ORIGINAL RESEARCH ARTICLE

Journal of

Cannabinoid Receptor 1 but not 2 Mediates Macrophage Phagocytosis by G(a)i/o/RhoA/ ROCK Signaling Pathway PING MAI, LEI TIAN, LE YANG, LIN WANG, LIN YANG,

AND

Cellular Physiology

LIYING LI*

Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China Phagocytosis is critical to macrophages linking innate and adaptive immune reaction. Cannabinoid receptor 1 (CB1) and 2 (CB2) mediate immune modulation. However, the role of cannabinoid receptors in macrophage phagocytosis is undefined. In this study, we found that two murine macrophage lines (J774A.1 and RAW264.7) and peripheral blood macrophages all expressed CB1 and CB2 by immunofluorescence-staining, real time RT-PCR and Western blot. Macrophage phagocytic activity was determined by quantifying fluorescent intensity of the engulfed BioParticles or fluorescence-activated cell sorting. mAEA (CB1 agonist) enhanced phagocytosis of macrophages, but JWH133 (CB2 agonist) had no influence. Pharmacological or genetic ablation of CB1 inhibited mAEA-enhanced phagocytosis, while CB2 had no such effects. Meanwhile, activation of CB1 increased GTP-bounding active form of small GTPase RhoA, but not Rac1 or Cdc42. AM281 (CB1 antagonist) and pertussis toxin (PTX, G(a) i/o protein inhibitor) decreased GTP-bound RhoA protein level with mAEA. In addition, PTX, C3 Transferase (RhoA inhibitor) or Y27632 (Rho-associated kinase ROCK inhibitor) attenuated CB1mediated phagocytosis. These results confirm that activation of CB1 regulates macrophage phagocytosis through G(a) i/o/RhoA/ROCK signaling pathway. Moreover, activation of CB1 induced significant up-regulation of CB1 expression by real time RT-PCR and Western blot analysis, but not CB2. It indicated the existence of a positive feedback between CB1 activation and CB1 expression. The up-regulation of CB1 was RhoA-independent but it may contribute to maintaining high phagocytic activity of macrophages for a longer time. In conclusion, CB1 mediates macrophage phagocytosis by G(a) i/o/RhoA/ROCK signal axis. These data further underline the role of CB1 in macrophage phagocytic process. J. Cell. Physiol. 230: 1640–1650, 2015. © 2014 Wiley Periodicals, Inc.

Macrophages display an important role in innate and adaptive immune reaction. They have strong phagocytic activities to process infectious agents (such as bacteria), dead cells, and debris, which will repair injured and inflamed tissue, lastly revive tissue homeostasis. Endocannabinoid system is involved in immune reaction, which mainly includes two receptors (cannabinoid receptor 1, CB1, and CB2) and two endogenous ligands (2-arachidonoylglycerol, 2-AG and NArachidonoylethanolamine, AEA). CB1 is abundantly expressed in nervous system, usually regulating synaptic neurotransmission and controlling psychoactive actions. Studies about immune system pay more attentions on CB2 because of its high expression in immune cells. Study by Ross et al. (2000) revealed that activation of CB2 inhibited lipopolysaccharide (LPS)-triggered nitric oxide production in murine RAW 264.7 macrophages. In addition, a recent report (Gui et al., 2013) showed that GW405833, a CB2 agonist, dose-dependently inhibited LPS-mediated proinflammatory cytokine release in murine peritoneal macrophages, including tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and high mobility group box-1 (HMGB1). An earlier study (Zhao et al., 2010) has found that CB2 signal reduced macrophages adhesion and infiltration, associating with the suppression of expressions of vascular cellular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and P-selectin. Another study by Raborn et al. (2008) indicated the cannabinoid delta-9tetrahydrocannabinol (THC)-mediated inhibition of macrophages migration was due to CB2. Moreover, research by Ramirez et al. (2013) pointed out that agonist of CB2 could limit human immunodeficiency virus (HIV) 1 infection in macrophages through attenuating HIV 1 replication. Recently, a study (Jourdan et al., 2013) found that CB1 was highly © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

coexpressed with CD68 (macrophage marker) in Zucker diabetic fatty rat. Pharmacological blockade of CB1 diminished macrophage infiltration into pancreatic islets and caused a shift of M1 phenotype to M2. Endocannabinoid anandamine (AEA) significantly increased the levels of inflammatory cytokines (such as IL-1b and TNF-a) in RAW 264.7 cells through CB1. Additionally, study (Sugamura et al., 2009) demonstrated that CB1 was also expressed in lesional macrophages of human coronary atheroma. During monocyte differentiation into macrophages, elevatation of biosynthetic enzyme

