git2–/– neutrophils (Fig. 6b). In contrast, there were onlymarginal differences (within the fluctuations of this assay system) forRac2 and Cdc42 activation in Git2–/– versus Git2+/– neutrophils(Fig. 6b). Rac1 and Rac2 are rapidly recruited to leading edges afterGPCR stimulation29. We found that polarized recruitment of Rac1 butnot Rac2 was mostly impaired in Git2–/– neutrophils (SupplementaryFig. 3 online).

DISCUSSION

Here we have shown that the GIT2 Arf–GTPase-activating proteinis necessary for the direction sensing and persistent chemotaxis ofGPCR-stimulated neutrophils as well as for the suppression of super-oxide anion production. At the molecular level, GIT2 is physicallyassociated with Gbg-mediated direction-sensing machinery and yet isalso involved in the regulation of Arf1 activity. GIT2 is important forPIP3 production as well as for the efficient activation of PAK1, Akt andRac1. In contrast, GIT2 is dispensable for polarized F-actin assembly,peripheral recruitment of p22phox and activation of Rac2 and Cdc42.In studies using Rac1- and Rac2-deficient neutrophils, it has beenshown that Rac1 is essential for chemotactic directionality28–32. Rac1 isessential for the accumulation of PIP3 and Akt at the leading edge ofactivated neutrophils, whereas Rac2 is necessary for F-actin assemblyand membrane recruitment of NADPH oxidase28–32. Our results areconsistent with those reports in terms of the functions of Rac1 andRac2 in neutrophils.

In resting neutrophils, GPCRs and their ‘downstream’ heterotri-meric G proteins may be positioned randomly at the cell surface. Apositive feedback loop initiated by GPCR stimulation may promotethe recruitment of several Gbg-signaling molecules to the leadingedge3,18,19. PI(3)Ks and their product PIP3 have been proposed to beimportant participants in this positive feedback loop18–20. Polarizedaccumulation of Gbg-signaling molecules at the leading edge is likelyto be important for direction sensing and the persistence of neutrophilchemotaxis. Our results indicate that GIT2 is located ‘downstream’ ofthe GPCR signaling pathway and yet is necessary for the recruitmentof Gbg-signaling proteins to the leading edge. Moreover, GIT2 itselfalso becomes recruited to the leading edge after GPCR stimulationand is necessary for efficient PIP3 production. Therefore, GIT2 may bea crucial component of the putative positive feedback loop regulatingthe polarized recruitment of Gbg-mediated direction-sensing machin-ery in GPCR-stimulated neutrophils. Notably, GIT2 deficiency andPI(3)K-g deficiency result in similar effects on neutrophil chemotaxis.Polarized F-actin assembly, the formation of the leading edge andrecruitment of p22phox to the leading edge still occur in the absenceof GIT2. Thus, F-actin assembly is not sufficient for polarizedrecruitment of Gbg-signaling molecules to the leading edge but maybe essential for polarized recruitment and activation of the phagocyteNADPH oxidase.

How is the negative regulation of Arf1 by GIT2 related to thesuppression of superoxide anion production after GPCR-signaling?Arf1 is involved mainly in the regulation of endoplasmic reticulum–to–Golgi vesicle trafficking and thus functions in intracellular secre-tory processes33. Both p22phox and gp91phox are thought to be locatedon secretory vesicle membranes and specific granules in resting cells1.In contrast, BFA-sensitive Arf proteins (which include all Arf isoformsexcept Arf6) are involved in fMLP-induced superoxide production6.Arf6 is involved mainly in the recycling of plasma membrane

components but not in secretory processes33. Here we have shownthat loss of GIT2 did not affect Arf6 activity. Therefore, althoughfurther analyses are required, our results, along with publishedresults25, suggest that GIT2 might regulate NADPH oxidase activityby regulating Arf1, which regulates the intracellular trafficking ofsecretory vesicles that contain subunits of NADPH oxidase. In theabsence of GIT2, Arf1 hyperactivation may result in superoxide anionoverproduction in response to GPCR stimulation. These resultssuggest that GIT2 links GPCR signaling NADPH oxidase and indicatethat NADPH oxidase is not regulated simply by factors ‘positively’regulating the oxidase activity. In contrast, a factor ‘suppressing’NADPH oxidase activity, such as GIT2, is necessary for the controlof superoxide production in neutrophils.Git2–/– mice often develop splenomegaly and granulocytic hyper-

plasia, although such granulocyte hyperproduction is not evident evenin Git2–/– mice that are healthy and do not have splenomegaly.Therefore, it is conceivable that GIT2 is not directly involved in theontogeny of granulocytes. Inefficient neutrophil infiltration intolesions in Git2–/– mice might lead to immunodeficiency and hyper-inflammation, which in turn induce abnormal extramedullary hema-topoiesis and increased granulocyte production.

