Rhoh/TTF Negatively Regulates Leukotriene Production in Neutrophils

RhoH/TTF Negatively Regulates Leukotriene Production in Neutrophils Arezoo Daryadel, Shida Yousefi, David Troi, Inès Schmid, Jan Schmidt-Mende, Carlo Mordasini, Clemens A. This information is current as Dahinden, Andrew Ziemiecki and Hans-Uwe Simon of October 2, 2021. J Immunol 2009; 182:6527-6532; ; doi: 10.4049/jimmunol.0803846 http://www.jimmunol.org/content/182/10/6527 Downloaded from

References This article cites 42 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/182/10/6527.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

*average by guest on October 2, 2021

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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

RhoH/TTF Negatively Regulates Leukotriene Production in Neutrophils1

Arezoo Daryadel,2* Shida Youseﬁ,2* David Troi,* Ine`s Schmid,* Jan Schmidt-Mende,* Carlo Mordasini,§ Clemens A. Dahinden,† Andrew Ziemiecki,3‡ and Hans-Uwe Simon4*

Leukotriene B4 (LTB4) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB4 production of neutrophils within minutes, a process called “priming”. In this study, we demonstrate that GM-CSF also limits the production of LTB4 by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient ؊/؊ (Rhoh ) mice demonstrated increased LTB4 production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB4 upon activation com- Downloaded from pared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses. The Journal of Immunology, 2009, 182: 6527–6532.

eutrophils play an important role in innate immune re- stances, priming is absolutely required for lipid mediator produc- sponses. They are rapidly recruited in tissues during in- tion of granulocytes, since most agonists are inactive when applied N fections, where they kill bacteria or at least inhibit their in the absence of hematopoietins (2, 3, 5, 6). On the other hand, http://www.jimmunol.org/ growth (1). They also generate several proinflammatory mediators. cytokine-mediated signal transduction is carefully controlled and 5 For instance, they generate leukotriene B4 (LTB4), which repre- of transient nature (7), and only little is known about such negative sents a chemotactic factor for neutrophils themselves (2, 3). In regulatory mechanisms in neutrophils and other granulocytes. addition, LTB4 activates other leukocytes, which express a high- Hemopoietic-specific Ras homologous/ translocation three four affinity receptor for this lipid mediator (4). By the generation of (RhoH/TTF) belongs to the rat sarcoma (Ras) homologous (Rho)

LTB4, neutrophils have the potential to largely amplify inﬂamma- subfamily of the Ras superfamily of small GTP-binding proteins. tory responses, which is required for effective elimination of mi- It was initially identiﬁed as a fusion transcript with Bcl-6 in lym-

crobes, but may also lead to tissue damage. Therefore, activation phoma cell lines (8). The expression of RhoH/TTF is restricted to by guest on October 2, 2021 of neutrophilic innate immune responses has to be tightly hemopoietic tissues (9). In contrast to other Rho family members, controlled. RhoH/TTF has no GTPase activity and remains, therefore, in a Functional responses of neutrophils to various agonists, includ- constitutive active GTP-bound state (9). This suggests that the ac- ing lipid mediators, complement factors, or chemokines, are in- tivity of RhoH/TTF is likely regulated by its gene expression only. creased by short-term exposure to hematopoietins, such as GM- The physiologic function of RhoH/TTF is largely unknown but CSF (2, 3). This effect of hematopoietins, called “priming”, is also may include the modulation of Rac GTPases (9–11). observed in basophils (5) and eosinophils (6). In many circum- We previously reported increased RhoH/TTF gene expression in neutrophils following GM-CSF stimulation, as assessed by a dif- *Institute of Pharmacology, †Institute of Immunology, and ‡Department of Clinical ferential gene display technique (12). In this study, we show that Research, University of Bern, Bern, Switzerland; and §Tiefenau Hospital Bern, Bern neutrophils are RhoH/TTF positive under inﬂammatory conditions Switzerland and that RhoH/TTF negatively regulates LTB4 production in these Received for publication November 17, 2008. Accepted for publication March cells. 4, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Reagents 1 This work was supported by grants 310000-107526 (to H.-U.S.) and 310000-112078 Human GM-CSF was purchased from Novartis Pharma. Mouse GM-CSF (to S.Y.) from the Swiss National Science Foundation as well as by the OPO as well as human IL-3, IL-8, and IFN-␥ were from R&D Systems Europe Foundation. and human complement factor C5a from MBL International. Mouse C5a 2 These authors share ﬁrst authorship. was from Hycult Biotechnology and mouse IL-3 from BD Biosciences. 3 Deceased. The remaining authors dedicate this work to the memory of Dr. Andrew LTB4 and the signaling inhibitors Go¨6976 (PKC inhibitor), PD98059 Ziemiecki. (MEK inhibitor), SB 203580 (p38 MAPK inhibitor), and LY294002 (PI3K 4 Address correspondence and reprint requests to Dr. Hans-Uwe Simon, Institute of inhibitor) were obtained from Calbiochem. Polyclonal anti-RhoGDI-2 Ab Pharmacology, University of Bern, Friedbu¨hlstrasse 49, CH-3010 Bern, Switzerland. was from Cell Signaling Technology. Polyclonal anti-Rac2 and anti-RhoA E-mail address: [email protected] Abs as well as anti-CD16 mAb were from Santa Cruz Biotechnology. 5 Anti-GAPDH mAb was from Chemicon International. HRP-conjugated Abbreviations used in this paper: LTB4, leukotriene B4; Cdc42, cell division cycle 42; CHX, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; secondary Abs were from Amersham Biosciences. FITC and tetramethyl- PKC, protein kinase C; Rac, Ras-related C3 botulinum toxin substrate; Ras, rat sar- rhodamine isothiocyanate-conjugated anti-mouse and anti-rabbit secondary coma; Rho, ras homologous; TTF, translocation three four. Abs were purchased from Molecular Probes (Invitrogen). Anti-Gr-1 mAb was from Miltenyi Biotec. Cycloheximide (CHX) and all other reagents Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 were, unless stated otherwise, from Sigma-Aldrich. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803846 6528 ROLE FOR RhoH/TTF IN NEUTROPHIL FUNCTION

