Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b

This information is current as Yu Li, Jingliang Yan, Pradip De, Hua-Chen Chang, Akira of September 25, 2021. Yamauchi, Kent W. Christopherson II, Nivanka C. Paranavitana, Xiaodong Peng, Chaekyun Kim, Veerendra Munugulavadla, Reuben Kapur, Hanying Chen, Weinian Shou, James C. Stone, Mark H. Kaplan, Mary C. Dinauer, Donald L. Durden and Lawrence A. Quilliam Downloaded from J Immunol 2007; 179:8322-8331; ; doi: 10.4049/jimmunol.179.12.8322 http://www.jimmunol.org/content/179/12/8322 http://www.jimmunol.org/ References This article cites 79 articles, 49 of which you can access for free at: http://www.jimmunol.org/content/179/12/8322.full#ref-list-1

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins1

Yu Li,2*¶ Jingliang Yan,*¶ Pradip De,ʈ Hua-Chen Chang,†‡§ Akira Yamauchi,§ Kent W. Christopherson II,** Nivanka C. Paranavitana,3*¶ Xiaodong Peng,§ Chaekyun Kim,§# Veerendra Munugulavadla,§ Reuben Kapur,*‡§ Hanying Chen,§ Weinian Shou,*§ James C. Stone,†† Mark H. Kaplan,†‡§ Mary C. Dinauer,†‡§ Donald L. Durden,ʈ and Lawrence A. Quilliam4*¶

The Ras-related Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical Downloaded from production, and cancer. To gain a better understanding of function in mammalian physiology, we deleted the Rap1a . Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some ,Rap1a؊/؊ embryos died in utero. , B cell, or myeloid cell development was not disrupted in Rap1a ؊/؊ mice. However macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human http://www.jimmunol.org/ neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a؊/؊ mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events. The Journal of Immunology, 2007, 179: 8322–8331.