Abbreviations: CBs, cannabinoid receptors; FBS, fetal bovine serum; PCR, polymerase chain reaction; PTX, Pertussis toxin; FACS, fluorescence-activated cell sorting; RNAi, RNA interference; LPS, lipopolysaccharide; ROCK, Rho-associated kinase. Ping Mai and Lei Tian contributed equally to this work. Conflict of interest: None. Contract grant sponsor: National Natural and Science Foundation of China; Contract grant number: 81170407. *Correspondence to: Prof. Liying Li, No.10 Xitoutiao, You An Men, Beijing 100069, P.R.China. E-mail: [email protected] Manuscript Received: 16 April 2014 Manuscript Accepted: 18 December 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 24 December 2014. DOI: 10.1002/jcp.24911

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(N-acylphosphatidyl-ethanolamine selective phospholipase D, NAPE-PLD) and decrease of degradative enzyme for AEA (fatty acid amidohydrolase, FAAH) suggested an increased level of AEA. Blockade of CB1 using different antagonists reduced the LPS-induced production of pro-inflammatory cytokines. These results show extensive and significant contributions of cannabinoid receptors (CBs) on macrophages. However, the effect of CBs especially CB1 on macrophages phagocytosis still remains undefined. Research (Niedergang and Chavrier, 2005) revealed that particle internalization required the local polymerization of actin filaments in phagocytic process and the reconstruction of cytoskeleton was mediated mainly by Rho GTPases. A review (Hanna and El-Sibai, 2013) showed the Rho family GTPases, including members of the RhoA, Rac1, and Cdc42, are critical regulators of cytoskeletal dynamics as ‘molecular switch’ of signal transduction (GTP-bound for active form and GDPbound for inactive form). The earlier studies (Massol et al., 1998; Castellano et al., 1999; Castellano et al., 2000) have found that in fragment crystallizable receptor (FcR)-mediated phagocytosis, Cdc42 controlled actin-rich pseudopod emission, and Rac1 was in charge of phagosome closure to permit particle entry. Meanwhile study by Chimini and Chavrier (2000) also noted that although RhoA was accumulated at nascent phagosome, it may not have direct effects on the FcR-mediated phagocytic process. In complement receptor 3 (CR3)-mediated phagocytosis, reports (Caron and Hall, 1998; Chimini and Chavrier, 2000) suggested the activity of RhoA was required, whereas inactivity of Cdc42 or Rac1 hardly affected CR3-mediated phagocytosis. Furthermore, recent studies (Morris et al., 2011; Asplund et al., 2013) showed that alcohol-attenuated J774A.1 phagocytosis may be associated with reduced expression of RhoA, and human neutrophils phagocytosis was also markedly attenuated through the inhibition of RhoA activation by anaphylatoxin C5a. The reduction or inactivation of RhoA prevented actin polymerization. Additionally, study by Qu et al. (2012) certified the inhibition of the Rho-associated kinase (ROCK, the downstream signal transducer of GTP-RhoA) signal reduced pahgocytic ability to erythrophagocytize in macrophages. The results suggest important roles of RhoA, Rac1, and Cdc42 in phagocytosis. The current study aimed to determine whether CBs affect macrophage phagocytosis, and to reveal which small GTPase participates in CBs-mediated phagocytosis. We found that two murine macrophages lines (J774A.1 and RAW264.7 cells) and peripheral blood macrophages (PBM) all expressed CB1 and CB2. Activation of CB1 enhanced the phagocytic ability of macrophages; whereas CB2 signal had no influence on it. Moreover, the elevated phagocytic ability of macrophages by CB1 depended on small GTPase RhoA signal and G(a) i/o protein. Rac1 and Cdc42 signals hardly participated in CB1mediated macrophage phagocytosis. Meanwhile, there was a homospecific positive feedback regulation that activation of CB1 up-regulated CB1 expression. CB1 up-regulation may contribute to maintaining macrophage phagocytosis during long-term activation of CB1.