Mice cycle), 95°C for 15 s and 58°C for 1 min (40 cycles). The Bio-Rad iQ5 2.0

Ϫ/Ϫ Standard edition optical system software was used to analyze real-time and Rhoh mice were generated and provided by Dr. C. Brakebusch (De- endpoint fluorescence. The fold induction values were obtained according partment of Molecular Pathology, University of Copenhagen, Copenhagen, to the ⌬⌬C method. Denmark) (13). For all experiments, 6- to 8-wk-old mice with a C57BL/6J T background were used. Mice were maintained under pathogen-free condi- Production of rRhoH/TTF and polyclonal anti-human tions. All animal experiments were reviewed and approved by the Animal RhoH/TTF Ab Experimentation Review Board of the State of Bern. The RhoH/TTF cDNA was obtained by RT-PCR using cDNA from Jurkat Cells cells. Primers were designed based on the published sequence (NCBI, accession no. NM_004310; Gene ID: 399) and synthesized by MWG Biotec. Mature human blood neutrophils were isolated from peripheral blood of Sequences were as follows: 5Ј-CCG CGG ATC CAT GCT GAG TTC healthy donors and cystic fibrosis patients by Ficoll-Hypaque centrifuga- CAT CAA-3Ј and 5Ј-CGC CGA ATT CTT AGA AGA TCT TGC ACT tion (14–17). Briefly, PBMC were separated by centrifugation on Ficoll- C-3Ј. These primers included restriction sites (underlined). The PCR prod- Hypaque (Seromed-Fakola). The lower phase, mainly granulocytes and uct was subcloned into pGEX-2T with GST (GE Healthcare Europe). The erythrocytes, was treated with erythrocyte lysis solution (155 mM NH Cl, 4 RhoH/TTF sequence was confirmed by sequence analysis. Recombinant 10 mM KHCO , and 0.1 mM EDTA (pH 7.3)). The resulting cell popu- 3 RhoH/TTF was produced and purified using Glutathione Sepharose 4B lations contained Ͼ95% mature neutrophils as assessed by staining with (GE Healthcare Europe). Polyclonal rabbit antisera were raised against Diff-Quik (Medion) and light microscopy analysis. purified GST-RhoH/TTF fusion protein (21). The production of anti-RhoH/ Mature mouse neutrophils were also isolated from wild-type and Ϫ Ϫ TTF Abs was controlled by immunoblotting using GST-RhoH/TTF and Rhoh / mice. Neutrophils were positively selected from bone marrow unrelated GST-fusion recombinant proteins. For confocal microscopic (obtained from femur and tibia of the hind legs) using anti-Gr-1 mAb as analysis, the anti-RhoH/TTF serum was affinity purified. described previously (18). The purity of the resulting mouse neutrophil populations was Ͼ90%. Immunoblotting Downloaded from Cell cultures Gel electrophoresis and immunoblotting were performed as described previously (18, 22, 23). Cells were lysed with 2ϫ loading buffer (Invitrogen) Human and mouse neutrophils were cultured at 1 ϫ 106/ml in complete and sonicated. After electrotransfer of the separated proteins, the filters culture medium (RPMI 1640 containing 10% FCS) and, where indicated, were incubated overnight with anti-RhoH/TTF (1/5000), anti-Rac2 treated with GM-CSF (50 ng/ml), IL-3 (50 ng/ml), IFN-␥ (250 U/ml), and (1/1000), anti-RhoA (1/1000), and anti-RhoGDI-2 (1/1000) Abs at 4°C in C5a (100 nM) for the indicated time periods. The signaling inhibitors TBS/0.1% Tween 20/5% nonfat dry milk. For loading controls, stripped Go¨6976 (500 nM), PD98059 (50 ␮M), SB203580 (30 ␮M), and LY294002 filters were incubated with anti-GAPDH (1/5000) mAb. Filters were http://www.jimmunol.org/ (10 ␮M) were added 30 min before cytokine stimulation of neutrophils. washed in TBS/0.1% Tween 20 for 30 min and incubated with the appro- CHX was used at 50 ␮g/ml. For LTB production, neutrophils were primed 4 priate HRP-conjugated secondary Ab (1/2000; Amersham Bioscience Eu- with GM-CSF or IL-3 for 30 min and subsequently stimulated with C5a for rope) in TBS/0.1% Tween 20/5% nonfat dry milk for 1 h. Filters were 30 min. developed by an ECL technique (ECL Kit; Amersham Biosciences) according to the manufacturer’s instructions. LTB4 immunoassay Confocal laser scanning microscopy LTB4 concentrations were measured in human and mouse neutrophil supernatants by using commercial ELISA kits (Assay design; LuBioScience) Indirect immunofluorescent stainings were conducted on 5-␮m-thick