he small GTPase Ras is an important regulator of cell but encoded by a separate gene, also exists (7) in addition to three growth and differentiation (1). It operates as a molecular Rap2 proteins that share ϳ65% identity with Rap1 (2, 8, 9). Like switch, alternating between inactive GDP and active Ras, these GTPases are activated by guanine nucleotide exchange by guest on September 25, 2021 T 5 GTP-bound states and is the prototype for more than 130 Ras- factors (GEFs) and turned off by GTPase activating proteins related proteins that regulate a myriad of biological functions (1). (GAPs). The activation of Raps by a large number of diversely Rap1a/Krev-1/Smg p21 was originally identified because of ho- regulated GEFs suggests that they play ubiquitous roles in cell mology with Ras (2), by purification of GTP binding proteins (3, biology triggered by multiple extracellular stimuli and associated 4), by copurification with the neutrophil NADPH oxidase (5), and signaling pathways, reviewed in Ref. 10–12. Although Rap1 and by its ability to revert the phenotype of Ras-transformed cells (6). 2 are regulated by the same GEFs and GAPs, until very recently A closely related , Rap1B, sharing 95% amino acid identity little was known about their biological functions. Even less is known about the functional redundancy of Rap1a and 1b. It was originally reported that Rap1 operates as an antagonist of † *Department of Biochemistry and Molecular Biology, Department of Microbiology Ras, competing with it for downstream effectors such as c-Raf-1. and Immunology, ‡Department of Pediatrics, Medical and Molecular Genetics, and §Wells Center for Pediatric Research, Indiana University School of Medicine, Indi- Although this can occur in certain cell types (13), Rap1 has more anapolis, IN 46202; ¶Walther Cancer Institute, Indianapolis, IN 46208; ʈAflac Cancer recently been found to play a number of additional roles. These Center and Blood Disorders, Department of Pediatrics, Emory University, Atlanta, GA 30322; #Inha University College of Medicine, Incheon, Korea; **Rush University include activation of the ERK/MAPK pathway via B-Raf, promo- Medical Center, Chicago, IL 60612; and ††Department of Biochemistry, University of tion of cell-matrix adhesion through inside-out activation of inte- Alberta, Edmonton, Alberta, Canada grins via RAPL and RIAM, and regulation of cell-cell junctions Received for publication June 26, 2007. Accepted for publication October 12, 2007. (reviewed in Refs. 12, 14, 15). Overactivation of Rap1 has also The costs of publication of this article were defrayed in part by the payment of page been recently associated with hemopoietic malignancy (16, 17). charges. This article must therefore be hereby marked advertisement in accordance Loss of the only rap1 gene in Drosophila is embryonic lethal (18). with 18 U.S.C. Section 1734 solely to indicate this fact. Similarly, loss of C3G/RapGEF1, the most ubiquitously expressed 1 This work was supported by an Indiana University School of Medicine Biomedical Research Grant, the Indiana Genomics Initiative of Indiana University, supported in Rap exchange factor is lethal to mice (19, 20). However, because part by Lilly Endowment Incorporated, and by National Institutes of Health Grants C3G also acts on additional Ras family proteins, it was not clear CA108647 and HL69974 (all to L.A.Q.). Y.L. was supported by American Heart Association predoctoral fellowship 0110245Z. 2 Current address: Amgen, One Amgen Center Drive Thousand Oaks, CA 91302. 3 Current address: Applied Biosystems, 2130 Woodward Street, Austin, TX 78744. 5 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; GAP, 4 Address correspondence and reprint requests to Dr. Lawrence A. Quilliam, Depart- GTPase activating protein; ES cells, embryonic stem cells; KO, knockout; WT, wild ment of Biochemistry and Molecular Biology, 635 Barnhill Drive, MS-4053, Indiana type; MEF, mouse embryo fibroblast; BMD, bone marrow-derived; University School of Medicine, Indianapolis, IN 46202. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 8323 whether this lethality was solely due to the loss of Rap1 activity fragment as template to confirm the long arm structure and correct inte- (21–24). gration 3Ј to the recombination site. PstI-digested ES cell genomic DNA To better understand the role of Rap1 in mammalian cell biol- was hybridized with a neo probe using a XhoI-SalI fragment from the neomycin resistance gene to confirm correct integration of 5Ј to the re- ogy and to address possible redundancy between the 1a and 1b combination site. Four ES cell clones were confirmed to contain the tar- Ϫ/Ϫ proteins, we generated a mouse lacking the gene. rap1A geted disruption and used to generate Rap1a KO mice by standard proce- mice initially exhibited no overt phenotype. However, upon back- dures (30) using C57BL/6J blastocysts and pseudopregnant females as crossing mice into the C57BL/6J strain, some embryonic lethality foster mothers. Chimeric mice were successfully generated from just one clone. These were screened by PCR and by Southern blotting for the pres- was observed. Because of the high abundance of Rap1a in human ence of the disrupted Rap1a allele and mated with C57BL/6J mice to platelets (25) and Rap1a in human neutrophils (26) where it asso- produce F1 heterozygous mice. Heterozygotes for the disrupted gene were ciates with the phagocyte NADPH oxidase (5, 27), we examined identified by PCR using digit samples along with three primers, the com- several properties of Rap1a-null white blood cells. Although there mon primer Rap1a 5Ј KO, the neo specific primer ADPR266, and the wild-type (WT) allele specific primer RapExon (5Ј-CCGTAAAATCTGT were no obvious defects in blood cell development, changes in Ј TCTCTC AAGTC-3 ). Confirmed heterozygous F1 mice were mated with macrophage adhesion, haptotaxis, and phagocytosis were observed siblings to generate null mutant mice. Mice were backcrossed six times and neutrophils produced less superoxide in response to formy- with C57BL/6J male and female mice to create F7 generation. F2 gener- lated peptide and phorbol ester challenge despite the presence of ation mice were used for characterization except where indicated. All mice Rap1b in these cells. These data support the role of Rap1 in cell were maintained in temperature- and humidity-controlled conditions with a 12-h light-12-h dark cycle and were allowed food and water ad libitum. All adhesion and migration, provide a unique model for studying Rap1 procedures described were approved by the Indiana University School of function, and suggest that the Rap1a and 1b proteins have both Medicine animal care and use committee. redundant and unique biological functions. Downloaded from Southern blot analysis Materials and Methods Genomic DNA from ES cells was prepared with the Puregene DNA iso- Generation of Rap1A targeting vector lation kit (Gentra). Genomic DNA was isolated from mouse tails by the proteinase K digestion method. Mouse tail pieces were clipped and di- A ϳ19 kb partial Rap1a genomic clone isolated from a 129Sv/J mouse gested in a solution containing 100 mM NaCl, 10 mM Tris-HCl (pH 7.8), genomic library (28) was sequenced to generate a restriction map and iden- 1% SDS, 200 ␮g of proteinase K per ml at 57°C overnight. After two tify exons. The clone was found to encode exons 3 through 6 of rap1A that chloroform-isoamyl alcohol (24/1) extractions, the DNA was precipitated http://www.jimmunol.org/ encode residues 43–184. To generate the targeting vector, a 0.95 kb PvuII- with ethanol. Genomic DNA (10–20 ␮g) digested with SpeIorPstI(ϳ10 NdeI fragment, located upstream of exon 4, was blunted and inserted into U/␮g) was resolved by electrophoresis in 0.5% agarose gels and transferred the SalI site of the pBluescript II/NeoTK vector (Fig. 1A) (29). This vector to nitrocellulose membranes (BA-85; Shleicher and Schuell). DNA frag- contains the phosphoglycerokinase promoter driving the neo gene and the ment probes were radiolabeled with 32P using the Megaprime DNA label- herpes simplex virus thymidine kinase gene both being expressed in the ing system (GE Biosciences). opposite direction to the Rap1a sequence. An 8.6 kb NdeI-StuI fragment downstream of exon 4 was inserted at the XhoI site of the vector. By this Flow cytometry strategy, exon 4 (codons 62–108) and surrounding sequence was replaced by the neomycin resistance gene. The targeting vector was linearized using All Abs were purchased from BD Biosciences. Surface expression on the NotI and electroporated into embryonic stem (ES) cells. indicated cell populations was analyzed by flow cytometry (FACScan or FACSCaliber; Becton Dickinson). The percent positive population was by guest on September 25, 2021 Generation of Rap1A knockout (KO) mice evaluated by the CellQuest program (BD Biosciences). Culture of ES cells and isolation of homologous recombinants were per- Blood cell proliferation assays formed according to standard protocols (30). Screening of 261 G418/gan- cyclovir double resistant ES clones for homologous recombinants was per- Cells were isolated from spleens of the indicated genotypes of mice. Cells formed by PCR methods as described (30) using 5Ј-TCTATCGCC were then plated in triplicate at 5 ϫ 104 cells/well in a round-bottom TTCTTGACGAGTTC-3Ј (ADPR266; in the neo gene) and 5[prime] 96-well plate in RPMI 1640 medium supplemented with 10% heat-inacti- CTTGCTCTCCTGTTACCTATAGGTGCC-3Ј (Rap1a 5Ј KO; in the vated FBS (HyClone) and stimulated with 2.5 ␮g/ml platebound anti-CD3 Rap1a gene) as primers (Fig. 1). Five positive clones were identified and (for T cells) or 2.5 ␮g/ml anti-IgM Ϯ 5 ng/ml IL-4 (for B cells) for 72 h. subjected to Southern blot analysis to confirm the structure of the targeted Cells were pulsed for the last 16–18 h with 1 ␮Ci per well of [3H]thymi- region. ES cell genomic DNA was digested with SpeI and hybridized with dine. The incorporation of [3H]thymidine was measured with a liquid scin- an external probe located at 3Ј of the long arm using a 1.1 kb StuI-BglII tillation counter.