and C3 Transferase (RhoA inhibitor) was from Cytoskeleton (Denver, CO). Y27632 (the inhibitor of ROCK), Pertussis toxin (PTX) and other common reagents were from sigma (St. Louis, MO). Cell lines culture Murine macrophages cell lines RAW264.7 (ATCC TIB71) and J774A.1 (ATCC TIB67) were cultured in DMEM (100 U/ml of penicillin and 100 mg/ml of streptomycin) containing 10% FBS at 37°C, in a humidified 5% CO2 atmosphere. Peripheral blood macrophages acquisition Murine peripheral blood with heparin treatment was subjected to density gradient (Histopaque-1077) centrifugation at 2,000 rpm for 20 min. The mononuclear cells were collected from the interface after centrifugation, washed twice with PBS, lastly cultured for 7 days in the presence of L929-conditioned 1640 medium (replacing culture medium at the third, fifth day). All animal work was performed under the ethical guidelines of the Ethics Committee of Capital Medical University. Immunofluorescence staining RAW264.7, J774A.1, and peripheral blood macrophages were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 (Amresco, OH) for 15 min. After blocked with 3% BSA (Roche, Switzerland), they were incubated with anti-F4/80 rat monoclonal antibody (1:100, Santa Cruz Biotechnology, CA), anti-CB1 or anti-CB2 rabbit polyclonal antibodies (1:50, Cayman Chemical, MI). Respectively, CyTM3-conjugate affinipure goat-antirat IgG or FITC-conjugate affinipure goat-anti-rabbit IgG (both 1:100, Jackson Immunoresearch, PA) was as secondary antibodies. At last, nuclei were stained with DAPI. Phagocytic activity of cells RAW264.7, J774A.1, and peripheral blood macrophages were plated into the 96-well plate with the aim of having 3,000 viable cells per well. Cells were starved for 4 h, and then exposed to different concentrations of mAEA for 1, 12, and 24 h with or without the pretreatment of inhibitors. pHrodoTM Zymosan A BioParticles (Invitrogen, Carlsbad, CA), which were added 2 ml PBS to make completely resuspend the particles, were transferred into a clean glass tube, and sonicated for 5 min, until all the fluorescent particles were homogeneously dispersed. When the cells have adhered, the culture medium were replaced with 100 ml of the prepared pHrodoTM BioParticles1 suspension quickly. Then the microplate was transferred to an incubator warmed to 37°C for 15–30 min. A fluorescence plate reader (Fluoroskan Ascent FL, Thermo), whose excitation and emission wavelengths were 485 nm and 530 nm respectively, was used to analyze the activity of phagocytosis by measuring fluorescent intensity. Furthermore, fluorescence-activated cell sorting (FACS) was also used to calculate the phagocytosis. The samples were analyzed on a BD AccuriTM C5 cytometer (BD Biosciences) using a 488 nm argon laser and 564–606 nm emission filter.

Materials and Methods Materials

Western blot analysis

Dulbecco’s modified Eagle’s medium (DMEM) and RPMI Medium 1640 were from GIBCO/Invitrogen (Grand Island, NY). FBS was from Hyclone/Thermo Scientific (Victoria, Australia). PCR reagents were from Applied Biosystems (Foster City, CA). mAEA (CB1 agonists), JWH133 (CB2 agonist), AM281 (CB1 antagonist), and AM630 (CB2 antagonist) were from TOCRIS/R&D (Minneapolis, MN). The pHrodoTM Zymosan A BioParticles (P35364 and P35365) was from Life Technologies (Carlsbad, CA)

Western blot analysis was performed with 50 mg of protein extract. Antibodies were as follows: anti-CB1 rabbit polyclonal antibody (1:100) and anti-CB2 rabbit polyclonal antibody (1:100); anti-GAPDH monoclonal antibodied (1:1000, Abcam, UK). ODYSSEY goat anti-rabbit IRDye 1 800 CW antibody (1:10000, LI-COR, NE) was used as secondary antibody. The bands were displayed using ODYSSEY and quantified by Odyssey v3.0 software. GAPDH was as reference.