Our results raise several important issues, such as the precisemechanism by which GIT2 acts to recruit the Gbg-signaling moleculesto F-actin assembled leading edges and how GIT2 is involved in theactivation of PI(3)K in chemotactic neutrophils. Why GIT2 is linkedto Rac1 regulation but not to Rac2 or Cdc42 regulation also remainsunknown. It has been shown that aPIX regulates Cdc42 but not Rac1or Rac2 in C5a-stimulated neutrophils3. The substrate specificityof aPIX can be changed by altering its monomeric versus dimericconfigurations or its association with Gbg effector molecules34.Because it is involved in the localized accumulation of aPIX andGbg, GIT2 may affect the substrate specificity of aPIX.

One additional issue that should be resolved is whether (and if so,how) GIT2-mediated suppression of Arf1 activity is involved inefficient chemotactic directionality and/or polarized recruitment ofGbg molecules. Dictyostelium provides an excellent model system forstudying chemotaxis in which GPCR-signaling and PIP3 productionare important35. However, a Git2 homolog does not exist in dictyos-telium36, which does not need to actively produce superoxide duringchemotaxis to kill its targets. Therefore, such issues concerning GIT2must be addressed in higher organisms, for example, by assessingimmune responses involving both chemotaxis and superoxide anion–mediated defense, as we have described here.

Resolving those issues will improve the understanding of the precisemechanisms underlying direction sensing and persistence as well ascontrol of superoxide anion production. In conclusion, our resultspresented here provide insight that may contribute to the develop-ment of disease therapeutics, such as those designed to treat inflam-matory disorders and allergic disorders, in which the dysfunction ofneutrophils, especially their ‘leaky’ or unnecessary hyperproductionof superoxide radicals at unexpected sites, is involved. Analyses ofthe possible genetic mutations and polymorphisms of human GIT2may also be interesting.

METHODSTargeted disruption of Git2. Git2 was isolated from a C57BL/6 mouse bacterial

artificial chromosome gene library. For construction of the targeting vector,

a neomycin-resistance cassette was inserted into the BspEI site in the second

exon, which contains the first ATG of Git2. The lengths of the homologous

regions were 8.1 kilobases and 3.6 kilobases in the Git2 targeting vector at the

5¢ and 3¢ sides of the neomycin-resistance cassette, respectively. For the

NATURE IMMUNOLOGY VOLUME 7 NUMBER 7 JULY 2006 729

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

production of Git2-deficient mice, TT2 embryonic stem cells were transfected

with the targeting vector as described37, and G418-resistant clones were

screened for homologous recombination by PCR. Embryonic stem clones

carrying the targeted Git2 allele were introduced into host embryos and were

used to produce chimeric mice. Chimeric mice with a high embryonic stem cell

contribution were crossed with C57BL/6 mice to produce Git2+/– mice. Git2–/–

mice were generated by intercrossing of Git2+/– mice. All mice were housed in a

specific pathogen–free facility and the protocols used for all animal experiments

in this study were approved by the Animal Research Committee of Osaka

Bioscience Institute (Osaka, Japan) and the RIKEN Kobe Animal Experiment

Committee (Kobe, Japan).

PCR, Southern blot and RT-PCR. Genomic DNA was prepared as described38

and the genotypes of the mice were determined by PCR. For Southern

hybridization, genomic DNA was digested with SphI, was separated by

electrophoresis through a 0.8% (weight/volume) agarose gel and was trans-

ferred to a Hybond XL membrane (GE Healthcare). Hybridization used a

1,080–base pair DNA fragment located outside the targeting vector. RT-PCR

analysis of mRNA expression used total RNA that was reverse-transcribed using

random primers, with 28 cycles of amplification (95 1C for 0.5 min, 55 1C for

0.5 min and 72 1C for 1 min). The primers used have been described39 or are

listed in Supplementary Table 2 online.

Antibodies. Rabbit polyclonal antisera to GIT2 was generated as described25.