A 1358 bp PCR product S P S N N Stu Bgl Rap1A gene 3 4 5 6 S Pst Pst Recombinant gene 3 Neo 5 6

WT KO 1107 bp PCR product C Ab B F2 progeny Controls Rap1A +/+ +/+ -/- +/- +/- Con WT H20 F1 WT Rap1 1358 bp Rap2 KO 1107 bp GAPDH FIGURE 1. Rap1a deletion strategy and detection. A, KO strategy showing replacement of exon 4 with neo resistance gene. Positions of genotyping primers are shown as well restriction used in gene manipulation or analysis. Bgl, BglII; N, NdeI; Pst, PstI; P, PvuII; S, SpeI; Stu, StuI. Numbered regions indicate location of exons, solid black bars show short and long arms used in homologous recombination. B, PCR analysis of Rap1a null mice. Using a common primer 5Ј of the targeted sequence (in exon 3) and 3Ј primers complementary to either exon 4 or the neoR gene, PCR detected Rap1a ϩ/ϩ (upper Ϫ Ϫ ϩ Ϫ band only) / (lower band only) or / (both bands) mice. Con, control plasmid template; F1, chimeric founder mouse. C, Western blot of Rap expression in mouse neutrophils. Cells were blotted for Rap1a, total Rap1, or Rap2 expression using specific Abs. GAPDH was used as an internal loading control. Data are shown for three separate Rap1aϩ/ϩ and Ϫ/Ϫ mice. 8324 CHARACTERIZATION OF Rap1a-DEFICIENT MOUSE MYELOID CELLS

