Small Rho Regulate Antigen Presentation in Dendritic Cells Galina V. Shurin, Irina L. Tourkova, Gurkamal S. Chatta, Gudula Schmidt, Sheng Wei, Julie Y. Djeu and Michael R. This information is current as Shurin of September 26, 2021. J Immunol 2005; 174:3394-3400; ; doi: 10.4049/jimmunol.174.6.3394 http://www.jimmunol.org/content/174/6/3394 Downloaded from

References This article cites 40 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/174/6/3394.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 26, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Small Rho GTPases Regulate Antigen Presentation in Dendritic Cells1

Galina V. Shurin,* Irina L. Tourkova,* Gurkamal S. Chatta,‡ Gudula Schmidt,§ Sheng Wei,¶ Julie Y. Djeu,¶ and Michael R. Shurin2*†

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms main- taining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased Downloaded from the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was signif- icantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo. The Journal of Immunology, 2005, 174: 3394–3400. http://www.jimmunol.org/ endritic cells (DC)3 perform an essential role in the ini- ture DC, which have a reduced potential for Ag uptake but have a tiation of innate and adaptive immunity. They are major high capacity for Ag presentation and T cell stimulation (3). This D contributors to host immunity against infection and ma- transition is accompanied by dramatic cytoplasmic reorganization, lignancy. DC are a heterogeneous group of cells derived from both characterized by a redistribution of MHC class II from intracellular myeloid and lymphoid precursors, which populate peripheral tis- compartments to the plasma membrane and up-regulation of sur- sues and lymphoid organs. Their main functions include the fol- face costimulatory molecules (CD80, CD86), CD40, MHC class I, lowing: 1) uptake and processing of different antigenic molecules, and T cell adhesion molecules (e.g., CD48 and CD58). DC also 2) migration from peripheral tissues to lymphoid organs, 3) Ag remodel their profile of chemokine receptors that facilitate migra- by guest on September 26, 2021 presentation in an MHC class I- and class II-restricted manner, and tion and homing to lymphoid organs (3). Finally, the cells also 4) production of cytokines and expression of costimulatory mol- extend long dendritic processes that further increase opportunities ecules critical for efficient activation of T cells (1, 2). for T cell capture and interaction. All of the above changes are DC exist in two functionally and phenotypically distinct states, crucial for DC function, and depend on regulation of assem- immature and mature (3). Immature DC are widely distributed bly, which in turn is mediated by the Rho family of GTPases, i.e., throughout the body and occupy sentinel positions in many non- Rho, Cdc42, and Rac (5–9). lymphoid tissues. They constantly sample their environment for The Rho GTPases form a subgroup of the of Ags by phagocytosis, macropinocytosis, and pinocytosis. Imma- 20- to 30-kDa GTP-binding proteins that have been shown to reg- ture cells express relatively low levels of MHC class I, class II, and ulate a wide spectrum of cellular function (10, 11). The mamma- costimulatory molecules (4, 5). After engulfing Ags and activation lian Rho-like GTPases comprise Ͼ20 distinct proteins, including by proinflammatory cytokines, immature DC differentiate into ma- RhoA, -B, -C, -D, and -E; Rac1 and -2, RacE, Cdc42, and TC10. Among all Rho GTPases, Rac1 (Ras-related C3 botulinum toxin substrate 1), Cdc42 (cell division cycle 42), and RhoA (Ras ho- Departments of *Pathology, †Immunology, and ‡Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA 15213; §Institute of Experimental and Clinical Phar- mologous member A) have been studied most extensively. Until macology and Toxicology, Albert Ludwig’s University, Freiburg, Germany; and ¶H. recently, members of the Rho subfamily were believed to be in- Lee Moffit Cancer Center, University of South Florida, Tampa, FL 33612 volved primarily in the regulation of cytoskeletal organization in Received for publication May 24, 2004. Accepted for publication January 3, 2005. response to extracellular growth factors. However, results from a The costs of publication of this article were defrayed in part by the payment of page number of laboratories over the past few years have revealed that charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Rho GTPases play a crucial role in diverse cellular events such as 1 This work was supported by National Institutes of Health Grant 2RO1 CA 084270 membrane trafficking, transcriptional regulation, cell growth con- (to M.R.S.). trol, endocytosis, differentiation, and (12, 13). 2 Address correspondence and reprint requests to Dr. Michael R. Shurin, Clinical Activity of Rho GTPases is regulated by signals originating Immunopathology, 5725 CHP-MT, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail from different classes of surface receptors including - address: [email protected] coupled receptors, tyrosine receptors, cytokine receptors, 3 Abbreviations used in this paper: DC, dendritic cell; GDI, guanine nucleotide dis- sociation inhibitor; dn, dominant negative; ca, constitutively active; VV, vaccinia and adhesion receptors (14). Like all members of the Ras super- virus; EGFP, enhanced GFP; ToxB, toxin B; CNF1, cytotoxic necrotizing factor 1; family, the Rho GTPases function as molecular switches, cycling PAK-1, p21-activated kinase-1; CRIB, Cdc42/Rac interactive binding; PBD, p21 binding domain; Rhotekin RBD, Rhotekin Rho binding domain; HPF, high power between an inactive GDP-bound state and an active GTP-bound field;WASP, Wiskott-Aldrich syndrome protein. state (15). The ratio of the two forms is regulated by the opposing