Antibodies to the following proteins were purchased from commercial sources:

aPIX, p22phox, Arf6 and PAK1, from Santa Cruz; GST, Gb, Cdc42, Rac1 and

Rac2, from Upstate Biotechnology; Akt and pS473Akt, from Cell Signaling;

PIP3, from Echelon; Arf1, from Abcam; pT423PAK, from Biosource Interna-

tional; Xpress, from Invitrogen; and Gr-1, from BD PharMingen. Carbocya-

nine-streptavidin as well as donkey antibody to rabbit, mouse or goat IgG

conjugated to horseradish peroxidase or carbocyanine were purchased from

Jackson ImmunoResearch.

Histochemistry. For hematoxylin-and-eosin staining, tissues were fixed in 4%

(volume/volume) formaldehyde in PBS, were embedded in paraffin and were

sectioned at a thickness of 5 mm. For immunohistochemical analysis, frozen

tissue sections (5 mm in thickness) were fixed in 4% (weight/volume)

paraformaldehyde in PBS and treated with 3% (volume/volume) H2O2, and

nonspecific sites were blocked for 30 min with 3% (volume/volume) goat

serum. Sections were then incubated with a rat antibody to mouse Gr-1

followed by MAX-PO (Nichirei). After color development with a diamino-

benzidine solution (Nichirei), sections were counterstained with hematoxylin.

Fungal infection. A. fumigatus was obtained from the Research Center for

Pathogenic Fungi and Microbial Toxicoses at Chiba University (Chiba, Japan).

Conidia were collected and were administered intranasally to 10- to 13-week-

old mice for 3 consecutive days (3 � 107 conidia in 50 ml saline per injection) as

described40. At 3 d after the last infection, mice were analyzed. Quantification

of fungal growth in lungs and Gomori methanamine silver staining were done

as described40,41.

Air pouch analysis. The air pouch analysis of local inflammation was done as

described42. Mice were anesthetized with ether and subcutaneous dorsal

pouches were created by injection of 5 ml of sterile air. After 3 d, pouches

were reinjected with 3 ml of air. Then, 6 d after the first injection, 1 ml of

0.5 mg/ml of human IL-8 (R&D Systems) in 0.5% (weight/volume) carbox-

ymethylcellulose in PBS was injected into the pouches. After 4 h, mice were

anesthetized and pouches were washed with 2 ml of PBS. Lavage fluid was

immediately cooled on ice, volume was recorded and neutrophils were counted.

Bone marrow neutrophils. Bone marrow neutrophils were prepared from 10- to

13-week-old mice by centrifugation through Percoll gradients as described43.

Purified neutrophils were suspended in Hank’s buffer (0.14 M NaCl, 5.4 mM

KCl, 1 mM Tris, 1.1 mM CaCl2, 0.4 mM MgSO4 and 1 mM HEPES, pH 7.2)

containing 5 mg/ml of BSA and were immediately assayed. We used Git2–/– mice

without splenomegaly for bone marrow neutrophil preparation and also for data

collection for the air pouch assay and fungal infection assay.

Chemotaxis. Transwell chemotaxis assays were done in 24-well transwell

chambers (pore size, 3.0 mm; Corning) as described12. Neutrophil chemotaxis

in response to fMLP (10 mM), C5a (9 nM) or IL-8 (50 nM) was measured for

90 min. For analysis of neutrophil chemotaxis in Zigmond chambers16, cover-

slips were placed on chambers and aliquots (0.1 ml) of a solution (Hank’s

buffer containing a 1:10 dilution of 10% (weight/volume) gelatin in H2O) were

added to one side of the chamber and the same solution containing fMLP

(10 mM) was added to the other. Analyses of the final position of a cell relative

to its starting position, the migration speed and the time interval between

direction changes were made by time-lapse videomicroscopic recording of cells

with LVR-3000AN/OL (Sony), as described3,20. Analysis of the chemotaxis of

HL60 cells is described in Supplementary Methods online.

Cell adhesion. These assays were done as described44. Neutrophils were placed

in flat-bottomed 96-well plates coated with fibrinogen (250 mg/ml) with or

without fMLP (10 mM), and the percentage of adherent cells relative to input

cells was determined by acid phosphatase assay.

F-actin assembly. This was measured as described30. Bone marrow neutrophils,

with or without stimulation for 30 s with 10 mM fMLP, were fixed by the

addition of 10 volumes of prewarmed 4.6% (volume/volume) formaldehyde in

PBS at 37 1C for 10 min and were permeabilized for 5 min at 25 1C with 0.1%

Triton X-100 in PBS. After being washed, cells were incubated for 30 min with

2 U/ml of Alexa Fluor 488–phalloidin (Molecular Probes) and were analyzed

on a FACSAria flow cytometer (Becton Dickinson). A minimum of 1 � 104

neutrophil events were routinely recorded and results are expressed as mean

cellular fluorescence. For normalization of data between experiments, baselines

of mean cellular fluorescence of untreated Git2+/– neutrophils were arbitrarily

assigned a value of 100%.