Th cell differentiation assay thermore, a ϳ16 kDa protein that could potentially have arisen CD4ϩ cells were purified from spleen and lymph node cells by MiniMacs from splicing of exons 3 and 5 was not detected. This was con- positive selection and plated in RPMI 1640 plus 10% FBS. Cells were sistent with the lack of Rap1 detection from an engineered Rap1a plated at 1 ϫ 106/ml stimulated with 4 ␮g/ml platebound anti-CD3, 0.5 cDNA, missing the equivalent 47 codons encoded by exon 4, upon ␮g/ml anti-CD28 in either Th1 (2 ng/ml IL-12 ϩ 10 ␮g/ml anti-IL-4) or transfection of 293T cells (residues 62–108; Y. Li and L. Quilliam, Th2 (10 ng/ml IL-4 ϩ 10 ␮g/ml anti-IFN-␥) culture conditions. After 5 ␮ unpublished observations). Western blotting with a pan Rap1 Ab days in culture, cells were washed and restimulated with 2 g/ml plate- ϳ bound anti-CD3 alone. Supernatants were collected after 24 h and tested that detects Rap1a and 1b, but not Rap2 indicated a 25% de- for levels of IL-4, IL-5, IL-13, and IFN-␥. crease in total Rap1 protein expressed in neutrophils following deletion of Rap1A. Only modest changes in neutrophil Rap2 were Macrophage isolation, cell migration, and phagocytosis detected (Fig. 1C). adhesion assays Ϫ/Ϫ Macrophages were prepared as previously described (31). Cell migration Rap1a mouse viability depended on background strain assays were performed on polycarbonate membranes using Transwell mi- Rap1aϪ/Ϫ mice were viable, healthy, and fertile, suggesting that gration chambers (pore size 8 ␮m; Costar) as previously described (31). The underside of the membrane to which cells migrate was coated with 20 the Rap1a gene is not required for mouse development or breeding. ␮g/ml of either vitronectin, or an ␣4␤1 integrin-binding fragment of fi- Segregation of the mutant Rap1a allele followed Mendelian inher- bronectin, H296, in PBS for1hat37°C. Surfaces were subsequently itance (24%:51%:25% ratio of ϩ/ϩ:ϩ/Ϫ:Ϫ/Ϫ from 446 mice blocked with heat-denatured BSA. Cell adhesion assays were performed born from heterozygote mating) with ϩ/ϩ and Ϫ/Ϫ mice weight ␮ using flat-bottom 96-well polystyrene plates coated with 20 g/ml of either and the organ/body weight ratio of their brain, heart, liver, and vitronectin or the H296 fibronectin peptide. Adherent cells were fixed with 3.5% formaldehyde and stained with 0.1% crystal violet. The stain was lungs being indistinguishable between genotypes (Y. Li and L. Downloaded from eluted with 10% acetic acid, and absorbance was determined at 600 nm Quilliam, unpublished observations). These mice were used for the with a microplate reader (31). Recombinant human fibronectin peptide initial characterization of Rap1a function. However after back- H296 was obtained from Collaborative Biomedical. Vitronectin was puri- crossing for six generations into the C57BL/6J mouse strain, the fied as described previously (31). Phagocytosis of serum opsinized-zymo- Ϫ/Ϫ san or IgG-coated sheep RBC was performed as described (32). number of Rap1a pups generated from heterozygote crosses was significantly reduced (30.3:54.8:14.9% ratio of ϩ/ϩ:ϩ/Ϫ: Chemotaxis Ϫ/Ϫ from 241 mice) with no sex bias. Dissection of pregnant http://www.jimmunol.org/ Ϫ Ϫ Chemotaxis assays were performed using 96-well chemotaxis chambers Rap1a / mice at E9.5–14.5 indicated that the lethality occurred (NeuroProbe) in accordance with the manufacturer’s instructions as de- much earlier than E9.5 due to no trace of embryos in the deciduas scribed previously (33) (H. Chen and W. Shou, unpublished observations). Superoxide anion measurement Normal T cell development and function in the absence of Purification of mature neutrophils from the bone marrow of 11–16 wk old Rap1a mice was performed by centrifugation through a Percoll gradient and His- topaque-1119 essentially as described (34). Superoxide response to 10 ␮M Rap1 is highly expressed in various cells of hematopoeitic lineage fMetLeuPhe was measured using chemiluminesence essentially as de- (13, 26, 36, 37). Therefore, we next examined whether the loss of scribed (35). Superoxide production in response to phorbol ester (200 Rap1a impacted T or B cell function. Rap1 is activated in T cells by guest on September 25, 2021 ng/ml phorbol myristate acetate) was determined by both reduction of cy- tochrome C and by chemiluminescence (34, 35). by TCR engagement variably resulting in decreased intracellular signals and enhanced integrin-mediated adhesion (13, 37–39). Detection of Rap1 proteins in mouse BM neutrophils However, the role of Rap1 in T cell development had not been Neutrophils were suspended in PBS to no greater than 5 ϫ 107/ml. A total examined. To determine whether Rap1a is required for normal T of 2.7 mM diisopropyl fluorophosphate, a serine protease inhibitor, was cell development, we first analyzed thymocytes from WT and 7 added and incubated for 10 min on ice. After two washes in PBS, 5 ϫ 10 Rap1aϪ/Ϫ mice. Thymi from both genotypes had similar cellular- cells were lysed in 1 ml IPB buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 20 ng/␮l chymostatin, 2 mM PMSF, 10 ity and flow cytometry revealed similar percentages of cells in the nM leupeptin, 1 mM 4-c2-aminoethyl benzenesulfomyl fluoride) with 30 CD4 and CD8 double-negative, double-positive, and single-posi- min incubation on ice. Cell debris were centrifuged down at 16,000 ϫ g for tive populations (Fig. 2A). Within the double negative population, 2 min at 4°C. Cleared lysates were mixed with 2ϫ sample buffer and boiled there were similar percentages of cells expressing CD25 and CD44 for 5 min. Thirty micrograms of lysate protein was loaded onto SDS-PAGE (H-C. Chang and M. Kaplan, unpublished observations). These gels. Rap1a protein was detected with mAb T22, a gift of Dr. Yoshimi Takai (Osaka University, Osaka, Japan) (26), and total Rap1a/1b by anti- results suggest that Rap1a-deficiency does not adversely affect T Rap1 R195 Ab. Rap2 protein was detected with anti-Rap2 from BD cell development. Biosciences. Analysis of peripheral blood and spleen by flow cytometry also revealed similar percentages of CD4ϩ and CD8ϩ cells with similar Results Ϫ/Ϫ cellularity observed in lymph node, peripheral blood, and spleen Generation of Rap1a mice between WT and Rap1aϪ/Ϫ mice (Fig. 2B and H-C. Chang and M. To explore the biological role of Rap1a, a targeting construct that Kaplan, unpublished observations). To determine whether T cells replaces the fourth exon of the murine GTPase with a neomycin were functional, WT and Rap1aϪ/Ϫ splenocytes were stimulated resistance gene cassette was developed and used to disrupt the with 2.5 ␮g/ml anti-CD3 for 48 h and proliferation was assessed Rap1a gene in ES cells by homologous recombination. Proper in- by [3H]thymidine incorporation. As shown in Fig. 2C, both WT tegration of the cassette was established by Southern blot (Y. Li and Rap1a-deficient T cells proliferated to a similar extent in re- and L. Quilliam, unpublished observations) and PCR analysis (Fig. sponse to anti-CD3. To further test T cell function, CD4ϩ T cells 1B). Embryonic stem cells were injected into mouse blastocysts. were purified from WT and Rap1a-deficient mice and differenti- Chimeric mice were then bred to obtain germline transmission of ated to Th1 and Th2 phenotypes using cytokine and Ab mixtures the disrupted allele. Heterozygous mice were further bred to obtain as described in Materials and Methods. The resulting populations Rap1a-deficient mice. Purified neutrophils were blotted for Rap1a were then washed, counted, and restimulated with anti-CD3 alone. expression using an isoform-specific mAb (26). Rap1a was not WT and Rap1a-deficient Th1 cells secreted similar levels of IFN-␥ detected in these cells (Fig. 1C), or in various organs derived from and minimal levels of Th2 cytokines (Fig. 2D). Conversely, Th2 Ϫ/Ϫ mice (Y. Li and L. Quilliam, unpublished observations). Fur- cells derived from both genotypes secreted similar levels of the The Journal of Immunology 8325

Th2 cytokines IL-4, IL-5, and IL-13 with little IFN-␥ (Fig. 2D). Together, these results indicate that Rap1a is not required for T cell development, migration to peripheral lymphoid organs, prolifera- tion, or Th cell differentiation.