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 3395

effect of guanine nucleotide exchange factors, which enhance the Rac1, caRac1, dnRhoA, or caRhoA. VV was used at a multiplicity of exchange of bound GDP for GTP, and the GTPase-activating pro- infection of 5. For the transfection, DC were harvested on day 6, washed teins, which increase the intrinsic rate of hydrolysis of bound GTP. twice in HBSS, and incubated at 37°C for 4–6 h with corresponding VV- based construct. The VV/EGFP construct was used to check the efficiency In addition, the Rho-like GTPases are regulated further by guanine of VV-based transfection by flow cytometry analysis. Transduction effi- nucleotide dissociation inhibitors (GDIs), which can inhibit both cacy of VV/Rho GTPase-transduced DC was also determined by Western the exchange of GTP and the hydrolysis of bound GTP (16). In blot and immunocytochemistry. addition to being an inhibitor of nucleotide dissociation, GDIs play In addition, we have used 1) a Rho family GTPase inhibitor, toxin B (ToxB) from Clostridium difficile (Calbiochem), which ribosylates ADP a crucial role in the shuttling of Rho GTPases between the cyto- and inactivates Rho, Rac, and Cdc42 (22), and 2) a Rho family GTPase plasm and membranes. For instance, Cdc42 function is also reg- activator, cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli, ulated by its subcellular localization, which depends on interac- which deamidates glutamine 63 of RhoA or 61 of Rac and Cdc42, render- tions with its GDI: active Cdc42 is bound to the membrane, ing ca GTPases (23). In some experiments, PMA was also used as a non- whereas inactive Cdc42 is localized in the cytosolic fraction (17). specific Rho GTPase activator (23). Before the functional assays, DC were pretreated with 1 ng/ml ToxB for 60 min, 250 ng/ml CNF1, or 60 ng/ml Although much is known about the Rho-type GTPase structure PMA for 30 min or medium (control). and , little is known about their regulation and function in immune cells. Rho GTPases have been demonstrated to p21-activated kinase-1 (PAK-1) p21-binding domain pull-down regulate migration, and chemotaxis in monocytes and macro- assay phages (18, 19). Endocytosis occurs via remodeling of the actin Affinity purification assays that monitor Cdc42, Rac, and Rho protein ac- and shares many of the core cytoskeletal components tivation are based on the fact that Rho proteins act as molecular switches,

involved in adhesion and migration. Small GTPases of the Rho family cycling between an inactive GDP-bound state and an active GTP-bound Downloaded from have been implicated in coordinating actin dynamics in response to state (24, 25). The Cdc42/Rac Activation Assay kit has been used in our experiments to assess specific binding and precipitation of GTP-Cdc42 and extracellular signals and during diverse cellular processes, including GTP-Rac from DC lysates (Upstate Biotechnology). The assay uses the endocytosis, but the mechanisms controlling their recruitment and Cdc42/Rac interactive binding (CRIB) region (also called the p21 binding activation in DC are not known. Because it has been recently reported domain (PBD)) of the Cdc42/Rac effector protein, PAK-1. The CRIB/PBD that DC developmentally regulate endocytosis at least in part by protein motif has been shown to bind specifically to the GTP-bound form of Rac or Cdc42, thus allowing isolation of active Cdc42 and Rac proteins. controlling levels of activated Cdc42 (3, 5), we have investigated in http://www.jimmunol.org/ In addition, Rho Activation Assay kit has been used for precipitation of this study whether Rho GTPases are involved in the regulation of GTP-Rho from DC lysates (Upstate Biotechnology). This assay uses the other DC functions. There are no published data on the role of Rho Rhotekin Rho binding domain (Rhotekin RBD), which specifically binds to GTPases in the regulation of Ag presentation by DC. We report on the and precipitates GTP-Rho (active Rho), but not GDP-Rho (i.e., inactive involvement of Cdc42, Rho, and Rac in Ag presentation by DC, using Rho) from the cell lysates (26). The facts that the PBD region of PAK and RBD region of Rhotekin have a high affinity for GTP-Cdc42, GTP-Rac, the OVA peptide as a model Ag. We have also investigated the role and GTP-Rho, respectively, and that PAK and Rhotekin binding results in of the Rho-type small GTPases (Rac1, Cdc42, and RhoA) in regulat- a significantly reduced intrinsic and catalytic rate of hydrolysis of Cdc42, ing DC adhesion and motility, by transducing DC with constructs, Rac, and Rho make it an ideal tool for affinity purification of GTP-Cdc42, encoding dominant-negative (dn) or constitutively active (ca) forms of GTP-Rac, or GTP-Rho from cell lysates (27). Therefore, 5–10 ␮g of PAK-

␮ by guest on September 26, 2021 Rho GTPases. We demonstrate that constitutive activation of Rho PBD agarose was added to 1 ml of cell lysate (200–1000 g of protein) from DC transfected with VV-based (dn) or (ca) mutant forms of Rho GTPases in DC differentially modifies DC Ag presentation, adher- GTPases for isolation of GTP-Cdc42, GTP-Rac, and GTP-Rho. Reaction ence, chemotaxis, and endocytosis. mix was gently rocked at 4°C for 60 min. The Rac and Cdc42 proteins bound to PAK-1 PBD, and Rho protein bound to Rhotekin RBD were Materials and Methods separated by SDS-PAGE. Proteins were transferred to polyvinylidene di- Mice fluoride membrane and probed with specific anti-Rac, -Cdc42, and -RhoA Abs (Upstate Biotechnology and BD Transduction Lab). The secondary Male BALB/c (H-2Kd, I-Ad) mice, 6–8 wk old, were obtained from Tac- Abs were HRP conjugated (1:100,000; Pierce). The immunoblot was pro- onic. Animals were maintained at Central Animal Facility at the University cessed and treated with chemiluminescent reagents (Pierce), and the bands of Pittsburgh according to standard guidelines. were visualized on Kodak film (Eastman Kodak). DC generation Western blot and immunocytochemistry Murine DC were generated as described previously (20). Briefly, mouse Expression of total Cdc42, Rac1, and RhoA proteins in DC lysates pre- hemopoietic progenitors were isolated from bone marrow and depleted of pared after VV-based transfections, was determined by Western blot. The RBC with lysing buffer (155 mM NH4Cl in 10 mM Tris-HCl buffer (pH cell lysates from nontransfected and VV/CD56-transfected DC were used 7.5), 25°C). The single-cell suspension was incubated with a mixture of as controls. For the Western blot and immunocytochemical detection of Abs followed by incubation with rabbit complement to deplete cells that tested small Rho GTPases, the following primary Abs were used: mouse express the lymphocyte Ags B220, CD4, and CD8. Cells were then cul- antiCdc42 (1:250; BD Transduction Lab), mouse anti-Rac (2 ␮g/ml; Up- ␮ tured overnight (37°C, 5% CO2) in six-well plates (Falcon) at the concen- state Biotechnology), and mouse anti-RhoA (3 g/ml; Upstate tration of 106/ml, in complete RPMI 1640 medium supplemented with 2 Biotechnology). mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 10 mM Percentage of DC overexpressing Cdc42, Rac1, or RhoA after VV- HEPES, 10% heat-inactivated FBS, 0.1 mM nonessential amino acids, and based transfection was determined by immunocytochemistry. Nontrans- 1 mM sodium pyruvate (Invitrogen Life Technologies). The nonadherent fected DC and CD56-transfected DC served as controls. Immunocyto- cells were collected and seeded at a concentration of 2 ϫ 105 cells/ml in chemical analysis of Cdc42, Rac1, and Rho expression was performed on six-well plates in complete RPMI 1640 medium in the presence of recom- the cytospinned (100 ϫ g; 5 min) cells, which were air-dried, fixed in binant mouse GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) (PeproTech). At ice-cold acetone, and permeabilized with saponin. day 6, DC were transduced with vaccinia virus (VV) encoding dn or ca forms of Rho GTPases (Cdc42, Rac, or Rho) proteins. Six hours later, DC Ag presentation assay were collected and used for further analyses. Murine bone marrow-derived DC were generated in cultures with GM-CSF VV-based transduction of murine DC with the Rho family of and IL-4 and transduced with VV encoding dn or ca mutant forms of small GTPases Cdc42, Rac, and Rho, or control protein as described above. To evaluate DC ability to present a specific Ag, we have tested capacity of modified DC VV-based constructs, encoding dnCdc42, caCdc42, dnRac1, caRac1, to present OVA (OVA-derived antigenic peptides) to syngeneic specific T dnRhoA, caRhoA, and control enhanced GFP (EGFP) and CD56 proteins cells. Ag presentation was assessed by the IL-2 production by the specific were prepared as described earlier (21). The DC (5 ϫ 106 cells) were OVA-recognizing T cell clone DO11.10 obtained from DO11.10 trans- transduced with VV-based constructs encoding dnCdc42, caCdc42, dn- genic mice (H-2Kd, I-Ad) expressing the DO11.10 TCR that is specific for 3396 Rho GTPases REGULATE DC FUNCTION