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Measurement of activity of small GTPases by pull-down assay J774A.1 macrophages with or without AM281 or PTX pretreatment were exposed to mAEA for different time, then J774A.1 macrophages were lysed in lysis buffer. Active RhoA, Rac1, and Cdc42 were extracted using pull-down and detection kit (Catalog No. 16116, 16118, and 16119, Thermo Scientific Pierce Biotechnology, IL). At last, active and total RhoA, Rac1, and Cdc42 were showed by Western Blot. RNA interference (RNAi) The siRNA targeting murine CB1 (L-042461–00) and CB2 (L062503–00) were from Dharmacon (Thermo Scientific, PA). Forty to fifty percent confluent J774A.1 macrophages were prepared in 60 mm dishes. Transient transfection of siRNA (40 nmol/L) was performed using Lipofectamine RNAiMAX (Invitrogen) as recommended by the manufacturer. Control cells were treated with 40 nmol/L RNAi negative control duplexes (scramble siRNA). After 48 h, cells were used to perform a further assay. Real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) Our earlier report (Li et al., 2011) described the procedures for extraction of total RNA from macrophages and real-time RT-PCR. Primers were as follows: CB1: sense, 50 GGCGGTGGCCGATCTC-30 ; antisense, 50 CGGTAACCCCACCCAGTTT-30 . CB2: sense, 50 AGCGCCCTGGAGAACATG-30 ; antisense, 50 AGCTGCTGATGAACAGGTACGA-30 . 18s rRNA: sense, 50 GTAACCCGTTGAACCCCATT-30 ; antisense, 50 CCATCCAATCGGTAGTAGCG-30 . Statistical analysis Results are expressed as mean  standard error of the mean (SEM). Statistical significance was determined by Student’s t-test or ANOVA. P < 0.05 was considered significant. Results J774A.1, RAW264.7 and peripheral blood macrophages express CB1 and CB2

Employing immunofluorescence staining, we found that murine macrophages lines (J774A.1 and RAW264.7) and peripheral blood macrophages all showed an abundant expression of F4/ 80(macrophage marker), CB1 and CB2 (Fig. 1A–C). RT-PCR and Western blot analysis further indicated that CB1 and CB2 were detectable in J774A.1, RAW264.7 and peripheral blood macrophages in mRNA and protein level, respectively (Fig. 1D and E). Activation of CB1 enhances phagocytic activity of J774A.1, RAW264.7 and peripheral blood macrophages

Review (Geissmann et al., 2010) has demonstrated phagocytosis is the critical function of macrophages in immune reaction. To reveal the effects of CBs on phagocytic activity of macrophages, we used pHrodo Red Zymosan A BioParticles Conjugates to perform the phagocytosis test. The BioParticles would emit fluorescence if they were ingested. Quantitative Fluorometry was used to analyze the activity of phagocytosis by the quantitation of fluorescent intensity. In representative phagocytic images (Fig. 2A), we could see that red fluorescent BioParticles aggregated around cellular nucleus of J774A.1 macrophages. mAEA (CB1 agonist) treatment apparently promoted J774A.1 phagocytosis for more engulfed particles. JOURNAL OF CELLULAR PHYSIOLOGY

Moreover, mAEA enhanced J774A.1 phagocytic activity in a dose-dependent manner (from 0.1 to 1 mM) (Fig. 2D). The strongest pro-phagocytic effect of mAEA emerged at 1 mM concentration. However, 10 mM mAEA showed lower prophagocytic effect compared with 1 mM, probably because cytotoxicity or other unclear effects were produced by the large dose of mAEA. Additionally, AM281 partially blocked mAEA-enhanced phagocytosis at 1 mM, and almost completely inhibited it at 10 mM concentration (Fig. 2D). In representative phagocytic images, AM281 evidently decreased the number of ingested particles by J774A.1 macrophages in mAEA existing (Fig. 2A). Similar effects were confirmed in RAW264.7 and peripheral blood macrophages as that in J774 macrophages (Fig. 2B, C, F, and G). Traditionally, mAEA is considered as an agonist of CB1, but mAEA also has low CB2 affinity. In order to further identify mAEA-enhanced phagocytosis was by CB1, not CB2, we used fluorescence-activated cell sorting (FACS) to analyze macrophage phagocytic ability in the condition of genetic and pharmacological ablations of CB1 or CB2. In representative FACS plots, the positive area plots (part right), which represent J774A.1 macrophages group with fluorescent particles uptake under mAEA excitation, were obviously higher than the vehicle. The quantitative proportion of mAEA-stimulated positive cells reached 40%, but the vehicle was only about 15% (Fig. 3A). siRNA was used to knock-down CB1 or CB2 (Fig. 3B). AM281 or siCB1 pretreatment significantly reduced the positive area plots, suggesting ablation of CB1 attenuates mAEA-enhanced phagocytosis of macrophages (Fig. 3C). However, pharmacological and genetic inactivation of CB2 had no influence on mAEA-induced phagocytosis (Fig. 3C). Statistical results made sure that mAEA-enhanced phagocytosis of J774A.1 cells was due to activation of CB1, but not CB2 (Fig. 3D). The mAEA-mediated phagocytosis of RAW264.7 and peripheral blood macrophages was inhibited by AM281 treatment, which was further verified by FACS (Fig. 3E and F). Activation of CB2 shows no effects on the phagocytic activity of macrophages