Normal B cell development and function in Rap1a-deficient mice Rap1 is also activated by BCR engagement (40). To determine whether Rap1a is required for B cell development, we first ana- lyzed B cells in the bone marrow of WT and Rap1a-deficient mice. Percentages of B220ϩ and B220ϩCD43ϩ populations were indis- tinguishable between the two genotypes (Fig. 3A). Similarly, the percentages of B220ϩ cells in the spleen, lymph nodes, and pe- ripheral blood of Rap1a-deficient mice did not indicate any defect in B cell development or migration into the periphery (Fig. 3B and unpublished observations). To test the function of Rap1a-deficient B cells, splenocytes from WT and Rap1AϪ/Ϫ mice were stimulated in vitro with anti-IgM in the presence or absence of IL-4. Similar levels of proliferation were observed in WT and Rap1A-deficient cultures stimulated Downloaded from with anti-IgM or anti-IgM plus IL-4 (Fig. 3C). Serum levels of IgM, IgG1, and IgG2a were also indistinguishable between WT and Rap1A-deficient cells (Fig. 3D). Together, these data indicate that Rap1A is not required for B cell development or function.

Normal macrophage and granulocyte development in Rap1a- http://www.jimmunol.org/ deficient mice Monocytes and polymorphonuclear leukocytes are stimulated by several factors that activate Rap1 and, thus, Rap1a may be required for development or function (41). To examine the content of these cells in marrow and spleen, we analyzed the expression of CD11b and Gr-1 on cells in the bone marrow and spleen of WT and Rap1a-deficient mice. As shown in Fig. 4, the percentages of ϩ ϩ ϩ CD11b and CD11b Gr-1 cells are similar between these ge- by guest on September 25, 2021 notypes, suggesting that Rap1a is not required for normal produc- tion of these cells.

Macrophages from Rap1aϪ/Ϫ mice exhibited increased haptotaxis, migration, and phagocytosis Rap1 has recently been implicated in inside-out signaling to reg- ulate integrin-mediated adhesion (12, 14, 42–45). To address whether Rap1a was required for macrophage adhesion, isolated bone marrow-derived cells were incubated in fibronectin peptide H296-coated 96-well plates for 4 h and bound cells were mea- ␣ ␤ sured. This is a measure of 4 1 integrin-dependent adhesion. As seen in Fig. 5A, Rap1a null cells bound to this fibronectin fragment less effectively. Similar results were found with adhesion to vitro- ␣ nectin, an v-specific extracellular matrix protein (P. De and D. Durden, unpublished observations). The levels of Rap1a and total Rap1 were measured using appropriate Abs. Although Rap1a, as FIGURE 2. Analysis of T cell development and function. A, Flow cy- expected, was absent from the cells, total Rap1 levels were similar tometric analysis of CD4 and CD8 expression on thymocytes from WT and to that of WT mice (Y. Li and L. Quilliam, unpublished observa- Rap1a-deficient mice. B, Flow cytometric analysis of CD4 and CD8 ex- tions). These findings suggest Rap1a and 1b play differential roles pression on WT and Rap1a-deficient spleen (top) and peripheral blood in signaling pathways required for provisional integrin dependent (bottom). C, Splenocytes from WT and Rap1a-deficient mice were stimu- migration. lated with 2.5 ␮g/ml anti-CD3 in microwells. Cultures were pulsed with To further address the role of Rap1a in integrin-mediated events, ϩ [3H]thymidine for the last 18 h of a 72 h incubation. D, CD4 cells were we generated mouse embryo fibroblasts (MEFs) from E13.5 em- purified from WT or Rap1a-deficient spleen and stimulated with 4 ␮g/ml bryos and immortalized them using SV40-T (46). A modest but ␮ platebound anti-CD3, 0.5 g/ml anti-CD28, or 10 ng/ml IL-4 plus 10 statistically significant reduction in adhesion was noted (30% re- ␮g/ml anti-IFN-␥, for Th2 cultures or 2 ng/ml IL-12 plus 10 ␮g/ml anti- duction in adhesion to fibronectin and collagen I, p Ͻ 0.001). IL-4 for Th1 cultures. After 5 days in culture, cells were restimulated with anti-CD3 alone and supernatants were recovered after 24 h for cytokine Because macrophages function by migrating to and phagocy- testing by ELISA. Data represent mean Ϯ SEM from six ϩ/ϩ and five tosing foreign material within the body, we examined whether the Ϫ/Ϫ mice. modest reduction in macrophage adhesion noted above affected their ability to function. Bone marrow-derived (BMD) and splenic 8326 CHARACTERIZATION OF Rap1a-DEFICIENT MOUSE MYELOID CELLS

FIGURE 4. Analysis of macrophage and granulocyte development. Flow cytometric analysis of CD11b and Gr-1 expression on bone marrow Downloaded from and spleen cells from WT and Rap1a-deficient mice.