d the peptide fragment of OVA323–339 in the context of I-A . This clone was kindly provided by Dr. B. Osborne (University of Massachusetts, Amherst, MA). All groups of transduced DC (5 ϫ 105 cells/ml) were pulsed with dialyzed whole OVA (Sigma-Aldrich; 1 mg/ml, experimentally established optimal dose for IL-2 production) for 24 h, collected, washed, and treated with medium (control), CNF1 (250 ng/ml; 30 min) or ToxB (1 ng/ml; 60 min). Then, DC were washed again and mixed (5 ϫ 103 cells) with syn- geneic T cell clone DO11.10 (2 ϫ 104 cells) in triplicate in 96-well U- bottom plates. Cell-free supernatants were collected 48 h later, and con- centrations of IL-2 were determined by ELISA (Endogen) according to the manufacturer’s instructions. Cell adhesion assay FIGURE 1. Genetic engineering of murine DC to express Rho GT- To evaluate the effect of Rho GTPases on DC adherence to extracellular Pases. Bone marrow-derived DC were transfected with VV and analyzed matrix in vitro, DC transfected with VV encoding dn or ca mutants of for the expression of different proteins. Transfection of DC with VV en- Cdc42, Rac, and Rho were washed and counted. In addition, CNF1 and Ͼ ToxB treatment was also performed as described above. A total of 2 ϫ 104 coding EGFP resulted in EGFP expression in 80% of cells. Flow cytom- cells in 120 ␮l of complete RPMI 1640 medium was placed in different etry analysis of positive cells was performed 6 h after DC transfection as wells on eight-chamber microscope slide coated with polylysine (Fisher described in Materials and Methods. Scientific). After 2-h incubation at 37°C, medium was gently aspirated, chambers were washed with warm medium, and dissembled, and adherent cells on the slides were fixed and stained with LeukoStat staining kit (Fisher Scientific). Enumeration of adherent DC was performed in at least Downloaded from 10 high power fields (HPF) using light microscopy independently by two by Western blot and immunocytochemistry. The results revealed investigators. that, as expected, DC from all experimental groups express detect- Chemotaxis assay able levels of endogenous total Cdc42, Rac1, and RhoA. The vi- rally expressed proteins were distinguished from endogenous con- Cell migration was measured in 48-well Transwell plates (5-␮m pores; Corning Costar). Recombinant murine MIP (MIP1␣; 10 ng/ml; PeproTech) stitutively expressed Cdc42, Rac1, and RhoA proteins by the and synthetic chemotactic peptide fMLP (10Ϫ7 M; Sigma-Aldrich) were appearance of the slower migrating specific bands containing myc- http://www.jimmunol.org/ diluted in RPMI 1640 medium contained 1% FBS (assay medium), and tag epitopes (21). The results of these studies are shown in Fig. 2 600-␮l aliquots were placed in the lower chamber of Transwell plates. and demonstrate 1) high levels of expression of virally encoded Assay medium was used to measure spontaneous migration of DC. All groups of transfected DC were diluted at 1 ϫ 106 cells/ml, and 100 ␮lof (ca) and (dn) mutant forms of Cdc42, RhoA, and Rac1 in VV- cell suspension was added to each Transwell insert. After 4-h incubation at transduced DC and 2) comparable levels of expression of both 37°C, the Transwell inserts were removed, and cells from the lower cham- mutant forms of Cdc42, Rac1, and RhoA. It is important to note ber were collected. Cells transmigrated through the 5-␮m pore size mem- that there are slight differences in the intensity of positive Western brane were acquired on FACScan (BD Biosciences) for 60 s. Data are reported as mean numbers of transmigrated cells from duplicate wells. blot bands between Cdc42, Rac1, and RhoA, which are due to the different affinity/avidity of specific Abs used in the described as- by guest on September 26, 2021 Endocytosis assay says. Finally, percentage of DC overexpressing Cdc42, Rac1, or The regulation of endocytotic activity of DC by the Rho family GTPases RhoA after VV-based transfection was determined by immunocy- was assessed by FITC-dextran-40 (2 mg/ml; Molecular Probes) uptake in tochemistry. We demonstrated that ϳ70–80% of DC were control DC and DC pretreated with 250 ng/ml CNF1 for 30 min, 60 ng/ml strongly positive for Cdc42, Rho, or Rac staining after their trans- PMA for 30 min, 1 ng/ml ToxB for 60 min, or medium (control). Active endocytosis of dextran 40 was measured for 30 min at 37°C, whereas fection with VV encoding dnCdc42, caCdc42, dnRac1, caRac1, background diffusion was determined at 4°C. Positive cells were analyzed dnRho, or caRho, respectively. In contrast, most of control DC by flow cytometry. exhibited only the background level of positive staining with no Statistical analysis more than 20–30% of DC displaying low-to-medium level of positivity. For a single comparison of two groups, Student’s t test was used after evaluation for normality. If data distribution was not normal, a Mann- Next, we demonstrated that VV/caCdc42-, VV/caRac-, and VV/ Whitney rank sum test was performed. For the comparison of multiple caRho-transduced DC express high levels of active GTP-bound groups, one- or two-way ANOVA was applied. For all statistical analysis, forms of Rho GTPases Cdc42, Rac1, and RhoA (Fig. 3). In con- the level of significance was set at a probability of 0.05 to be considered trast, all three dn mutant forms of Cdc42, Rac1, RhoA, as well as significant. All experiments were repeated at least two to three times. Data are represented as the mean Ϯ SEM. control transfected and nontransfected DC did not exhibit any de- tectable binding to the fusion proteins, suggesting the absence or Results very low levels of active GTP-bound forms of Cdc42, Rac1, and Expression of Rho GTPases in murine DC RhoA. It is important to note that there are slight differences in the The most common method to investigate Rho GTPases in cells is intensity of positive Western blot bands between Cdc42, Rac1, and to microinject dn or ca forms into tested cells (9, 18, 28). However, RhoA, which are due to the different affinity/avidity of specific Abs this methodology cannot be used to study certain functions of DC used in the assays. However, the results of these studies demon- that require a great number of cells, such as Ag presentation, ad- strate that 1) DC were efficiently transfected with ca and dn forms herence, etc. To solve this problem, we have established a new of all tested small Rho GTPases and 2) transfection was functional, method of DC modification using a vector-based approach with i.e., (ca) mutants express active Rho GTPases, whereas (dn) mu- VV-based constructs encoding dnCdc42, caCdc42, dnRac, caRac, tants and control nontransfected DC express no active GTP-bound dnRho, caRho, and control EGFP and CD56 proteins. First, using proteins. Thus, VV transfection of DC with Rho GTPases can be VV/EGFP, we have demonstrated that transduction efficacy of DC used to study the regulation of DC function. reaches 82% as was determined by a FACScan analysis (Fig. 1). In the next series of experiments, we evaluated whether trans- Protein expression was maximal in 4–8 h. duction of DC with dn or ca mutant forms of Rho GTPases reg- Next, expression of total Cdc42, Rac1, and RhoA proteins in DC ulates DC adherence, migration, chemotaxis, endocytosis, and Ag lysates prepared after VV-based transfections, was also determined presentation. The Journal of Immunology 3397