para- according to the manufacturer’s recommendations. formaldehyde-fixed, paraffin-embedded tissue sections from appendicitis Gene expression profiling and ulcerative colitis patients (18, 24). Slides were dried at 52d°C for 2 h by guest on October 2, 2021 and deparaffinized using NeoClear Solution (Merck), ethanol (100, 90, 80, The transcriptional repertoire of mature human blood neutrophils cultured 60, and 40%), and distilled water at room temperature. Following micro- for7hinthepresence and absence of GM-CSF was analyzed using HG- wave treatment in buffer solution (10 mM sodium citrate (pH 6.0)), slides U95Av2 GeneChip arrays (Affymetrix) as described previously (19). Each were washed in distilled water. To prevent nonspecific binding, slides were array was performed in triplicate. incubated in blocking solution (33% human IgG polyvalent, 33% normal goat serum, 33% BSA (7.5% in PBS) and 1% human IgG whole molecule) RT-PCR at room temperature for 1 h. Immunostainings with primary Abs were performed at 4°C overnight. RhoH/TTF was stained by using affinity-pu- ϫ 7 Neutrophils (1 10 ) were washed with PBS and total cellular RNA was rified anti-RhoH/TTF Ab (colitis: 1/20; appendicitis: 1/25). To specifically ␮ isolated using TRIzol solution (Invitrogen). Approximately 1 g of total detect neutrophils in these tissues, we used mouse anti-CD16 mAb (colitis: RNA was reverse-transcribed using oligo-p(dT)15 priming (Promega) and 1/20; appendicitis: 1/25). Following incubation with primary Abs, tissues Superscript Reverse Transcriptase (Invitrogen). Primers for human RhoH/ were incubated with appropriate FITC- and tetramethylrhodamine isothio- Ј TTF (5 -CCG GAA GCT TTA GAA GAT CTT GCA CTC ATT GAT cyanate-conjugated secondary Abs (1/400) in the dark at room temperature Ј Ј GGA G-3 and 5 -GGC TGG ATC CAT GCT GAG TTC CAT CAA GTG for 1 h. The anti-fading agent Dako Mounting Medium (DakoCytomation) Ј CGT GTT G-3 ) amplifications were synthesized (MWG Biotec). The cy- was added. Slides were covered by coverslips and analyzed by confocal cling parameters for RhoH/TTF cDNA amplification were as follows: 94°C laser scanning microscopy (LSM 510; Carl Zeiss) equipped with argon and for 5 min; 25 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 30 s; helium-neon lasers. and 72°C for 7 min. Primers for GAPDH were described previously (20). RhoH/TTF (580 bp) and GAPDH (417 bp) PCR products were separated Statistical analysis on 1% agarose gels and visualized by means of ethidium bromide staining. The identity of the RhoH/TTF PCR product was confirmed by sequence ANOVA followed by Tukey’s HSD test was used to compare mean levels. analysis. A p value of Ͻ0.05 was considered statistically significant. Mean levels are presented together with SEM. Quantitative real-time PCR (TaqMan) One microgram of total RNA from mouse neutrophils isolated with SV Results Total RNA Isolation System (Promega) was used for RT-PCR (ImProm-II GM-CSF rapidly induces RhoH/TTF expression in mature Reverse Transcription System; Promega) using random primer for ampli- neutrophils fication. After cDNA synthesis, 25 ng of cDNA were used for additional analysis. Primers were designed based on the published sequence of mouse To gain an understanding of the molecular processes that occur Rhoh (National Center for Biotechnology Information (NCBI), accession during infection in neutrophils, we screened 12,599 genes in these no. NM_001081105; Gene ID: 74734) and synthesized by Microsynth Lab- cells for changes in gene expression following 7-h GM-CSF stim- oratory. Sequences were as follows: 5Ј-CTC AAT CAA GTG CGT GCT ulation (19). There were four genes, which were up-regulated GGT AG-3Ј and 5Ј-GAT ACT TCT GAA GGC GTC GTT GC-3Ј. The Ͼ reporter dye was SYBR green. The PCR buffer was from Bio-Rad. The run 20-fold: SOCS-1, RhoH/TTF, CD69, and CD44 (Fig. 1A). was performed on the iQ5 Multicolor Real-Time PCR detection system RhoH/TTF mRNA was induced 23.43-fold as assessed by the used from Bio-Rad. The cycling conditions were as follows: 95°C for 15 min (1 microarray assay. The full microarray data set is provided at the The Journal of Immunology 6529