Protein-binding assay. The cDNAs encoding Gb1, Gg2, PAK1 and GIT2 were

amplified by PCR from first-strand cDNA prepared from mRNA of human fetal

brain (BD Clontech); cDNA encoding aPIX was from the KIAA0006 plasmid

(a gift from T. Nagase, Kazusa DNA Research Institute, Kisarazu, Japan). GIT2

cDNA was ligated in-frame with the C terminus of the GST tag sequence of

pEBG45, and cDNAs encoding Gb1, Gg2, PAK1 and aPIX were each ligated in-

frame with the C terminus of the Express-His tag sequence of pcDNA 3.1 His

C (Invitrogen). Cos-7 cells were transfected with those plasmids using Polyfect

(Qiagen) according to the manufacturer’s instructions. Cell lysates were pre-

pared with a solubilizing buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM

Tris-HCl, pH 7.4, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml

of aprotinin, 2 mg/ml of leupeptin and 3 mg/ml of pepstatin A), and protein-

binding assays were done by precipitation of GST fusion proteins from 1 mg of

cell lysates using glutathione-beads (GE Healthcare) coupled with immunoblot

analysis of the coprecipitated proteins using an antibody to Express tag, as

described25. Antibodies retained on filters were visualized with enzyme-linked

chemiluminescence (GE Healthcare).

Immunofluorescence microscopy. Bone marrow neutrophils placed in an

fMLP gradient in a Zigmond chamber for 15 min as described above were

fixed for 15 min at 37 1C with 4% (weight/volume) paraformaldehyde in PBS

and were permeabilized for 10 min at 25 1C with 0.1% (volume/volume) Triton

X-100 (Sigma) in PBS. Immunofluorescence staining was then done, followed

by visualization with a laser confocal microscope (model 510; Carl Zeiss)

as described25.

Superoxide production. Cytochrome C reduction by bone marrow neutrophils

was measured by spectroscopy (V-560, Jasco) at 550 nm and is expressed as

nanomoles of O2– produced over 5 min by 1 � 106 cells, as described15. For

this, neutrophils were incubated with fMLP (10 mM), BFA (60 mg/ml), C5a

(9 nM), phorbol 12-myristate 13-acetate (0.5 mM) or IgG-opsonized zymosan

(10 vesicles/cell). In situ detection of ROS produced by neutrophils was done

with nitroblue tetrazolium (Sigma) as described46. Analysis of the superoxide

production of HL60 cells is described in Supplementary Methods online.

Phagocytosis. Texas Red–zymosan A (Molecular Probes) was opsonized with

mouse IgG (Molecular Probes) as described47. Bone marrow neutrophils were

incubated with opsonized zymosan beads and then fixed, and the numbers of

730 VOLUME 7 NUMBER 7 JULY 2006 NATURE IMMUNOLOGY

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

beads phagocytosed per cell were assigned scores manually by fluorescence

microscopy (IX70; Olympus).

Small GTPase activity. Bone marrow neutrophils were stimulated with 10 mM

fMLP or were left untreated. GTP-bound Arf1 and Arf6 were then precipitated

with 50 mg of a GST-bound peptide of amino acids 1–226 of the Arf-binding

protein GGA3 and were detected by immunoblot with specific antibodies

as described48. GTP-bound Rac1, Rac2 and Cdc42 were similarly determined

with GST–p21-binding domain as described49. The amounts of these GTPases

in total cell lysates were simultaneously determined by immunoblot with

specific antibodies.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank M. Hiraishi, Y. Shibata and M. Iwahara for help; H.A. Popiel (OsakaUniversity, Suita, Japan) for critical reading of the manuscript; and R.Y. Tsien(University of California, La Jolla, California) for monomeric red fluorescentprotein. Supported by Grants-In-Aid from the Ministry of Education, Science,Sports and Culture of Japan (16370090 to H.S.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureimmunology/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

1. Cross, A.R. & Segel, A.W. The NADPH oxidase of professional phagocytes: prototype ofthe NOX electron transport chain systems. Biochim. Biophys. Acta 1657, 1–22(2004).