FIGURE 2. Determination of the efficiency of DC transduction with VV/Rho GTPases by Western blot. DC were infected with rVV expressing ca or dn forms of Rac1, RhoA, Cdc42, and irrelevant protein (CD56) for6hat37°C. Nontransfected DC were used as control as well. The infected DC were then collected, and cell lysates were subjected to Western blot analysis using anti-Rac1 (left panel), RhoA (middle panel), or Cdc42 Abs. Western blot analysis revealed high and comparable levels of expression of virally encoded (ca) and (dn) mutant forms of Rac1, RhoA, and Cdc42 in VV-transduced DC. The myc-tagged virally expressed proteins were differentiated from endogenous Rac1, RhoA, and Cdc42 by the appearance of a slower migrating band. The same membranes was stripped and reprobed with anti-␤-actin Ab to check equal loading.

Regulation of DC adhesion by small GTPases from the Rho Regulation of Ag presentation in DC by the Rho family GTPases Downloaded from family In the next series of studies, we have used the same approach to The results demonstrating the effect of Rho GTPases on DC ad- activate or block Rho GTPase in DC (Fig. 5A). The results show hesion in vitro are shown in Fig. 4. Thus, the Rho GTPase activator that CNF1 increased, whereas ToxB decreased the ability of DC to CNF1 significantly up-regulates adherence of both control (non- present OVA-derived peptides to OVA-specific T cell clones, as transduced) and CD56 (control protein)-transduced DC (23.6 Ϯ determined by IL-2 production by activated T cells (359 Ϯ 40 and 3.4 and 18.5 Ϯ 2.3 vs 13.2 Ϯ 1.7 and 14.1 Ϯ 0.8 cells/HPF, 128 Ϯ 10 vs 248 Ϯ 26 pg/ml/48 h in controls; p Ͻ 0.05). Both http://www.jimmunol.org/ respectively; p Ͻ 0.05), whereas Rho GTPase inhibitor ToxB sig- caCdc42 and caRho up-regulated Ag presentation by DC (up to nificantly inhibits DC adhesion in both groups (7.0 Ϯ 0.5 and ϳ450 pg/ml), which could be blocked by ToxB. dnCdc42 and 5.2 Ϯ 0.4 cells/HPF; p Ͻ 0.05). ToxB also inhibited adhesion of dnRho decreased Ag presentation, although the effect of dnRho did the caCdc42-transduced DC (6.9 Ϯ 1.1 cells/HPF; p Ͻ 0.05) (Fig. not reach statistical significance. Surprisingly, both ca and dn 4A). Transduction of DC with dnCdc42 markedly decreased forms of Rac blocked DC Ag presentation ( p Ͻ 0.05), suggesting (5.5 Ϯ 0.4 cells/HPF), whereas transduction with caCdc42 that Rac might mediate different effector pathways in DC than strongly increased (19.5 Ϯ 1.8 cells/HPF) adhesion of DC ( p Ͻ Cdc42 and Rho. Together, these data demonstrate that Rho 0.05). Furthermore, CNF1 only slightly up-regulated DC adhesion, GTPases are involved in Ag presentation by DC, although the which was blocked by the overexpression of dnCdc42, whereas effector mechanisms remain to be elucidated. by guest on September 26, 2021 ToxB did not further decrease it. Taken together with the fact that CNF1 did not further up-regulate adhesion of caCdc42-transduced Regulation of DC motility by the Rho family GTPases DC, these data suggest that Cdc42 plays a major role among Rho GTPases in the regulation of DC adhesion. This was confirmed in The effects of Rho, Rac, and Cdc42 on DC chemotaxis and spon- the second set of experiments where the effect of the different Rho taneous migration in a Transwell system are shown in Fig. 5B. GTPases on DC adhesion was compared (Fig. 4B). The transduc- Constitutive activation of Cdc42 in DC significantly inhibits both Ϯ Ϯ tion of DC with caRho caused only a modest elevation of DC spontaneous (7,380 568 vs 12,260 1,112 cells/min in control; Ͻ Ϯ adherence (14.4 Ϯ 1.1 vs 8.8 Ϯ 0.8 in control; p Ͻ 0.05). This p 0.05) and chemokine-induced DC migration: 8,040 590 vs Ϯ effect was blocked by ToxB, but not by caRac. Thus, among the 30,240 2,674 cells/min for chemotactic peptide fMLP and Ϯ Ϯ three tested Rho GTPases, activation of Cdc42 in DC displayed the 9,780 867 vs 39,414 4,532 cells/min for CC chemokine ␣ Ͻ highest potential for augmenting DC function. MIP1 ( p 0.001). Interestingly, the dnCdc42 mutant did not modulate DC migration in either assay. Unexpectedly, all tested mutant forms of Rac and Rho (except dnRAC) significantly inhibit both spontaneous migration and chemotaxis of transduced DC. Together, our results demonstrate differential requirements for the Rho family GTPases in DC motility and suggest that a dynamic regulation of Rac and Rho may be required for DC chemotaxis.