FIGURE 1. GM-CSF rapidly induces RhoH/TTF in mature neutrophils. A, Relative expression of genes induced upon 7-h GM-CSF stimulation as assessed by microarray analysis. B, Time-dependent RhoH/TTF mRNA expression upon GM-CSF stimulation as assessed by RT-PCR. C, RhoH/TTF protein expression upon 8-h stimulation with different cytokines as assessed by immunoblotting. D, Time-dependent RhoH/TTF protein expression upon GM-CSF stimulation as assessed by immunoblotting. In contrast to RhoH/TTF, levels of RhoGDI-2 were not regulated by GM-CSF. E, RhoH/TTF, RhoA, and Rac2 protein expression upon 6-h GM-CSF stimulation as assessed by immunoblotting. F, Pharmacological inhibition of PKC, PI3K, and p38 MAPK Downloaded from blocks GM-CSF-induced RhoH/TTF protein expression as assessed by immunoblotting. Mean levels of three independent experiments (A) and representative examples of at least three independent experiments in (B–F) are shown.

GEO repository (www.ncbi.nlm.nih.gov/geo/info/linking.html; ac- To test whether activation of protein kinase C (PKC), PI3K, cession no. GSE15139). To confirm these data and to generate a and/or MAPK pathways are involved in GM-CSF-mediated RhoH/ http://www.jimmunol.org/ kinetic of RhoH/TTF mRNA induction, we used a RT-PCR tech- TTF expression in neutrophils, neutrophils were preincubated with nique. Freshly isolated neutrophils had no detectable RhoH/TTF defined kinase inhibitors. Go¨6976, an inhibitor of PKC, mRNA expression but induced RhoH/TTF mRNA upon GM-CSF SB203580, a selective inhibitor of p38 MAPK, and LY294002, an stimulation within 30 min. Maximal levels of RhoH/TTF mRNA inhibitor of PI3K, blocked RhoH/TTF expression (Fig. 1F), sug- were seen after 1 h and appeared to decline thereafter (Fig. 1B). gesting that PKC, p38 MAPK, and PI3K pathways are involved in These data confirmed our previous work, demonstrating increased the transcriptional activation of the gene. In contrast, PD98059, an RhoH/TTF mRNA expression in GM-CSF-treated neutrophils as inhibitor of MEK proximal to p42/44 MAPKs had no blocking assessed by Northern blot analysis (12). effect. by guest on October 2, 2021 To determine whether the induction of RhoH/TTF mRNA is followed by increased protein production, we performed immuno- blot analysis using an anti-RhoH/TTF Ab, which we generated in Neutrophils express RhoH/TTF under inflammatory conditions the course of this study. This Ab detected RhoH/TTF protein at the Because RhoH/TTF is markedly up-regulated under conditions of expected size of 21.3 kDa in GM-CSF-treated neutrophils (Fig. GM-CSF exposure, we hypothesized that neutrophils under in- 1C). Freshly isolated neutrophils contained only little or no de- flammatory conditions should express higher levels of RhoH/TTF. tectable RhoH/TTF protein. In contrast to GM-CSF, both IL-8 and Indeed, compared with normal neutrophils, blood neutrophils from IFN-␥ did not induce RhoH/TTF protein. We also studied RhoH/ patients with cystic fibrosis had increased RhoH/TTF protein lev- TTF protein expression in cultured neutrophils in the presence and els (Fig. 2A). Moreover, to demonstrate RhoH/TTF expression in absence of GM-CSF in a time-dependent manner. Depending on neutrophils under in vivo inflammatory conditions, we analyzed the blood donor, elevated levels of RhoH/TTF protein were de- neutrophils in tissue sections of patients suffering from appendi- tected between 1 and 3 h upon GM-CSF stimulation in neutrophils, citis and ulcerative colitis, respectively. Neutrophils were identi- and maximal levels of RhoH/TTF protein were seen after 4–8 h fied using anti-CD16 mAb. Neutrophils expressed RhoH/TTF in (Fig. 1D). Culturing of the cells in the absence of GM-CSF had no these tissues, and it appeared that RhoH/TTF was located both in effect on RhoH/TTF levels (data not shown). the cytosol and at the cell membrane (Fig. 2B). In the ulcerative In contrast to RhoH/TTF protein, RhoGDI-2, a Rho GDP dis- colitis sections, we also obtained evidence for strong RhoH/TTF sociation inhibitor (25), was strongly expressed in freshly isolated expression in infiltrating CD16-negative cells, most likely neutrophils, and its expression was independent from GM-CSF eosinophils. exposure (Fig. 1D). Similarly, RhoA and Rac2 were both expressed in freshly isolated neutrophils and were not inducible by GM-CSF (Fig. 1E). These data also demonstrated the specificity of LTB4 is decreased in neutrophils from cystic fibrosis patients the anti-RhoH/TTF Ab, which did not recognize RhoA and Rac2. and increased in mouse neutrophils defective in Rhoh

We also tested the anti-RhoH/TTF Ab in several cell lines. HL-60, Human neutrophils are known to generate LTB4 upon priming EOL-1, Jurkat, and the B cell lymphoma lines SUDHL-6 and with GM-CSF and subsequent short-term stimulation with C5a (2).

SUDHL-7 expressed RhoH/TTF protein, whereas HeLa, T293, Indeed, signiﬁcant LTB4 production was seen in GM-CSF-primed and the small cell lung cancer cell lines H82 and SW2 did not (data cells only, whereas single stimulation with neither GM-CSF nor not shown). These data are in agreement with previously published C5a resulted in a functional response (Fig. 3, left panel). Interest- work, suggesting that RhoH/TTF expression is restricted to hemo- ingly, neutrophils from patients suffering from cystic ﬁbrosis were poietic tissues (9) and, at the same time, demonstrate the validity defective in LTB4 production upon GM-CSF priming and subse- of the generated anti-RhoH/TTF Ab. quent C5a stimulation (Fig. 3, right panel). 6530 ROLE FOR RhoH/TTF IN NEUTROPHIL FUNCTION

FIGURE 4. Prevention of GM-CSF-induced RhoH/TTF by CHX is as- Downloaded from

sociated with increased LTB4 production. A, C5a-induced LTB4 production by neutrophil populations pretreated with GM-CSF for the indicated times FIGURE 2. RhoH/TTF is expressed in neutrophils under inﬂammatory in the presence and absence of CHX. B, Time-dependent RhoH/TTF pro- conditions. A, RhoH/TTF protein expression in freshly isolated blood neu- tein expression upon GM-CSF stimulation in the presence and absence of trophils from normal healthy individuals compared with cystic ﬁbrosis (CF, CHX as assessed by immunoblotting. Cells in A and B were from the same n ϭ 3) as assessed by immunoblotting. B, Confocal microscopy. Tissue experiment, which is representative of three independent experiments.

