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Impairs Cell Adhesion and Migration PI3K Signaling andγLeads to Defective p110

Cyclooxygenase-2 Deficiency in Macrophages

Íñiguez and Manuel FresnoManuel D. Díaz-Muñoz, Inés C. Osma-García, Miguel A.

http://www.jimmunol.org/content/191/1/395doi: 10.4049/jimmunol.12020022013;

2013; 191:395-406; Prepublished online 3 JuneJ Immunol 

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2.DC1http://www.jimmunol.org/content/suppl/2013/06/03/jimmunol.120200

Referenceshttp://www.jimmunol.org/content/191/1/395.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2013 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Cyclooxygenase-2 Deficiency in Macrophages Leadsto Defective p110g PI3K Signaling and Impairs CellAdhesion and Migration

Manuel D. Dıaz-Munoz,1,2 Ines C. Osma-Garcıa,1 Miguel A. Iniguez,3 and Manuel Fresno3

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of

immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon

thioglycolate challenge, CD11b+ F4/80+ macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-22/2

mice. In vivo migration of cox-22/2 macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the

mesothelium, was reduced in response to LPS. In vitro migration of cox-22/2 macrophages toward MCP-1, RANTES, MIP-1a, or

MIP-1b, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in

chemokine receptor expression. Remarkably, cox-22/2 macrophages showed a deficiency in focal adhesion formation, with

reduced phosphorylation of paxillin (Tyr188). Interestingly, expression of the p110g catalytic subunit of PI3K was severely reduced

in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that

the paxillin/p110g-PI3K/Cdc42/Rac1 axis is defective in cox-22/2 macrophages, which results in impaired cell adhesion and

migration. The Journal of Immunology, 2013, 191: 395–406.

Leukocyte migration to the site of infection is a well-characterized process required to build the immune re-sponse (1, 2). Less is known about the egression of im-

mune cells from tissues. Emigration is a key component of normalphysiology required for leukocyte recirculation and immune sur-veillance (3, 4). Egression of leukocytes, including neutrophils,monocytes, and dendritic cells (DCs), is indispensable during theadaptive immune response to transport and present Ags in secondaryimmune organs (5–8). Furthermore, clearance of inflammatoryconditions, such as atherosclerosis (5, 9) or acute peritonitis,occurs, in part, as a result of macrophage emigration (10, 11).Nonetheless, very little is known about the molecular basiscontrolling this mechanism.

PGs are a group of bioactive lipid mediators that regulate im-portant responses in many physiological and pathological processes,including inflammation, cancer, angiogenesis, and cardiovasculardiseases. PGs have also been implicated in lymphocyte develop-ment and function (12–15). They are generated by cyclooxygenases(Cox-1 and Cox-2) that metabolize arachidonic acid into PGH2, asubstrate of different PG synthases, to produce prostanoids (PGE2,PGF2a, PGI2, PGD2 and TXA2). Cyclooxygenases are the targets ofnonsteroidal anti-inflammatory drugs. Cox-1 is primarily involvedin cell homeostasis, whereas Cox-2 is tightly associated with PGproduction during inflammation and cancer. In macrophages, Cox-2is upregulated in response to several proinflammatory stimuli, suchas LPS, IFN-g, and TNF-a, among others (16–18).Multiple lines of evidence support the role of Cox-2 and Cox-

2–derived prostanoids as key modulators of the immune response.Cox-2–knockout mice show reduced inflammation, fever, and ill-ness in response to infection by bacteria and virus (19, 20). Cox-2deficiency enhances antitumor responses by promoting Foxp3expression and CD4+ CD25+ T regulatory cells (21, 22). PGE2,PGF2a, and PGD2 were reported to modulate cell migration ofneutrophils, monocytes, DCs, and T cells. PGD2 is a chemotacticagent for DCs and Th2 cells, whereas PGF2a was shown to induceneutrophil migration (23, 24). PGE2 enhances hematopoietic cellhoming and modulates monocyte response to chemokines (25,26). PGE2 can also favor migration of DCs to lymph nodes, likelyby inducing MMP9 expression (27–29).Expression of chemokine receptors on the cell membrane reg-