Regulation of endocytotic activity of DC by the Rho family GTPases FIGURE 3. Transfection of DC with VV encoding ca Cdc42, Rac1, and Because it has already been reported that DC developmentally reg- RhoA (caDC), but not dn Cdc42, Rac1, and RhoA (dnDC), dramatically ulate endocytosis by controlling levels of activated Cdc42 (5), we increased the levels of active GTP-bound Cdc42, Rac1, and RhoA. Active next demonstrate that Rho GTPases are involved in the regulation forms of small Rho GTPases was isolated from DC lysates by affinity of DC endocytotic activity in our test system (Fig. 6). The results purification on the CRIB region of the Cdc42/Rac effector protein, PAK-1 indicate that uptake of dextran 40, which reflects mannose recep- and Rhotekin RBD. After separation of beads with bound GTP-Cdc42, GTP-Rac, and GTP-Rho by centrifugation, the pellet was denatured in a tor-mediated endocytosis in DC, was significantly up-regulated (up SDS-loading buffer, and Rho GTPases were determined by Western blot. to 170% and up to 144%) by the Rho GTPase activators CNF1 and GST-Cdc42, GST-Rac, GST-Rho (a higher m.w. because of GST), and rat PMA, respectively (Fig. 6), and reduced 2- to 4-fold by a Rho cerebellum served as a positive control. The results of one representative GTPase inhibitor, ToxB (Fig. 6, p Ͻ 0.05). Our results also reveal experiment are shown (n ϭ 3). that transduction of DC with caCdc42 increases their endocytotic 3398 Rho GTPases REGULATE DC FUNCTION

FIGURE 4. Regulation of DC adhesion by small GTPase from the Rho family. Murine bone marrow-derived DC were generated in cultures with GM-CSF and IL-4 and harvested on day 6. After washing, DC were transduced with VV encoding dn or ca mutants of Cdc42, Rac and Rho. Four hours later, DC were washed, and 2 ϫ 104 cells in 120 ␮l of complete medium were placed on a microscope slide with 8 chambers. After 2-h incubation at 37°C, medium was aspirated, and adherent cells were fixed and stained with LeukoStat staining kit. Enumeration of adherent DC was performed in at least 10 HPF using light microscopy. The results are shown as the mean Ϯ SEM (n ϭ 3). A, Modulation of Cdc42-mediated effect on DC adherence by Rho GTPase p Ͻ 0.05 (one-way ANOVA). B, Inhibition of Rho GTPase-induced up-regulation ,ء .(inhibitor ToxB (1 ng/ml) and Rho GTPase activator CNF1 (250 ng/ml .(p Ͻ 0.05 (one-way ANOVA ,ء .of DC adherence by ToxB Downloaded from

activity up to 150%, whereas transduction with dnCdc42 decreases it by 40% ( p Ͻ 0.05). In summary, our data demonstrate that DC adhesion, Ag pre- sentation, migration, chemotaxis, and endocytosis are differentially regulated by the Rho family GTPases. http://www.jimmunol.org/