neutrophils from patients with appendicitis and ulcerative colitis demon- http://www.jimmunol.org/ strated evidence for RhoH/TTF expression. Neutrophils were detected using anti-CD16 mAb (arrows). Note the yellow ring-like staining pattern, vented GM-CSF-induced RhoH/TTF protein expression in neutro- suggesting that RhoH/TTF is at least partially located at the cell membrane. phils (Fig. 4A). Interestingly, reduced amounts of LTB4 production In both the ulcerative colitis and appendicitis sections, we also observed were associated with increased RhoH/TTF expression at all time CD16-negative cells, which strongly expressed RhoH/TTF (at least some points up to 5 h after GM-CSF stimulation (Fig. 4B). Taken to- of them appear to be eosinophils, marked by arrowheads). For both appendicitis and ulcerative colitis, representative data from at least ﬁve in- gether, these data pointed to the possibility that RhoH/TTF could dependent experiments are shown. indeed play a limiting role in LTB4 production of neutrophils. The precise investigation of signaling mechanisms in neutrophils has been difﬁcult because the cells are fragile and undergo by guest on October 2, 2021

To obtain first evidence whether such a defective LTB4 produc- rapid spontaneous apoptosis. Plasmid transfection appeared to be tion could be due to increased RhoH/TTF expression, we per- impossible in neutrophils (26), limiting the common approaches to formed a time-course experiment using normal neutrophils in the study signaling pathways in these cells. Therefore, to investigate a presence and absence of a protein synthesis inhibitor (CHX). C5a possible function of RhoH/TTF in neutrophil’s leukotriene pro- alone did not significantly induce LTB4 production of freshly iso- duction, we assessed the capacity to generate LTB4 in neutrophils lated neutrophils. However, pretreatment with GM-CSF for 30 min derived from wild-type and RhohϪ/Ϫ mice. Similar to human neu- and subsequent C5a stimulation resulted in the generation of sig- trophils, wild-type mouse neutrophils rapidly induced the Rhoh nificant amounts of LTB4 (Fig. 4A). CHX had no effect at this time gene following GM-CSF stimulation (Fig. 5A). Neutrophils from point. In the absence of CHX, we observed that this priming effect RhohϪ/Ϫ mice demonstrated no detectable Rhoh mRNA expres- was less after 1-h GM-CSF stimulation and was practically lost at sion in these experiments (data not shown). Freshly isolated neu- the 2-h time point (Fig. 4A). In contrast, in the presence of CHX, trophils from wild-type mice generated similar amounts of LTB4 neutrophils kept the capacity to generate LTB4 after C5a stimula- after GM-CSF priming and subsequent C5a stimulation (mean tion (Fig. 4A). In these experiments, we monitored RhoH/TTF ex- level: ϳ15,000 pg/ml) as seen using human neutrophils. Moreover, pression at all time points. As expected, RhoH/TTF was rapidly there was no significant difference between neutrophils derived detectable (within 1 h) after GM-CSF stimulation, and CHX pre- from wild-type and RhohϪ/Ϫ mice (Fig. 5B), consistent with the

FIGURE 3. LTB4 production by normal and cystic fibrosis neutrophils. GM-CSF priming and subsequent C5a stimulation resulted in significant LTB4 production of normal neutrophils Ͼ (mean LTB4 levels were 10,000 pg/ml in supernatants, n ϭ 10). In contrast, neutrophils from cystic fibrosis patients did almost not re- spond under the same stimulation conditions Ͻ ϭ (mean LTB4 levels were 400 pg/ml, n 6). Values are means Ϯ SEM of the indicated num- p Ͻ ,ءءء .bers of independent experiments 0.001. The Journal of Immunology 6531

Discussion Ras-like proteins constitute a protein superfamily that can be sub- divided into five main families, one of these is the family of Rho proteins (27). The Rho family members are defined by the presence of a Rho-specific insert located between the G4 and the G5 boxes and involved in mediating protein-protein interactions (28). Rho proteins have been shown to participate in multiple signaling pathways (29). Most functional data have been reported for Rac1/2, RhoA, and cell division cycle 42 (Cdc42). In contrast, little is known about the function of the many other members of the Rho family, such as RhoH/TTF. Recently published work suggests that RhoH/TTF plays a key role in TCR signaling (13, 30). For instance, it has been demonstrated that RhoH/TTF is required for recruitment of ZAP70 to the TCR. Consequently, RhoH/TTF deficiency resulted in decreased activation of phospholipase C␥1 and impaired calcium influx (13), as well as in a T cell proliferation defect (30). These data suggest that RhoH represents a positive regulator of TCR signaling.