Review by Tanasescu and Constantinescu (2010) showed CB2 is recognized the predominant expression on immune cells and plays an important role in inflammatory process. We also found J774A.1, RAW264.7, and peripheral blood macrophages all expressed copious CB2. Nevertheless, the results of phagocytosis assay revealed that JWH133 (CB2 agonist) had no influence on J774A.1 macrophage phagocytic activity at common doses (0.1, 0.5 and 1 mM) and a flushing dose (10 mM) (Fig. 2E). Phagocytic images showed JWH133 had hardly changed the uptake of fluorescent particles by macrophages compared with the control (Fig. 2A). There was a similar phenomenon in RAW264.7 and peripheral blood macrophages (Fig. 2B, C, F, and G). The elevated phagocytic activity of J774A.1 macrophages by CB1 depends on G(a) i/o protein and small GTPase RhoA signal, but not Rac1 or Cdc42

Review by Chimini and Chavrier (2000) showed small GTPases Rho family proteins play critical roles in macrophage phagocytosis through controlling actin dynamics. Pull-down analysis displayed that mAEA markedly increased active GTPbound RhoA protein levels of J774A.1 macrophages in 5, 15, 30 min, 12 and 24 h and the highest level appeared in 15 min (Fig. 4A). However, Rac1-GTP and Cdc42-GTP proteins changed slightly (Fig. 4A). This mAEA-elevated GTP-bound RhoA was attenuated by AM281 or siCB1 treatment but not siCB2 (Fig. 4B and C). Furthermore, PTX (G(a) i/o protein

CANNABINOID RECEPTORS/MACROPHAGE PHAGOCYTOSIS

Fig. 1. Cannabinoid receptors (CBs) are expressed in J774A.1, RAW264.7, and murine peripheral blood macrophages (PBM). Representative images of immunofluorescence staining for F4/80 (macrophage marker, red), CB1 or CB2 (green) were shown in J774A.1 macrophages (A), in RAW264.7 macrophages (B), and in PBM (C). (D) CBs PCR products were size-fractionated in a 2% agarose gel. (E) Western blot analysis for expression of CBs. Brain and spleen tissue as positive controls, respectively. Nuclei were stained with DAPI. Scale bars: 25 mm.

inhibitor) also decreased GTP-bound RhoA protein levels with mAEA (Fig. 4B). These results suggested that mAEA firstly triggers the activation of G(a) i/o-coupled CB1, then GTPbound RhoA signal is augmented, eventually the stronger phagocytosis is driven. Rho-associated protein kinase ROCK is known as the downstream effector of GTP-Rho protein and Y27632 is the inhibitor of ROCK. In our study, PTX, C3 Transferase, or Y27632 pretreatment attenuated mAEAenhanced phagocytosis, which further confirmed that G(a) i/o/ RhoA/ROCK signal axis was essential for CB1-mediated phagocytosis (Fig. 4D). Activation of CB1 up-regulates CB1 expression

To gain further insight into the involvement of CBs in phagocytosis, we detected the changes of CBs expressions in JOURNAL OF CELLULAR PHYSIOLOGY

J774A.1 and RAW264.7 macrophages with the activation of CB1 by real time RT-PCR and Western blot. The mRNA level of CB1 significantly increased in 4 and 8 h with mAEA stimulation (Fig. 5A and B). The protein level of CB1 was upregulated in 12 and 24 h (Fig. 5C and D). In addition, activation of CB1 hardly influenced CB2 expression (Fig. 5E–H). Meanwhile, phagocytosis was markedly up-regulated by 12 and 24 h treatment of mAEA (Fig. 6A and B). We also detected the GTP-RhoA level of J774A.1 macrophages with long-term stimulation of mAEA (12 and 24 h), and found that GTP-RhoA level was still kept in a higher level after reaching the highest in mAEA-stimulated 15 min (Fig. 4A). These data suggested that the process of CB1-triggered RhoA activation is rapid, CB1/ RhoA/ROCK signal-enhanced phagocytosis can be kept for a longer time and up-regulated CB1 might be involved in this process. Through measuring the mRNA and protein levels of