more effectively. Using flow cytometric analysis, surface expres- ␣ ␣ ␤ sion levels of V (CD51), 5 (CD49e), Mac-1 (CD11b), 2 http://www.jimmunol.org/ ␣ (CD18), and 4 (CD49d) integrins or the macrophage marker F4/80 were not significantly different between WT and Rap1aϪ/Ϫ cells (J. Yan, V. Munugulavadla, and R Kaqur, unpublished ob- servations) suggesting that altered integrin levels were not respon- sible for the reduced adhesion. Rap1 has been implicated in phagocytosis in both Dicteostelium and macrophages and localized to endocytic vesicles as well as the specific granules of the neutrophil (47–50). Therefore, we next Ϫ Ϫ compared the ability of macrophages from WT and Rap1a / by guest on September 25, 2021 mice to take up IgG-coated sheep RBCs. The phagocytic index (number of ingested particles/100 macrophages) was greatly in- creased in Rap1a null cells implicating Rap1a in receptor signaling and/or phagocytosis (Fig. 5C). There was no difference noted in the percentage of macrophages forming sheep RBC rosettes nor the number of sheep RBCs bound per spleen-derived or BMD macrophage between Rap1aϪ/Ϫ and WT mice (P. De and D. Durden, unpublished observations). The above macrophage phagocytic activity had been measured in response to Fc␥R stim- ulation that is mediated by Rac- and Cdc42-induced engulfment of particles (51). To examine the response to integrin activation, ␳ FIGURE 3. Analysis of B cell development and function. A, Flow cy- which is alternatively mediated by (51), we further examined the tometric analysis of B220 and CD43 expression in bone marrow from WT ability of macrophages to engulf C3bi-opsonized sheep RBCs. and Rap1a-deficient mice. B, Flow cytometric analysis of B220 and CD3 Surprisingly, there was no significant difference in the phagocyto- expression on spleen and peripheral blood cells from WT and Rap1a-de- sis of C3bi-opsonized cells in macrophages derived from Rap1a ficient mice. C, Splenocytes from WT and Rap1a-deficient mice were stim- ϩ/ϩ vs Ϫ/Ϫ mice, neither in terms of the percentage of cells ulated with 2.5 ␮g/ml anti-IgM in the presence or absence of 5 ng/ml IL-4 taking up RBCs nor the number of RBC taken up by individual in microwells. Cultures were pulsed with tritiated thymidine for the last macrophages (Fig. 5D). Cell surface expression of Fc␥RII/III 18 h of a 72 h incubation. D, Serum was isolated from WT and Rap1a- (CD16/32), as measured by fluorescence staining intensity, was deficient mice. Concentrations of individual Ig isotypes were tested by not significantly different between Rap1a ϩ/ϩ and Ϫ/Ϫ macro- ELISA. phages. Furthermore, all the above macrophage studies yielded comparable data using second or seventh generation backcross into macrophages were isolated and random movement (haptotaxis) ex- the C57BL/6J mouse strain. amined following plating on 8-␮m porous filters. As shown in Fig. 5B, BMD macrophages from Rap1aϪ/Ϫ mice migrated consider- Loss of Rap1a resulted in decreased chemotaxis ably faster than those obtained from Rap1aϩ/ϩ littermates. Similar Inside-out signaling involves the activation of integrin-extracellu- results were obtained using either vitronectin or the H296 fi- lar matrix binding from within the cell. Several reports have im- bronectin-derived peptide (Fig. 5B) and were qualitatively similar plicated Rap1 in promoting increased affinity and/or avidity of in splenic cells (PD and DLD, not shown). This suggests that the integrins depending on the system studied (12). G-protein-coupled reduced adhesion to matrix may have permitted cells to migrate chemokine receptors promote integrin activation and Rap1 has The Journal of Immunology 8327 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Loss of Rap1a influences chemotaxis of leukocytes. The ability of B220ϩ splenic B cells (upper), CD4ϩ splenic T cells (middle), or ScaIϩ/lin- bone marrow hematopoeitic stem cells (lower) from WT and Rap1aϪ/Ϫ mice to migrate toward CCL21 or CXCL12 chemokines was determined using a Transwell assay. Data represent mean Ϯ SEM for cells -p Ͻ 0.05 with a two ,ء .(derived from six mice (four for bone marrow tailed Student’s t test.

been implicated as a downstream effector in several G protein- mediated events (44, 52, 53). We therefore examined the ability of T cells, B cells, and myeloid cells to migrate toward SDF1/ CXCL12 and CCL21 chemokines. As seen in Fig. 6, a decrease in basal and chemokine-induced migration was observed upon loss of Rap1a. A similar decrease in chemotaxis was seen in Rap1aϪ/Ϫ BMD T and B cells as well as macrophages (K. Christopherson, J.

through fibronectin H296 peptide-coated Transwell filters was measured at indicated time points. C, Phagocytosis of IgG-coated sheep RBC by BMD macrophages was measured at the indicated time points after exposure. D, FIGURE 5. Loss of Rap1a affects macrophage haptotaxis, adhesion and Phagocytosis of C3bi-opsonized sheep RBCs by macrophages was mea- Ϫ/Ϫ Ϯ phagocytosis. A, Adhesion of WT and Rap1a macrophages to the (a4b1 sured 30 min after exposure. All data are mean SD for cells derived from integrin binding) fibronectin H296 peptide or vitronectin-coated cell cul- three mice/genotype and are representative of at least four independent ture wells was determined 90 min post plating following fixing and staining experiments (two experiments using a total of four mice were pooled for of adherent cells. B, Haptotaxis of WT and Rap1aϪ/Ϫ macrophages 5D). 8328 CHARACTERIZATION OF Rap1a-DEFICIENT MOUSE MYELOID CELLS