In contrast to these ﬁndings obtained in T cells, RhoH/TTF has Downloaded from been described as a negative regulator of proliferation and engraft- ment of hemopoietic progenitor cells, perhaps due to the induction of apoptosis in these cells (10). We did not observe differences in the kinetics of neutrophil apoptosis between wild-type and RhohϪ/Ϫ mice (data not shown). When acting as a negative reg-

ulator, RhoH/TTF was suggested to antagonize Rac1/2-mediated http://www.jimmunol.org/ signaling pathways (9, 10, 31). This assumption was supported by a study performed in Jurkat cells, in which retroviral RhoH/TTF gene transfer resulted in inhibition of Rac1 signaling (32). Clearly, besides differences in the cell types used and signaling pathways investigated, it cannot be excluded that differences in the method- FIGURE 5. LTB production by wild-type and Rhoh-deficient mouse 4 ological approaches account, at least partially, for the different neutrophils. A, Rhoh mRNA expression in freshly isolated and GM-CSF- stimulated wild-type mouse neutrophils. The time periods of GM-CSF conclusions that have been made regarding RhoH/TTF functions stimulation are indicated. Values are means Ϯ SEM of two independent in the different studies. by guest on October 2, 2021 experiments. In control experiments, we verified that RhohϪ/Ϫ mice had no We report in this study that RhoH/TTF is a negative regulator of detectable Rhoh mRNA expression. B, GM-CSF priming and subsequent LTB4 production in neutrophils. In these cells, RhoH/TTF is tran- C5a stimulation resulted in significant LTB4 production of neutrophils de- scriptionally regulated by GM-CSF, which plays a key role in an- Ϫ/Ϫ ϳ rived from wild-type and Rhoh mice (mean LTB4 levels were 15,000 tibacterial defense mechanisms. We also show that RhoH/TTF is pg/ml in supernatants). Stimulation with GM-CSF or C5a alone did not expressed in neutrophils under inflammatory conditions, such as result in significant LTB production (data not shown). C, Time-dependent 4 cystic fibrosis, ulcerative colitis, and appendicitis. Rho family IL-3- and C5a-stimulated LTB4 production of neutrophils derived from wild-type and RhohϪ/Ϫ mice following GM-CSF pretreatment. The re- members have previously been reported to exert important func- Ϫ/Ϫ leased LTB4 levels from Rhoh compared with wild-type neutrophils tions in neutrophils. For instance, Cdc42 is translocated to lipid were higher at all indicated time points. Stimulation with GM-CSF, IL-3, rafts, where it activates p38 MAPK upon LPS-induced activation or C5a alone did not result in significant LTB4 production (data not in these cells (33). Similarly, we obtained evidence that a propor- shown). Same results were seen in another independent experiment. tion of RhoH/TTF is located at the cell membrane of neutrophils under inflammatory conditions (Fig. 2B), pointing to the possibility that it translocates from the cytosol to the cell membrane upon activation. Indeed, RhoH/TTF has a conserved polybasic domain observation that resting human neutrophils express no or only very and a CAAX prenylation site (9, 34), which allows membrane little RhoH/TTF. targeting (35). How RhoH/TTF exerts its regulatory role in neu- Because both human and mouse neutrophils consistently ex- trophils remains to be investigated. pressed more RhoH/TTF after GM-CSF stimulation, we also per- Rac2, another member of the Rho family, was found to be es- formed LTB4 release experiments in mouse neutrophils treated sential for primary (azurophilic), but not secondary and tertiary with GM-CSF for several hours. Because the priming effect of granule release of neutrophils (36). In addition, Rac2 was found to GM-CSF on human neutrophils did not last longer than 1 h (Fig. be crucial for superoxide production and chemotaxis in neutrophils 4), neutrophils were primed with IL-3 and subsequently stimulated (34). These data are in agreement with the increased susceptibility with C5a. Because maximal levels of Rhoh mRNA were seen after toward bacterial and fungal infections in functional Rac2-deficient 3-h GM-CSF stimulation (Fig. 5A), we performed these experi- mice and humans (37–39). Moreover, RhoA has been studied in ments