2. Murphy, P.M. The molecular biology of leukocyte chemoattractant receptors. Annu. Rev.Immunol. 12, 593–633 (1994).

3. Li, Z. et al. Directional sensing requires Gbg-mediated PAK1 and PIXa-dependentactivation of Cdc42. Cell 114, 215–227 (2003).

4. Bokoch, G.M. Biology of the p21-activated kinase. Annu. Rev. Biochem. 72, 743–781(2003).

5. Dana, R.R., Eigsti, C., Holmes, K.L. & Leto, T. A regulatory role for ADP-ribosylationfactor 6 (ARF6) in activation of the phagocyte NADPH oxidase. J. Biol. Chem. 275,32566–32571 (2000).

6. Kaldi, K. et al. Contribution of phopholipase D and a brefeldin A-sensitive ARF tochemoattractant-induced superoxide production and secretion of human neutrophils.J. Leukoc. Biol. 71, 695–700 (2002).

7. Bagrodia, S. et al. A tyrosine-phosphorylated protein that binds to an importantregulatory region on the cool family of p21-activated kinase-binding proteins. J. Biol.Chem. 274, 22393–22400 (1999).

8. Premont, R.T., Claing, A., Vitale, N., Perry, S.J. & Lefkowits, R.J. The GIT family ofADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2through alternative splicing. J. Biol. Chem. 275, 22373–22380 (2000).

9. Schmalzigaug, R. & Premont, R. in ARF Family GTPases 159–183 (Kluwer Academic,Dordrecht, 2003).

10. Zhao, Z.S., Manser, E., Loo, T.H. & Lim, L. Coupling of PAK-interacting exchange factorPIX to GIT1 promotes focal complex disassembly. Mol. Cell. Biol. 20, 6354–6363(2000).

11. Romani, L. Immunity to fungal infections. Nat. Rev. Immunol. 4, 1–23 (2004).12. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorpho-

nuclear leucocytes. J. Exp. Med. 115, 453–466 (1962).13. Hirsch, E. et al. Central role for G protein-coupled phosphoinositide 3-kinase g in

inflammation. Science 287, 1049–1053 (2000).14. Li, Z. et al. Roles of PLC-b2 and -b3 and PI3Kg in chemoattractant-mediated signal

transduction. Science 287, 1046–1049 (2000).15. Sasaki, T. et al. Function of PI3Kg in thymocyte development, T cell activation, and

neutrophil migration. Science 287, 1040–1046 (2000).16. Zigmond, S.H. Orientation chamber in chemotaxis. Methods Enzymol. 162, 65–72

(1988).17. Phee, H., Abraham, R.T. & Weiss, A. Dynamic recruitment of PAK1 to the immuno-

logical synapse is mediated by PIX independently of SLP-76 and Vav1. Nat. Immunol.6, 608–617 (2005).

18. Weiner, O.D. et al. A PtdInsP3- and Rho GTPase-mediated positive feedback loopregulates neutrophil polarity. Nat. Cell Biol. 4, 509–513 (2002).

19. Wang, F. et al. Lipid products of PI(3)Ks maintain persistent cell polarity and directedmotility in neutrophils. Nat. Cell Biol. 4, 513–518 (2002).

20. Hannigan, M. et al. Neutrophils lacking phosphoinositide 3-kinase g show loss ofdirectionality during N-formyl-Met-Leu-Phe-induced chemotaxis. Proc. Natl. Acad. Sci.USA 99, 3603–3608 (2002).

21. Brazil, D.P., Yang, Z.Z. & Hemmings, B.A. Advances in protein kinase B signalling:AKTion on multiple fronts. Trends Biochem. Sci. 29, 233–242 (2004).

22. Dekker, L.V. & Segal, A.W. Signals to move cells. Science 287, 982–985 (2000).23. Brock, C. et al. Roles of Gbg in membrane recruitment and activation of p110g/p101

phosphoinositide 3-kinase g. J. Cell Biol. 160, 89–99 (2003).24. Chardin, P. & McCormick, F. Brefeldin A: the advantage of being uncompetitive. Cell

97, 153–155 (1999).25. Mazaki, Y. et al. An ADP-ribosylation factor GTPase-activating protein Git2-short/

KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletalorganization. Mol. Biol. Cell 12, 645–662 (2001).

26. Vitale, N. et al. GIT proteins, A novel family of phosphatidylinositol 3,4,5-trispho-sphate-stimulated GTPase-activating proteins for ARF6. J. Biol. Chem. 275, 13901–13906 (2000).

27. Zhang, Q., Cox, D., Tseng, C.C., Donaldson, J.G. & Greenberg, S. A requirement forARF6 in Fcg receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 273,19977–19981 (1998).