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Fig. 2. The activation of CB1, not CB2 enhances macrophages phagocytic activity. Cells were treated with mAEA for 1 h and fluorescent BioParticles were used for macrophage phagocytic activity assay. Representative images of J774A.1 (A), RAW264.7 (B), or PBM (C) macrophages with fluorescent BioParticles uptake in indicated condition. (D) mAEA-induced J774A.1 phagocytosis assay with or without AM281 (CB1 antagonist). (E) Phagocytic activity assay of J774A.1 with JWH133 (CB2 antagonist) treatment. Phagocytic activity assays of RAW264.7 (F) and PBM (G) in indicated condition. Scale bars: 25 mm. *P < 0.05 compared with control, #P < 0.05 compared with mAEA-treated cells alone in the same concentration.

CB1, we found RhoA inhibitor (C3 Transferase) did not reduce CB1 up-regulation in response to mAEA, suggesting CB1 upregulation is not related to RhoA activity (Fig. 6C–F). Discussion

Review (Fonseca et al., 2013) demonstrated that researches about the contributions of CBs in inflammation paid more JOURNAL OF CELLULAR PHYSIOLOGY

attentions to CB2, but not CB1, because of CB2 predominant expression in the immune cells. However, in this study, CB1 and CB2 are both highly expressed in J774A.1, RAW264.7, and murine peripheral blood macrophages. CB1, but not CB2 shows a powerful effect on phagocytic activity of murine macrophages. Furthermore, the CB1-mediated phagocytic ability requires the signals transduction of G(a) i/o protein and small GTP-binding protein RhoA. However, Rac1 and Cdc42

CANNABINOID RECEPTORS/MACROPHAGE PHAGOCYTOSIS

Fig. 3. The mAEA-enhanced phagocytosis of macrophages depends on CB1, but not CB2. Cells were treated with mAEA for 1 h and fluorescence-activated cell sorting (FACS) was used for macrophage phagocytic activity assay. (A) Representative FACS histograms are shown in indicated conditions. Basal: J774A.1 macrophages without fluorescent BioParticles treatment; Vehicle: Fluorescent BioParticles-treated J774A.1 macrophages with vehicle; mAEA: Fluorescent BioParticles-treated J774A.1 macrophages with mAEA 1 mM. The percentages represent the proportion of positive cells with fluorescent particles uptake. (B) The protein levels with CB1 and CB2 RNA interference (RNAi) by Western blot. (C) FACS histograms in indicated conditions. AM281: CB1 antagonist, 10 mM; AM630: CB2 antagonist, 10 mM. (D) FACS quantitative assay of phagocytosis. Representative FACS histograms of RAW264.7 (E) and PBM (F) phagocytosis in indicated conditions. *P < 0.05 compared with control, #P < 0.05 compared with mAEA-treated alone.

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Fig. 4. CB1 mediates phagocytosis through G(a) i/o/RhoA/ROCK signaling pathway. Total and active GTP-bound Rac1, Cdc42, and RhoA proteins were measured by pull-down assay. (A) J774A.1 macrophages were exposed to mAEA (1 mM) for 5, 15, 30 min, or 12, 24 h, the representative images of Western blots and quantitative assay were shown. J774A.1 macrophages were exposed to mAEA for 15 min with PTX (pertussis toxin, G(a) i/o inhibitor, 20 ng/ml) or AM281 (10 mM) pretreatment (B), with siCB1 or siCB2 treatment (C). (D) The effects of mAEA on J774A.1 phagocytosis with PTX, C3 Transferase (RhoA inhibitor) or Y27632 (the inhibitor of Rho-associated kinase) pretreatment. *P < 0.05 compared with control, #P < 0.05 compared with mAEA-treated alone.

signals have no effects on this process. In addition, the activation of CB1 raises CB1 expression. Review by Pandey et al. (2009) showed that many inflammatory cells express both CB1 and CB2, but CB2 JOURNAL OF CELLULAR PHYSIOLOGY

expressions are usually tens times higher than CB1. Cellular state affects CB1 expression. The earlier study by Klein et al. (1998) has found activated macrophages expressed more CB1. In addition, study by Han et al. (2009) indicated that phorbol