in 3-, 5-, and 8-h pretreated neutrophils. Strikingly, under neutrophils and is believed to be involved in integrin functions of these conditions of GM-CSF pretreatment and IL-3 priming, neu- neutrophils (40). RhoA may also induce stress fibers formation, Ϫ/Ϫ trophils from Rhoh mice generated significantly more LTB4 which affects the efficacy of signal transduction via surface recep- after C5a stimulation compared with neutrophils from wild-type tors (41). In a recent study, it was suggested that RhoA interacts mice (Fig. 5C). with Cdc42, exerting both positive and negative signaling effects 6532 ROLE FOR RhoH/TTF IN NEUTROPHIL FUNCTION depending on the activation stage of neutrophils (42). Taken to- and nonapoptotic death signals into neutrophils depending on the proinflamma- gether, Rho family members are expressed in neutrophils and are tory cytokine environment. Blood 106: 1423–1431. 17. von Gunten, S., A. Schaub, M. Vogel, B. M. Stadler, S. Miescher, and H.-U. crucial for several functions of these cells. They interact with each Simon. 2006. Immunologic and functional evidence for anti-Siglec-9 autoanti- other and other signaling molecules; however, the molecular in- bodies in intravenous immunoglobulin preparations. Blood 108: 4255–4259. teractions between Rho family members and their effectors are 18. Conus, S., R. Perozzo, T. Reinheckel, C. Peters, L. Scapozza, S. Yousefi, and H.-U. Simon. 2008. Caspase-8 is activated by cathepsin D initiating neutrophil largely not understood. apoptosis during the resolution of inflammation. J. Exp. Med. 205: 685–698. In summary, this study demonstrates elevated levels of RhoH/ 19. Martinelli, S., M. Urosevic, A. Daryadel, P. A. Oberholzer, C. Baumann, M. F. Fey, R. Dummer, H.-U. Simon, and S. Yousefi. 2004. Induction of genes TTF in neutrophils in association with GM-CSF exposure in vitro mediating interferon-dependent extracellular trap formation during neutrophil and in vivo. Therefore, GM-CSF does not only exhibit well known differentiation. J. Biol. Chem. 279: 44123–44132. proinflammatory activities, it also induces the expression of genes 20. Conus, S., A. Bruno, and H.-U. Simon. 2005. Leptin is an eosinophil survival that limit inflammatory responses, such as suppressor of cytokine factor. J. Allergy Clin. Immunol. 116: 1228–1234. 21. Gregory, P., A. Ziemiecki, G. Zu¨rcher, U. Brodbeck, and P. Bu¨tikofer. 2003. signaling 1 (see Fig. 1A) and RhoH/TTF. RhoH/TTF negatively Anti-mouse GPI-PLD antisera highlight structural differences between murine and bovine GPI-PLDs. Biol. Chem. 384: 1575–1582. regulates agonist-mediated LTB4 production. Further studies are 22. Kostylina, G., D. Simon, M. F. Fey, S. Yousefi, and H.-U. Simon. 2008. Neu- needed to understand the molecular mechanisms of transcriptional trophil apoptosis mediated by nicotinic acid receptors (GPR109A). Cell Death activation of the RhoH/TTF gene. Moreover, the intracellular Differ. 15: 134–142. movement of RhoH/TTF and its binding partners as well as the 23. Yousefi, S., R. Perozzo, I. Schmid, A. Ziemiecki, T. Schaffner, L. Scapozza, T. Brunner, and H.-U. Simon. 2006. Calpain-mediated cleavage of Atg5 switches exact signaling mechanisms and possible additional cellular func- autophagy to apoptosis. Nat. Cell Biol. 8: 1124–1132. tions that are regulated by RhoH/TTF in neutrophils remain to be 24. Altznauer, F., S. Martinelli, S. Yousefi, C. Thu¨rig, I. Schmid, E. M. Conway, investigated. M. H. Scho¨ni, P. Vogt, C. Mueller, M. F. Fey, et al. 2004. Inflammation-associated cell-cycle-independent block of apoptosis by survivin in terminally differ- Downloaded from entiated neutrophils. J. Exp. Med. 199: 1343–1354. Disclosures 25. Lelias, J. M., C. N. Adra, G. M. Wulf, J. C. Guillemot, M. Khagad, D. Caput, and The authors have no financial conflict of interest. B. Lim. 1993. cDNA cloning of human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins. Proc. Natl. Acad. Sci. USA 90: 1479–1483. References 26. Downey, G. P., J. R. Butler, H. Tapper, L. Fialkow, A. R. Saltiel, B. B. Rubin, 1. Nathan, C. 2006. Neutrophils and immunity: challenges and opportunities. Nat. and S. Grinstein. 1998. Importance of MEK in neutrophil microbicidal respon-