Discussion Rho-related small GTPases, including Cdc42, Rac, and Rho, are known to regulate actin reorganization in response to different ex- tracellular cues in several adherent cell types (29, 30). Recently, other cellular functions have also been ascribed to these proteins, i.e., transcriptional regulation, growth control, endocytosis, and exocytosis (5–7, 9). However, little is known about the role of by guest on September 26, 2021 small Rho GTPases in the regulation of DC function. Thus, we have investigated the role of Rho GTPases in regulating DC ad- hesion, Ag presentation, migration, chemotaxis, and endocytosis. Rho family proteins are known to regulate cell adhesion in mac- rophages and fibroblasts. The most common method to examine the role of Rho GTPases in cells is to microinject dn or ca forms into test cells (18). For example, Allen et al. (31) demonstrated that using Bac1 macrophages, Cdc42 and Rac are required for the as- sembly of adhesion sites to the extracellular matrix. Cdc42 has been implicated in VLA-4-dependent adhesion of macrophages (19). Rac2 appears to be critical for hemopoietic stem cell adhe- FIGURE 5. Regulation of OVA Ag presentation by DC (A) and che- sion both in vitro and in vivo (32). In this study, we demonstrate moattractant-induced DC migration (B) by the Rho family GTPases. Bone for the first time that the Rho GTPase activator CNF1 significantly marrow-derived DC were transfected with VV encoding caCdc42, up-regulates adherence of both control (nontransduced) and CD56 dnCdc42, caRho, dnRho, caRac, dnRac, or control CD56 protein (cntr). (control protein)-transduced DC, whereas a Rho GTPase inhibitor Because no significant differences between control protein-transfected DC and nontransfected DC were detected, only control transfection-related re- ToxB significantly inhibits DC adhesiveness in both groups. Fur- sults are shown in this figure. A, All groups of transduced DC were pulsed thermore, we showed that Cdc42 played a major role among other with dialyzed whole OVA (1 mg/ml) for 24 h, collected, washed, and Rho GTPases in the regulation of DC adhesion in vitro, because treated with medium (control), CNF1 (250 ng/ml; 30 min), or ToxB (1 transduction of DC with caCdc42 strongly increased DC adhesion ng/ml; 60 min). Then, DC were washed and mixed (5 ϫ 103 cells) with to extracellular matrix. Rho GTPase inhibitor ToxB significantly syngeneic D11.10 T cell clone (2 ϫ 104) in triplicate in 96-well U-bottom down-regulates adhesion of caCdc42-transfected DC, demonstrat- plates. Cell-free supernatants were collected 48 h later, and concentrations ing a specificity for this effect. However, transduction of DC with p Ͻ 0.05 vs control DC (one-way caRho caused only a modest elevation of DC adhesion, and caRac ,ء .of IL-2 were determined by ELISA ANOVA). The results from a representative experiment are shown as had no effect. mean Ϯ SEM (n ϭ 3). B, Migration and chemoattraction of modified DC This effect may be explained by the formation or contraction of were studied using 48-well Transwell plates with 5-␮m pores as described in Materials and Methods. Spontaneous DC migration (medium) or mi- dendrites by different Rho GTPases. For example, Swetman et al. gration toward fMLP (10Ϫ7 M) and MIP1␣ (10 ng/ml) were tested in a 4-h (9) showed that DC dendrite formation is directed by members of assay with 106 DC/ml. Cells transmigrated through the membrane, and the Rho GTPase family, with the Cdc42 acting predominantly to were enumerated by FACScan three times for 60 s. Results are shown as promote cellular extension and spreading, and Rho, conversely, p Ͻ 0.05 vs control DC (one-way ANOVA; n ϭ 3). acting to drive contraction and detachment of the dendrites. Using ,ء .mean Ϯ SEM The Journal of Immunology 3399

caRho-transduced DC to present OVA peptides to the specific OVA-recognizing T cell clone DO11.10 obtained from transgenic

mice expressing the TCR specific for the OVA323–339 peptide. We have shown that CNF1, an activator of small Rho GTPases, in- creased, whereas ToxB, an inhibitor of Rho GTPases, decreased the ability of DC to present the OVA to specific T cells. Further- more, caCdc42 and caRho increased Ag presentation by DC, and the effect was prevented by ToxB. dnCdc42 and dnRho decreased Ag presentation. Surprisingly, both ca and dn forms of Rac blocked DC Ag presentation, which suggests that Rac may serve as a down-regulator of Cdc42 and Rho either directly or indirectly. It is possible that increased Ag presentation by DC with activated Rho GTPases is mediated by increased endocytosis. It is known that DC developmentally regulate endocytosis by controlling lev- els of activated Cdc42 (5). We have confirmed these data by show- ing that uptake of FITC-dextran, i.e., receptor-mediated endocy- tosis, was significantly up-regulated by Rho GTPase activators CNF1 and PMA and reduced by Rho GTPase inhibitor ToxB.

Transfection of DC with caCdc42 increased their endocytotic ac- Downloaded from tivity, whereas transfection with dnCdc42 decreased it. Similarly, it was reported that treatment of immature spleen-derived DC (8) or bone marrow-derived DC (5) with ToxB completely blocked the macropinocytotic uptake of fluorescent fluid-phase markers, a form of high-volume, nonspecific endocytosis involving the extension of