CANNABINOID RECEPTORS/MACROPHAGE PHAGOCYTOSIS

Fig. 5. The activation of CB1 up-regulates CB1 expression in J774A.1 and RAW264.7 macrophages. Serum-starved J774A.1 or RAW264.7 cells were stimulated with mAEA for 2, 4, 8, 12, and 24 h, respectively. Expressions of CB1 and CB2 were determined by real-time RT-PCR and Western blot. (A, B) Changes of CB1 mRNA were shown in indicated condition. (C, D) Representative images of Western blot and quantitative assay were shown. (E–H) CB2 mRNA and protein were determined in indicated conditions. *P < 0.05 compared with vehicle-treated alone in the same time.

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Fig. 6. The long-term treatment of mAEA significantly enhances macrophage phagocytosis and CB1 up-regulation is independent of RhoA activity. Serum-starved cells were stimulated with mAEA 1 mM for 1, 12, and 24 h, respectively. Phagocytic activity assay of J774A.1 (A) and RAW264.7 (B) were shown. (C and D) CB1 mRNA for 8 h mAEA with or without C3 Transferase pretreatment were determined by real-time RT-PCR. (E-F) Representative images of Western blots and quantitative assay of CB1 for 12 h mAEA with or without C3 Transferase pretreatment were shown. *P < 0.05 compared with vehicle-treated alone in the same time.

ester, an inducer of macrophage differentiation markedly elevated CB1 expression in isolated human macrophages. Here, we found two murine macrophages lines (J774A.1 and RAW264.7) and primary murine peripheral blood macrophages highly expressed both CB1 and CB2. Recent studies (Tanasescu and Constantinescu, 2010; Kaplan, 2013) have also identified the CB1 expressions of macrophages in different tissues under various pathological conditions. Research by Pryce et al. (2003) found that mice deficient CB1 developed more severe neurodegeneration following inflammatory attack in experimental allergic encephalo JOURNAL OF CELLULAR PHYSIOLOGY

myelitis, supporting a significant neuroprotected role of CB1. Furthermore, reports (Sacerdote et al., 2005; Martín-Moreno et al., 2011) revealed cannabidiol, the main nonpsychotropic component of marijuana, and other synthetic cannabinoids promoted microglial cells migration depending on CBs, and cannabidiol dose-dependently inhibited formy-met-leu-phe (fMLP)-triggered macrophages migration by CB2. Another study by Ross et al. (2000) also has found activation of CB2 reduced lipopolysaccharide-mediated nitric oxide release. Importantly, study by Sacerdote et al. (2005) noted that the blockade of CB1 or CB2 prevented cannabidiol-mediated the