Rev. Immunol. 6: 173–182. siveness. J. Immunol. 160: 434–443. http://www.jimmunol.org/ 2. Dahinden, C. A., J. Zingg, F. E. Maly, and A. L. de Weck. 1988. Leukotriene 27. Wennerberg, K., and C. J. Der. 2004. Rho-family GTPases: it’s not only Rac and production in human neutrophils primed by recombinant human granulocyte/ Rho (and I like it). J. Cell Sci. 117: 1301–1312. macrophage colony-stimulating factor and stimulated with the complement com- 28. Freeman, J. L., A. Abo, and J. D. Lambeth. 1996. Rac “insert region” is a novel ponent C5a and FMLP as second signals. J. Exp. Med. 167: 1281–1295. effector region that is implicated in the activation of NADPH oxidase, but not 3. DiPersio, J. F., P. Billing, R. Williams, and J. C. Gasson. 1988. Human granu- PAK65. J. Biol. Chem. 271: 19794–19801. locyte-macrophage colony-stimulating factor and other cytokines prime human 29. Ridley, A. J. 2001. Rho family proteins: coordinating cell responses. Trends Cell neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis. Biol. 11: 471–477. J. Immunol. 140: 4315–4322. 30. Gu, Y., H.-D. Chae, J. E. Siefring, A. C. Jasti, D. A. Hildeman, and 4. Serhan, C. N., and S. M. Prescott. 2000. The scent of a phagocyte: Advances on D. A. Williams. 2006. RhoH GTPase recruits and activates Zap70 required for T leukotriene B4 receptors. J. Exp. Med. 192: F5–F8. cell receptor signaling and thymocyte development. Nat. Immunol. 7: 1182–1190. 5. Bischoff, S. C., A. L. de Weck, and C. A. Dahinden. 1990. Interleukin-3 and 31. Gu, Y., Y. Zheng, and D. A. Williams. 2005. RhoH GTPase—a key regulator of granulocyte-macrophage colony-stimulating factor render human basophils re- hematopoietic cell proliferation and apoptosis? Cell Cycle 4: 201–202. by guest on October 2, 2021 sponsive to low concentrations of complement component C3a. Proc. Natl. Acad. 32. Cherry, L. K., X. Li, P. Schwab, B. Lim, and L. B. Klickstein. 2004. RhoH is Sci. USA 87: 6813–6817. required to maintain the integrin LFA-1 in a nonadhesive state on lymphocytes. 6. Takafuji, S., S. C. Bischoff, A. L. de Weck, and C. A. Dahinden. 1991. IL-3 and Nat. Immunol. 5: 961–967. IL-5 prime normal human eosinophils to produce leukotriene C4 in response to 33. Fessler, M. B., P. G. Arndt, S. C. Frasch, J. G. Lieber, C. A. Johnson, soluble agonists. J. Immunol. 147: 3855–3861. R. C. Murphy, J. A. Nick, D. L. Bratton, K. C. Malcolm, and G. S. Worthen. 7. Gadina, M., D. Hilton, J. A. Johnston, A. Morinobu, A. Lighvani, A. Zhou, 2004. Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and Y. J. Visconti, and J. J. O’Shea. 2001. Signaling by type I and II cytokine re- inflammatory functions of the human neutrophil. J. Biol. Chem. 279: ceptors: ten years after. Curr. Opin. Immunol. 13: 363–373. 39989–39998. 8. Dallery, E., S. Galie`gue-Zouitina, M. Collyn-d’Hooghe, S. Quief, C. Denis, 34. Filippi, M.-D., C. E. Harris, J. Meller, Y. Gu, Y. Zheng, and D. A. Williams. M. P. Hildebrand, D. Lantoine, C. Deweindt, H. Tilly, C. Bastard, and 2004. Localization of Rac2 via the C terminus and aspartic acid 150 specifies J. P. Kerckaert. 1995. TTF, a gene encoding a novel small G protein, fuses to the superoxide generation, actin polarity and chemotaxis in neutrophils. Nat. Immu- lymphoma-associated LAZ3 gene by t(3,4) chromosomal translocation. Onco- nol. 5: 744–751. gene 10: 2171–2178. 35. Hancock, J. F. 2003. Ras proteins. Different signals from different locations. Nat. 9. Li, X., X. Bu, B. Lu, H. Avraham, R. A. Flavell, and B. Lim. 2002. The hema- Rev. Mol. Cell Biol. 4: 373–384. topoiesis-specific GTP-binding protein RhoH is GTPase deficient and modulates 36. Abdel-Latif, D., M. Steward, D. L. Macdonald, G. A. Francis, M. C. Dinauer, and activities of other Rho GTPases by an inhibitory function. Mol. Cell. Biol. 22: P. Lacy. 2004. Rac2 is critical for neutrophil primary granule exocytosis. Blood 1158–1171. 104: 832–839. 10. Gu, Y., A. C. Jasti, M. Jansen, and J. E. Siefring. 2005. RhoH, a hematopoietic- specific Rho GTPase, regulates proliferation, survival, migration, and engraft- 37. Roberts, A. W., C. Kim, L. Zhen, J. B. Lowe, R. Kapur, B. Petryniak, A. Spaetti, ment of hematopoietic progenitor cells. Blood 105: 1467–1475. J. D. Pollock, J. B. Borneo, G. B. Bradford, et al. 1999. Deficiency of the he- 11. Chae, H.-D., K. E. Lee, D. A. Williams, and Y. Gu. 2008. Cross-talk between matopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormal- RhoH and Rac1 in regulation of actin cytoskeleton and chemotaxis of hemato- ities in neutrophil function and host defense. Immunity 10: 183–196. poietic progenitor cells. Blood 111: 2597–2605. 38. Ambruso, D. R., C. Knall, A. N. Abell, J. Panepinto, A. Kurkchubasche, 12. Yousefi, S., P. R. Cooper, B. Mueck, S. L. Potter, and G. Jarai. 2000. cDNA G. Thurman, C. Gonzalez-Aller, A. Hiester, M. deBoer, R. J. Harbeck, et al. representational difference analysis of human neutrophils stimulated by GM-CSF. 2000. Human neutrophil immunodeficiency syndrome is associated with an in- Biochem. Biophys. Res. Commun. 277: 401–409. hibitory Rac2 mutation. Proc. Natl. Acad. Sci. USA 97: 4654–4659. 13. Dorn, T., U. Kuhn, G. Bungartz, S. Stiller, M. Bauer, J. Ellwart, T. Peters, 39. Williams, D. A., W. Tao, F. Yang, C. Kim, Y. Gu, P. Mansfield, J. E. Levine, K. Scharffetter-Kochanek, M. Semmrich, M. Laschinger, et al. 2007. RhoH is B. Petryniak, C. W. Derrow, C. Harris, et al. 2000. Dominant negative mutation important for positive thymocyte selection and T-cell receptor signaling. Blood of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human 109: 2346–2355. phagocyte immunodeficiency. Blood 96: 1646–1654. 14. Daigle, I., S. Yousefi, M. Colonna, D. R. Green, and H.-U. Simon. 2002. Death 40. Williams, M. A., and J. S. Solomkin. 1999. Integrin-mediated signaling in human receptors bind SHP-1 and block cytokine-induced antiapoptotic signaling in neu- neutrophil functioning. J. Leukocyte Biol. 65: 725–736. trophils. Nat. Med. 8: 61–67. 41. Anderson, S. I., N. A. Hotchin, and G. B. Nash. 2000. Role of the cytoskeleton 15. Altznauer, F., S. Conus, A. Cavalli, G. Folkers, and H.-U. Simon. 2004. Cal- in rapid activation of CD11b/CD18 function and its subsequent down-regulation pain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in in neutrophils. J. Cell Sci. 113: 2737–2745. neutrophil apoptosis. J. Biol. Chem. 279: 5947–5957. 42. Fessler, M. B., P. G. Arndt, I. Just, J. A. Nick, K. C. Malcolm, and G. S. Worthen. 16. von Gunten, S., S. Yousefi, M. Seitz, S. M. Jakob, T. Schaffner, R. Seger, 2007. Dual role for RhoA in suppression and induction of cytokines in the human J. Takala, P. M. Villiger, and H.-U. Simon. 2005. Siglec-9 transduces apoptotic neutrophil. Blood 109: 1248–1256.