membrane ruffles. The involvement of specific Rho family members http://www.jimmunol.org/ was dissected by microinjection of dn inhibitory versions of the different Rho GTPases. Administration of dnRac1 or dnCdc42 blocked macropinocytosis in the immature DC. Significantly, in mature DC, which have down-regulated macropinocytosis, microin- jection of activated Cdc42 stimulated endocytosis, indicating that the machinery for macropinocytosis controlled by Cdc42 remains intact after maturation (5). These results implicate both Cdc42 and Rac in the regulation of endocytosis in DC and strongly suggest that the shutdown in endocytosis seen during maturation is caused by inacti- by guest on September 26, 2021 vation of Cdc42 (6). Thus, Cdc42-mediated up-regulation of endocy- tosis may play a role in increased Ag presentation by DC. Additional mechanisms are also likely to be involved. For instance, using a specific Rho inhibitor exoenzyme C3 and a specific Rho-associated kinase inhibitor Y-27632, it has been demonstrated that inactivation of Rho in DC was associated with inhibited interaction between DC and CD4ϩ T cells and ϳ80% reduction of T cell stimulatory capacity in allogeneic MLR, although the surface expression of MHC, co- FIGURE 6. Regulation of endocytotic activity in DC by the Rho family stimulatory, and adhesion molecules were unaffected (7, 33). Finally, GTPases. Receptor-mediated endocytosis of murine DC was assessed by because intracellular transport of secretory vesicles and exocytosis is FITC-labeled dextran 40 uptake using FACScan analysis as described in Materials and Methods. DC were pretreated with 250 ng/ml CNF1, 60 also regulated by small Rho GTPases (34–36), one can hypothesize p Ͻ 0.05 that transport of MHC class I/peptide complexes to the cell surface ,ء .ng/ml PMA, 1 ng/ml ToxB, or medium (control) for 30 min vs control (ANOVA). A, Representative from three independent experi- and increased production of DC cytokines may up-regulate T cell ments. B, The results are shown as the mean Ϯ SEM from three indepen- activation by DC as well. However, this hypothesis requires further dent experiments. experimental verification. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced (fMLP and MIP1␣) DC migration. Inter- a specific Rho inhibitor exoenzyme C3 from Clostridium botuli- estingly, the dnCdc42 mutant did not modulate DC migration, num and a specific Rho-associated kinase inhibitor Y-27632, it has which suggests that the inhibitory effect of active Cdc42 may in- been demonstrated that both C3 and Y-27632 markedly reduce volve activation of other members of the Rho family known to actin polymerization in parallel with the disappearance of den- play a role in the regulation of chemotaxis and migration in other drites in DC (7, 33). Furthermore, DC microinjected with a ca cell types. Unexpectedly, all tested mutant forms of Rac and Rho mutant of Cdc42, which lacks intrinsic GTPase activity, show ex- (except dnRac) significantly inhibited both migration and chemo- aggerated filopodial activity, whereas dnCdc42, which depletes the taxis of transduced DC. Taken together, these data suggest a com- cell of guanine nucleotide exchange factors, inhibits spike forma- plex mechanism and multiple roles for Rho GTPases in the regu- tion and membrane ruffles (28). lation of DC chemotaxis and migration. This is in accordance with Next, we have demonstrated the role of Rho GTPases in the the published data on macrophages, where Allen at al. (18) have regulation of MHC class II Ag presentation and chemotaxis in DC. recently reported that Rho and Rac are required for the process of To evaluate the ability of DC to present specific Ag, we have tested macrophage migration, whereas Cdc42 is required for cells to the capacity of dnCdc42-, caCdc42-, dnRac-, caRac-, dnRho-, or respond to chemokines but is not essential for cell locomotion 3400 Rho GTPases REGULATE DC FUNCTION and migration. The authors also reported that caRho, caRac, and 9. Swetman, C. A., Y. Leverrier, R. Garg, C. H. Gan, A. J. Ridley, D. R. Katz, and caCdc42 reduce macrophage translocation, and/or migration and B. M. Chain. 2002. Extension, retraction and contraction in the formation of a dendritic cell dendrite: distinct roles for Rho GTPases. Eur. J. Immunol. 32:2074. activated Rho may act antagonistically to Rac and Cdc42. Simi- 10. Van Aelst, L., and C. D’Souza-Schorey. 1997. Rho GTPases and signaling net- larly, inhibition of endogenous Rho and Rac proteins also prevents works. Genes Dev. 11:2295. chemokine-induced cell migration, both in DC and in macrophages 11. Leonard, D., M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione. 1992. The identification and characterization of a GDP-dissocia- (18). In another report, Weber et al. (19) have demonstrated that tion inhibitor (GDI) for the CDC42Hs protein. J. Biol. Chem. 267:22860. CC chemokines, including MIP1␣, or caCdc42 induce formation 12. Aspenstrom, P. 1999. The Rho GTPases have multiple effects on the actin cy- of filopodia-like projections in monocytes. Both caCdc42 and toskeleton. Exp. Cell Res. 246:20. 13. Boettner, B., and L. Van Aelst. 2002. The role of Rho GTPases in disease de- dnCdc42 mutants inhibited CC chemokine-induced monocyte mi- velopment. Gene 286:155. gration, implicating Cdc42 activity and its effector functions in 14. Kjoller, L., and A. Hall. 1999. Signaling to Rho GTPases. Exp. Cell Res. 253:166. chemotaxis (19). Cdc42 has been also involved in membrane ruf- 15. Boguski, M. S., and F. McCormick. 1993. Proteins regulating Ras and its rela- tives. Nature 366:643. fling induced by fMLP and PMA (37). 16. Olofsson, B. 1999. Rho guanine dissociation inhibitors: pivotal molecules in Additional evidence of the important role of Rho GTPases in cellular signalling. Cell. Signal. 11:545. DC function was obtained from the investigation of DC harvested 17. Johnson, D. I. 1999. Cdc42: an essential Rho-type GTPase controlling eukaryotic . Microbiol. Mol. Biol. Rev. 63:54. from Wiskott-Aldrich syndrome protein (WASP)-null animals and 18. Allen, W. E., D. Zicha, A. J. Ridley, and G. E. Jones. 1998. A role for Cdc42 in WASP-null humans. WASP is a member of a recently defined family macrophage chemotaxis. J. Cell Biol. 141:1147. of proteins that are involved in the transduction of signals to the actin 19. Weber, K. S., L. B. Klickstein, P. C. Weber, and C. Weber. 1998. Chemokine- induced monocyte transmigration requires -mediated cytoskeletal changes. cytoskeleton (28). WASP is uniquely expressed on hemopoietic cells, Eur. J. Immunol. 28:2245. and is the specific effector of Cdc42 (38). WASP-deficient DC have 20. Shurin, M. R., P. P. Pandharipande, T. D. Zorina, C. Haluszczak, V. M. Subbotin, Downloaded from marked abnormalities of the cellular cytoskeleton (including short- O. Hunter, A. Brumfield, W. J. Storkus, E. Maraskovsky, and M. T. Lotze. 