28. Li, S. et al. Chemoattractant-stimulation Rac activation in wild-type and Rac2-deficientmurine neutrophils: preferential activation of Rac2 and Rac2 gene dosage effect onneutrophil functions. J. Immunol. 169, 5043–5051 (2002).

29. Filippi, M.D. et al. Localization of Rac2 via the C terminus and aspartic acid 150specifies superoxide generation, actin polarity and chemotaxis in neutrophils. Nat.Immunol. 5, 744–751 (2004).

30. Roberts, A.W. et al. Deficiency of the hematopoietic cell-specific Rho family GTPaseRac2 is characterized by abnormalities in neutrophil function and host defense.Immunity 10, 183–196 (1999).

31. Glogauer, M. et al. Rac1 deletion in mouse neutrophils has selective effects onneutrophil functions. J. Immunol. 170, 5652–5657 (2003).

32. Sun, C.X. et al. Rac1 is the small GTPase responsible for regulating the neutrophilchemotaxis compass. Blood 104, 3758–3765 (2004).

33. Roth, M.G. in GTPase 176–197 (Oxford Univ Press, New York, 2000).34. Feng, Q., Baird, D. & Cerione, R.A. Novel regulatory mechanisms for the Dbl family

guanine nucleotide exchange factor Cool-2/a-Pix. EMBO J. 23, 3492–3504(2004).

35. Comer, F.I. & Parent, C.A. PI 3-kinases and PTEN: how opposites chemoattract. Cell109, 541–544 (2002).

36. Eichinger, L. et al. The genome of the social amoeba Dictyostelium discoideum. Nature435, 43–57 (2005).

37. Yagi, T. et al. A novel ES cell line, TT2, with high germline-differentiating potency. Anal.Biochem. 214, 70–76 (1993).

38. Suda, Y. et al. Driven by the same Ig enhancer and SV40 T promoter ras induced lungadenomatous tumors, myc induced pre-B cell lymphomas and SV40 large T gene avariety of tumors in transgenic mice. EMBO J. 6, 4055–4065 (1987).

39. Kitamura, H. et al. IL-6-STAT3 controls intracellular MHC class II ab dimmerlevel through cathepsin S activity in dendritic cells. Immunity 23, 491–502(2005).

40. Bellocchio, S. et al. The contribution of the Toll-like/IL-1 receptor superfamily to innateand adaptive immunity to fungal pathogens in vivo. J. Immunol. 172, 3059–3069(2004).

41. Grocott, R.G. A stain for fungi in tissue sections and smears using Gomori’s methen-amine-silver nitrate technic. Am. J. Clin. Pathol. 25, 975–979 (1955).

42. Romano, M. et al. Role of IL-6 and its soluble receptor in induction of chemokines andleukocyte recruitment. Immunity 6, 315–325 (1997).

43. Mocsai, A., Ligeti, E., Lowell, C.A. & Berton, G. Adhesion-dependent degranulation ofneutrophils requires the Src family kinases Fgr and Hck. J. Immunol. 162, 1120–1126(1999).

44. Lowell, C.A., Fumagalli, L. & Berton, G. Deficiency of Src family kinases p59/61hck andp58c-fgr results in defective adhesion-dependent neutrophil functions. J. Cell Biol. 133,895–910 (1996).

45. Mayer, B.J., Hirai, H. & Sakai, R. Evidence that SH2 domains promote processivephosphorylation by protein-tyrosine kinases. Curr. Biol. 5, 296–305 (1995).

46. Hirai, K., Moriguchi, K. & Wang, G.Y. Human neutrophils produce free radicals from thecell-zymosan interface during phagocytosis and from the whole plasma membranewhen stimulated with calcium ionophore A23187. Exp. Cell Res. 194, 19–27(1991).

47. Gakidis, M.A. et al. Vav GEFs are required for b2 integrin-dependent functions ofneutrophils. J. Cell Biol. 166, 273–282 (2004).

48. Luton, F. et al. EFA6, exchange factor for ARF6, regulates the actin cytoskeleton andassociated tight junction in response to E-cadherin engagement. Mol. Biol. Cell 15,1134–1145 (2004).

49. Benard, V. & Bokoch, G.M. Assay of Cdc42, Rac, and Rho GTPase activation by affinitymethods. Methods Enzymol. 345, 349–359 (2002).

NATURE IMMUNOLOGY VOLUME 7 NUMBER 7 JULY 2006 731

A R T I C L E S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y