CANNABINOID RECEPTORS/MACROPHAGE PHAGOCYTOSIS

increase of IL-12 and the decrease of IL-10 in murine peritoneal macrophages. There was also a study (Han et al., 2009) suggesting that CB1 mediated pro-inflammatory responses by phosphorylation of p38-mitogen-activated protein kinase (p38MAPK) following reactive oxygen species (ROS) production and pro-inflammatory cytokines (TNF-a and MCP-1) synthesis, while CB2 regulated ROS negatively. These results indicate the critical roles of CBs in migration and inflammatory cytokines and chemokines secretion of macrophages. This study showed that CB1 but not CB2 was involved in phagocytosis, which is another important function of macrophages. CB1 agonist mAEA enhanced the phagocytic ability of macrophages but CB2 agonist JWH133 had no influence. The ablations of pharmacological and genetic CB1 reversed mAEA-enhanced phagocytosis but barely influenced spontaneous phagocytosis of macrophages. This implies that CB1 signal augments macrophage phagocytic activity but it is not essential for macrophage basic phagocytic action. mAEA has also very weak affinity with CB2. However, CB2 antagonist and knockdown both had no effects on mAEA-enhanced phagocytosis. These results certify that CB1 signal strengthens macrophages phagocytic ability. Nevertheless, an earlier study by Shiratsuchi et al. (2008) showed that endocannabinoid 2-AG stimulated macrophage phagocytosis of zymosan via CB2. The reason might be that distinct macrophages were used, macrophages cell lines and ICR mice peripheral blood macrophages in our experiments, yet C57BL/6 mice peritoneal and alveolar macrophages in the study of Shiratsuchi et al. The reagents of activating CB1 also were different, mAEA, used in our experiments, is synthetic as a CB1 selective agonist, but endogenous 2-AG, which has more complicated biological activity, was employed in the study of Shiratsuchi et al. Cannabinoid receptors are 7-transmembrane G-proteincoupled receptors, which are linked predominantly to G(a) i/o protein and thus inhibit adenylate cyclase. A review (Howlett et al., 2004) showed activations of CBs induce complex signal transduction such as ion channel, phospholipase C and mitogen activated protein kinase (MAPK). In addition to negatively regulating adenylate cyclase, review and study (Chen et al., 2010; Kaplan, 2013) indicated activation of CB1 stimulates cAMP accumulation in some situations, suggesting G(a) s protein binding. Inhibition of G(a) i/o protein signal by PTX reduced CB1-mediated phagocytosis. The result demonstrates that the signal of CB1 coupling G(a) i/o protein triggers the elevation of macrophages phagocytic ability. Rho family proteins, mainly including Rho, Rac, and Cdc42, have a central role in cell membrane and cytoskeleton remodeling during macrophage phagocytosis. The earlier reports (Caron and Hall, 1998; Massol et al., 1998) showed RhoA, Rac1, and Cdc42 all accumulated at the nascent phagosome in FcR-mediated phagocytosis, and common blockade of these three GTPases signals inhibited phagocytic activity through FcR. During FcR-mediated phagocytosis, Cdc42 and Rac1 control actin rearrangement at distinct stages. Cdc42 controls pseudopod extension and Rac mediates pseudopod fusion and phagosome closure. There was another study by Caron and Hall (1998) demonstrating that inactivation of RhoA signal inhibited CR3-mediated phagocytosis but Cdc42 and Rac blockade seemed to have little influence during this process. Moreover, review by Bishop and Hall (2000) showed RhoA signal mediates the activation of serine/ threonine kinases ROCK. ROCK promotes the assembly of stress fibres and focal adhesions. Significantly, research (Mizutani et al., 2009) described that through the phosphorylation of myosin light chain (MLC) and the inhibition of MLC phosphatases, ROCK stimulates and enhances actinmyosin contractility. Recent review by Hanna and El-Sibai (2013) demonstrated downstream effectors of Rho include mammalian homolog of diaphanous (mDia), LIM-kinases and JOURNAL OF CELLULAR PHYSIOLOGY

members of the ezrin–radixin–moesin (ERM) proteins, which mediate actin nucleation and actin cytoskeleton remodeling. In our study, activation of CB1 augmented RhoA signal without the changes of Rac1 and Cdc42 signals. Specific blockade of ROCK (the downstream target of RhoA) inhibited CB1mediated phagocytosis, suggesting RhoA/ROCK signal is involved in this process. In addition, PTX treatment to macrophages reduced CB1-mediated RhoA signal and phagocytosis. Interestingly, study (Zimmerman et al., 2013) has also indicated that the up-regulated cAMP levels decreased levels of active RhoA. Our results imply that the decreased cAMP by G(a) i/o-coupled CB1 magnifies RhoA/ROCK signal, which promotes actin-myosin contractility and cytoskeleton remodeling, thus boosting the phagocytic activity of macrophages. We also found that CB1 protein in macrophages was up-regulated by 12 or 24 h treatment of mAEA. CB1 upregulation is independent on RhoA signal, but the increased CB1 may contribute to keeping high phagocytic activity of macrophages for a longer time. The mechanism of CB1 upregulation needs to be studied further. The earlier reports (Varga et al., 1998; Matias et al., 2002; Liu et al., 2003) have revealed that macrophages produce endocannabinoids (AEA and 2-AG) by LPS stimulation. Our results demonstrate that activation of CB1 enhances macrophage phagocytic activity and a positive feedback exists between CB1 activation and CB1 expression. Therefore, we might believe that, in inflammatory reaction (specifically in bacterial infection), endocannabinoids promote macrophages to ingest pathogens by CB1 coupling G(a) i/o protein and RhoA/ ROCK signal pathway. Furthermore, CB1-mediated high phagocytic activity would be sustained for up-regulated CB1 expression in a positive feedback manner. In conclusion, our study identifies a novel role of CB1 during macrophage phagocytosis, which promotes phagocytic activity by G(a) i/o/ RhoA/ROCK signal axis. Acknowledgment

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