1997. FLT3 ligand induces the generation of functionally active dendritic cells in mice. ened filopodia) and are severely compromised in chemotaxis and their Cell. Immunol. 179:174. ability to migrate (7, 39). WASP-null DC have no podosome (spe- 21. Jiang, K., D. L. Gilvary, P. K. Epling-Burnette, C. Ritchey, J. Liu, R. J. Jackson, cialized adhesion structures with ␤ -integrin clusters) and a reduced E. Hong-Geller, and S. Wei. 2003. Regulation of Akt-dependent cell survival by 2 Syk and Rac. Blood 101:3240. ability to adhere to ICAM1-coated surfaces (28). The disturbances in 22. Aktories, K. 1997. Rho proteins: target for bacterial toxins. Trends Microbiol. motility of WASP-null DC might relate to abnormalities of Cdc42- 5:282. http://www.jimmunol.org/ WASP-mediated filopodia formation, and they are consistent with 23. Lerm, M., M. Pop, G. Fritz, K. Aktories, and G. Schmidt. 2002. Proteasomal deg- radation of cytotoxic necrotizing factor 1-activated . Infect. Immun. 70:4053. suggestions that these structures are essential for chemotaxis (18, 31). 24. Zhou, K., Y. Wang, J. L. Gorski, N. Nomura, J. Collard, and G. M. Bokoch. 1998. In fact, it has been recently reported that chemokine CCL19 induces Guanine nucleotide exchange factors regulate specificity of downstream signaling rapid dendritic extension of murine DC, which was Rac and Cdc42, from Rac and Cdc42. J. Biol. Chem. 273:16782. 25. Benard, V., B. P. Bohl, and G. M. Bokoch. 1999. Characterization of rac and but not Rho, dependent (40). The authors postulated that the chemo- cdc42 activation in chemoattractant-stimulated human neutrophils using a novel kine-induced dendritic extension was positively regulated by Rac or assay for active GTPases. J. Biol. Chem. 274:13198. Cdc42, but negatively regulated by Rho. Together, these data and our 26. Burbelo, P. D., D. Drechsel, and A. Hall. 1995. A conserved binding motif de- fines numerous candidate target proteins for both Cdc42 and Rac GTPases. results demonstrate differential requirements for the Rho family GT- J. Biol. Chem. 270:29071. Pases in DC motility and suggest that a dynamic regulation of Rac and 27. Zhang, B., J. Chernoff, and Y. Zheng. 1998. Interaction of Rac1 with GTPase- by guest on September 26, 2021 Rho may be required for DC chemotaxis. activating proteins and putative effectors: a comparison with Cdc42 and RhoA. J. Biol. Chem. 273:8776. In summary, members of the Rho GTPases family, Cdc42, Rho, 28. Thrasher, A. J. 2002. WASp in immune-system organization and function. Nat. and Rac, have emerged as key coordinators of signaling pathways Rev. Immunol. 2:635. leading to remodeling of the actin cytoskeleton, transcriptional regu- 29. Ridley, A. J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. lation, and the cell cycle; i.e., the processes that play a critical role in Cell 70:389. cell adhesion, Ag presentation, migration, chemotaxis, and endocyto- 30. Govind, S., R. Kozma, C. Monfries, L. Lim, and S. Ahmed. 2001. Cdc42Hs sis. Our results demonstrate that Ag processing and presentation by facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin. J. Cell Biol. 152:579. DC is regulated by the Rho GTPase family members, and these data 31. Allen, W. E., G. E. Jones, J. W. Pollard, and A. J. Ridley. 1997. Rho, Rac and may serve as a basis for the characterization of Rho GTPase effector Cdc42 regulate actin organization and cell adhesion in macrophages. J. Cell Sci. molecules that modulate DC function and have therapeutic potential. 110:707. 32. Yang, F. C., S. J. Atkinson, Y. Gu, J. B. Borneo, A. W. Roberts, Y. Zheng, J. Pennington, and D. A. Williams. 2001. Rac and Cdc42 GTPases control he- Disclosures matopoietic stem cell shape, adhesion, migration, and mobilization. Proc. Natl. The authors have no financial conflict of interest. Acad. Sci. USA 98:5614. 33. Kobayashi, M., E. Azuma, M. Ido, M. Hirayama, Q. Jiang, S. Iwamoto, References T. Kumamoto, H. Yamamoto, M. Sakurai, and Y. Komada. 2001. A pivotal role of Rho GTPase in the regulation of morphology and function of dendritic cells. 1. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of J. Immunol. 167:3585. immunity. Nature 392:245. 34. Nassar, N., R. A. Cerione, and E. Hong-Geller. 2000. Cdc42 and Rac stimulate 2. Steinman, R. M. 2003. Some interfaces of dendritic cell biology. APMIS 111:675. exocytosis of secretory granules by activating the IP /calcium pathway in RBL- 3. Mellman, I., and R. M. Steinman. 2001. Dendritic cells: specialized and regulated 3 2H3 mast cells. Cell 100:345. antigen processing machines. Cell 106:255. 35. Ellis, C., G. J. Clark, F. Tamanoi, and W. Guo. 2001. Spatial regulation of the 4. Inaba, K., S. Turley, T. Iyoda, F. Yamaide, S. Shimoyama, C. Reis e Sousa, exocyst complex by Rho1 GTPase. Methods Enzymol. 333:217. R. N. Germain, I. Mellman, and R. M. Steinman. 2000. The formation of im- munogenic major histocompatibility complex class II-peptide ligands in lysoso- 36. Perret-Menoud, V., S. Gattesco, S. Magnin, I. Pombo, U. Blank, R. Regazzi, and mal compartments of dendritic cells is regulated by inflammatory stimuli. J. Exp. C. Frantz. 2002. Involvement of Rho GTPases and their effectors in the secretory Med. 191:927. process of PC12 cells. Biochem. J. 362:273. 5. Garrett, W. S., L. M. Chen, R. Kroschewski, M. Ebersold, S. Turley, 37. Cox, D., P. Chang, Q. Zhang, P. G. Reddy, G. M. Bokoch, and S. Greenberg. S. Trombetta, J. E. Galan, and I. Mellman. 2000. Developmental control of en- 1997. Requirements for both Rac1 and Cdc42 in membrane ruffling and phago- docytosis in dendritic cells by Cdc42. Cell 102:325. cytosis in leukocytes. J. Exp. Med. 186:1487. 6. Nobes, C., and M. Marsh. 2000. Dendritic cells: new roles for Cdc42 and Rac in 38. Thrasher, A. J., S. Burns, R. Lorenzi, and G. E. Jones. 2000. The Wiskott-Aldrich antigen uptake? Curr. Biol. 10:R739. syndrome: disordered actin dynamics in haematopoietic cells. Immunol. Rev. 7. Burns, S., A. J. Thrasher, M. P. Blundell, L. Machesky, and G. E. Jones. 2001. 178:118. Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS 39. Binks, M., G. E. Jones, P. M. Brickell, C. Kinnon, D. R. Katz, and A. J. Thrasher. protein, and differentiation. Blood 98:1142. 1998. Intrinsic dendritic cell abnormalities in Wiskott-Aldrich syndrome. Eur. 8. West, M. A., A. R. Prescott, E. L. Eskelinen, A. J. Ridley, and C. Watts. 2000. J. Immunol. 28:3259. Rac is required for constitutive macropinocytosis by dendritic cells but does not 40. Yanagawa, Y., and K. Onoe. 2002. CCL19 induces rapid dendritic extension of control its downregulation. Curr. Biol. 10:839. murine dendritic cells. Blood 100:1948.