Yan, and L. Quilliam, unpublished observations). This suggests was still a significant amount of Rap1b expressed in Rap1aϪ/Ϫ that the Rap1a isoform may be responsible for coupling chemokine mouse neutrophils (Fig. 1C). signals to integrin activation. Similar findings were observed with F2 and F7 generation C57BL/6J backcrossed mice. Thus despite Discussion alterations in embryonic development, the examined leukocyte Rap1 has been implicated in a number of biological events and is functions remained unaltered. regulated by a broad spectrum of biological stimuli and pathways (10, 12–14). Furthermore, over-activation of Rap1 by KO of the Rap1a was required for an efficient oxidative burst SpaI Rap GAP or a RasGRP2 translocation or expression of a Rap1a is abundantly expressed in human neutrophils and copuri- mutant transgene, has been recently reported to promote several fies with the p22 subunit of the NADPH oxidase (3, 5, 26). It was myeloproliferative disorders (16, 17, 59). Loss of the single Rap1 previously reported that both activating and inhibitory Rap1 mu- gene in Drosophila was lethal at the larval stage, suggesting its tants influence the activity of the phagocyte NADPH oxidase (54, fundamental role in development (18). However, mammals have 55). We therefore examined the effect of Rap1a loss on oxidase two copies of Rap1 (1a and 1b) and, although there is data indi- activity of BMD neutrophils. Following stimulation with fMLP, cating some differential expression, most notably high Rap1b ex- Ϫ cells from WT mice rapidly produced O2 peaking at 30 s and pression in platelets (25), little was known about the specific func- slowly returned to baseline (Fig. 7A). In contrast, neutrophils from tions of the two isoforms. Therefore, to better understand the KO mice had a slower, milder response. The onset of neutrophil biological functions of mammalian Rap1, we deleted the mouse superoxide production in response to PMA is typically slower but Rap1a gene. The results of this study demonstrated that loss of overall NADPH oxidase activity is much more pronounced and Rap1a affected cell adhesion, migration, phagocytosis, and the ox- Downloaded from sustained. Although the sustained induction of superoxide produc- idative burst of leukocytes. This occurred in cells that expressed tion was not statistically distinguishable between WT and significant levels of Rap1b protein, suggesting that the two Rap1 Rap1aϪ/Ϫ neutrophils, the initial rate of superoxide production isoforms are not redundant. Additionally, defects in early embry- was significantly reduced in Rap1a Ϫ/Ϫ mice (Fig. 7B). This was onic development were observed that were strain dependent and seen using isoluminol or cytochrome C assays to measure super- exhibited only partial penetrance. oxide production. These data support previous findings for a role The Mendelian ratio of mice born from heterozygote crosses of http://www.jimmunol.org/ of Rap1 in NADPH oxidase regulation but indicate that, unlike mixed lineage (C57BL/6J:129Sv) animals was normal, consistent Rac2 (56–58), Rap1a is not an essential component of the with the findings of a recent independent report of a Rap1aϪ/Ϫ complex. Analysis of Rap1a and total Rap1 indicated that there mouse (60). However, our findings of embryonic lethality in

FIGURE 7. Loss of Rap1a dimin- ishes the oxidative burst of BMD neu- A ␮ trophils. , The ability of 10 M fMLP by guest on September 25, 2021 to stimulate superoxide production in neutrophils derived from WT or Rap1aϪ/Ϫ mice was determined using HRP catalyzed activation of isolumi- nol. Data in upper panel show time courses of superoxide production from cells derived from a typical control and least responsive Ϫ/Ϫ mouse cells. The maximum response (second panel) and total superoxide produced (third panel) were reduced while the time to reach maximum response was delayed (lower panel) upon averaging data (mean Ϯ SE) from 12 mice/genotype (data ob- tained from four independent experi- -p Ͻ 0.02 (t test) for compar ,ء .(ments isons between ϩ/ϩ and Ϫ/Ϫ mouse- derived cells. B, Stimulation with 200 ng/ml PMA promoted superoxide pro- duction in cells derived from WT or Rap1aϪ/Ϫ mice measured by isolumi- nol chemiluminescence. Upper panel shows a typical time course. Pooled data in second panel shows a weaker overall maximal response in Rap1aϪ/Ϫ neutrophils. The third panel shows a weaker initial (first 3 min) response to PMA. The lower panel shows a similar amount of superoxide was produced over 30 min in response to phorbol es- ter stimulation. Mean Ϯ SE, n ϭ 12 per p Ͻ 0.01 to phorbol ester ,ء .genotype stimulation. The Journal of Immunology 8329

C57BL/6J back-crossed mice suggests that levels or timing of, for servations). However, this finding can support either common or example, Rap1b expression may compensate for loss of Rap1a in divergent function for Rap1 isoforms. the129Sv but not the C57BL/6J strain. 129Sv-Rap1aϪ/Ϫ mice Because Rap1 has been demonstrated to activate various inte- were not established from chimeric founders. Loss of mice during grins, the reduction in macrophage binding to fibronectin and vi- early development is consistent with embryonic morphogenesis tronectin was logical and consistent with reduced adhesion of defects in Drosophila (18) and may be related to the role of Rap1 MEFs from C3G KO mice (19, 20). Although C3G can also reg- in cell-cell and/or cell-matrix adhesion (14, 15). Interestingly, the ulate Rap1b, Rap2, TC10, and R-Ras (21–24), our data suggest noncanonical Wnt pathway was recently demonstrated to regulate that the effect of GEF loss was at least in part due to decreased zebrafish and Xenopus gastrulation by activating casein kinase ␧. Rap1a activation. It is likely that the modest observed decrease in This kinase then bound and phosphorylated the Rap GAP, SpaI, adhesion in the current study was responsible for the enhanced resulting in Rap1-GTP accumulation (61). It is therefore possible macrophage haptotaxis as was observed with C3GϪ/Ϫ fibroblasts that loss of Rap1a in mice resulted in insufficient Rap1 activity (19, 20). This finding is supported by the decreased adhesive ca- during gastrulation to mediate Wnt-regulated morphogenesis. pacity of primary hemopoietic cells in this study and impaired During the completion of this study, Chrzanowska-Wodnicka et polarization of T cells from Rap1aϪ/Ϫ mice reported by Duch- al. (62) reported that Rap1b KO resulted in 85% embryonic and niewicz et al. (60). Similar binding of cell surface integrins was perinatal lethality. This difference from Rap1a null mice may, at observed on macrophages isolated from Rap1a ϩ/ϩ and Ϫ/Ϫ least in part, be due to abnormal bleeding associated with reduced mice in the current study, suggesting that altered adhesion was not platelet function following Rap1b loss (62). Using the Rap1a Ϫ/Ϫ because of changes in integrin composition. mice described in the current study, it was established that Rap1A- In contrast to nondirectional haptotaxis, chemotaxis involves di- Downloaded from null platelets have normal aggregation responses (62), again sup- rected migration in response to a chemical gradient. Because Rap1 porting differential roles for the Rap1a and 1b isoforms in higher signaling has been found downstream of G protein coupled recep- eukaryotes. Six of the nine residues that are divergent between tors (52, 53) and to couple CXCR4 to integrins (44), the reduced Rap1a and 1b are located close to the C terminus. This is remi- chemotactic response to CXCL12 or CCL21 was anticipated and niscent of Rac 1 and 2 where 50% of divergent residues occur in was consistent with our findings. That the chemokines still elicited

the hypervariable domain adjacent to the prenylated CAAX cys- a significant response is presumably due to the continued presence http://www.jimmunol.org/ teine. C-terminal tail swaps between Rac1 and 2 have demon- of Rap1b. Alternatively, Rap2 has also been implicated in chemo- strated that these residues confer unique properties to these pro- kine signaling (71). The decreased basal migration on plastic in teins (63, 64). Whether, like Rac1 vs Rac2, these differences Fig. 6 cannot be attributed to increased adhesion as Rap1aϪ/Ϫ between Rap1a and 1b are due to their divergent C termini remains macrophages bound slightly less than WT cells to tissue culture to be established. Because the hypervariable region can influence plastic (10% difference, p Ͻ 0.005) but still exhibited reduced the subcellular locale of Ras proteins (65–67), one might antici- basal and chemokine-induced migration (J. Yan, unpublished pate unique subcellular distribution. However, our findings ex- observation). pressing GFP-fusion proteins in Cos7 cells (Y. Li, J. Yan, and L. Because Rap1 controls integrin-mediated events through inside- Quilliam, unpublished observations) and those of Pizon et al. (48) out signaling (14) and specifically can control functional activation by guest on September 25, 2021 suggest that the two proteins have similar intracellular localization. of the integrin-type CR3 receptor (␣M␤2) (47), we anticipated that This may however differ in specialized cells or situations. Most loss of Rap1a would impair phagocytosis of complement-coated Rap1a or 1b specific Abs are of poor quality, detect only unproc- particles. Because no effect of Rap1a deletion was observed, it is essed protein, or are no longer available (68–70) making differ- possible that Rap1a and 1b act redundantly in this process or that ential expression and localization of endogenous proteins difficult it is preferentially mediated by Rap1b. Fc␥R-mediated phagocy- to monitor. We are currently attempting to generate isoform spe- tosis of Ig-coated particles is a Cdc42/Rac2-dependent process in cific anti-sera to address such issues. macrophages (72). Because we have previously implicated Rap1 in Analysis of blood cells revealed that Rap1a was not required for Rac activation (73), it was again anticipated that loss of Rap1a development, migration to peripheral organs, or many mature might attenuate uptake of IgG-coated red cells. Interestingly, functions of T, B, or myeloid cells. This is consistent with a recent Rap1a-null macrophages exhibited enhanced Fc␥R-mediated study by Duchniewicz et al. (60) who independently knocked out phagocytosis. We and others have found that activation of Rap1 by Rap1a. Duchniewicz et al. reported that primary hemopoietic cells its upstream GEFs is spatially regulated, resulting in selective ac- isolated from the spleen or thymus had a diminished adhesive ca- tivation of downstream targets at specific intracellular locales (74– pacity on ICAM and fibronectin substrates. In addition, polariza- 78). It is tempting to speculate that in the absence of Rap1a, Rap1b tion of T cells from Rap1aϪ/Ϫ mice after CD3 stimulation was can be more effectively activated in a locale that leads to phago- impaired. These cumulative data suggest that Rap1 isoforms are, at cytosis. For example, activation of Rac by Vav2 at the cell pe- least in part, redundant. Total levels of Rap1 were only reduced by riphery to promote cell spreading is dependent on Rap1-GTP (73). 25% in Rap1AϪ/Ϫ neutrophils, suggesting that Rap1b either is the Drosophila macrophage function is dependent on the Rap GEF major isoform or is up-regulated to compensate for the loss of “Dizzy” (79). Because the localization of the mammalian homo- Rap1A. In support of the former notion, PCR analysis of reverse logue of Dizzy, PDZ-GEF1/RapGEF2, is regulated by its Rap1- transcribed neutrophil mRNA (Y. Li and L. Quilliam, unpublished GTP product (75), loss of Rap1a might lead to PDZ-GEF redis- observations) suggested that there was at least a two-fold excess of tribution to more effectively activate Rap1b and enhance Rap1b over Rap1a message. Therefore it is all the more interesting phagocytosis. The development of Rap1 isoform-specific Abs will that there were defects in the ability of Rap1aϪ/Ϫ cells to adhere help address this possibility. Because Fc␥R signaling in myeloid ␣ and migrate. This finding further supports the notion that Rap1 cells and v integrin-mediated haptotaxis in macrophages requires isoforms serve distinct as well as common functions. Upon cross- Rac2 and the Syk tyrosine kinase, but not Rac1 (31), preferential ing, Rap1aϪ/Ϫ mice with the Rap1bϩ/Ϫ mice generated by Chr- interaction of Rap1a vs 1b with the Syk-Rac2 signaling axis in zanowska-Wodnicka et al. (62), Rap1a/1b double KO was more myeloid cells might occur. Future experiments will examine the lethal than loss of either single isoform; no surviving null offspring impact of Rap1a loss on integrin-induced actin polymerization and were obtained (N. Paianavitana and L. Quilliam), unpublished ob- macrophage polarization. 8330 CHARACTERIZATION OF Rap1a-DEFICIENT MOUSE MYELOID CELLS

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