ulates cell migration. Migration of macrophages is primarilymodulated by CCR1, CCR2, CXCR31, and CCR5, whereas DCmigration is primarily mediated by CCR7 (30). PGs can regulatechemokine and chemokine receptor expression, although the roleof PGs in cell migration might depend on the cell type analyzed.PGE2 is an inducer of CCR7 expression and DC migration inresponse to CCL19 and CCL21 (31), whereas it downregulatesCCR5 (26) in macrophages. Moreover, control of migration ofimmune cells by PGs is involved in the progression of somediseases. Thus, decreased migration of Langerhans cells in the

Departamento de Biologıa Molecular, Centro de Biologıa Molecular Severo Ochoa(CSIC-UAM), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid,Spain; and Instituto de Investigacion Sanitaria Princesa, Universidad Autonoma deMadrid, Cantoblanco, 28049 Madrid, Spain

1M.D.D.-M. and I.C.O.-G. contributed equally to this work.

2Current address: The Babraham Institute, Babraham, Cambridge, U.K.

3M.A.I. and M.F. contributed equally to the direction of this work.

Received for publication July 27, 2012. Accepted for publication April 23, 2013.

This work was supported in part by grants from Comunidad Autonoma de Madrid(S2010/BMD-2332), the Cardiovascular Red Tematica de Investigacion en Enferme-dades Cardiovasculares and Red de Investigacion Cooperativa en EnfermedadesTropicales Networks of the Instituto de Salud Carlos III (RD06/0014/1013 andRD06/0021/0016), the European Union (EICOSANOX LSH-CT-2004-005033), andMinisterio de Ciencia e Innovacion (SAF2007-61716 and SAF2010-18733 to M.F.and BFU2010-21055 and SAF2011-23971 to M.A.I.). I.C.O.-G. holds a predoctoralfellowship from Fondo de Investigacion Sanitaria. Centro de Biologıa Molecular SeveroOchoa receives institutional funding from the Fundacion Ramon Areces.

Address correspondence and reprint requests to Prof. Manuel Fresno, Centro deBiologıa Molecular Severo Ochoa, Universidad Autonoma de Madrid, Nicolas Cabrera,1, Cantoblanco, 28049 Madrid, Spain. E-mail address: mfresno@cbm.uam.es

The online version of this article contains supplemental material.

Abbreviations used in this article: Cox, cyclooxygenase; DC, dendritic cell; FAK,focal adhesion kinase; PAK, p21-activated kinase; PBD, p21-binding domain; PFA,paraformaldehyde.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202002

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absence of PGE2 signaling leads to decreased contact hypersen-sitivity (32).Integrin interaction with their ligands (i.e., ICAM-1) induces

reorganization of the actin cytoskeleton and cell polarization,which are indispensable for cell migration (33). Actin polymeri-zation is modulated by GTPases, including Cdc42, Rho, Ras, andRac. Deficient activation of GTPases results in defective actinpolymerization and failure in cell migration (1, 34, 35). Recently,it was shown that PGE2 modulates podosome stability in DCs(36). PGE2 signals through the EP2/EP4 receptors and switchesCdc42 on and Rac-1 off. This switch promotes podosome disso-ciation and focal adhesion formation (37). Cdc42, Rac, and Rhoare direct targets of PI3K (38, 39). Among the PI3K family ofproteins, class I PI3Ks were associated with leukocyte migration(40). In particular, the p110g catalytic subunit plays an importantrole in regulating the migration of macrophages (41, 42).In this study, we analyzed the impact of Cox-2 deficiency on

macrophage migration and adhesion. Our results suggest that Cox-2 may play an important role in these processes, likely by mod-ulating p110g PI3K–mediated cell signaling. These results mayhelp to clarify the role of Cox-2 during the onset and progressionof inflammation, especially as a modulator of cell trafficking.

Materials and MethodsAnimals and reagents

B6;129S7-Ptgs2tm1Jed/J (cox-22/2) mice were purchased from The JacksonLaboratory. B6/129S wild type (cox-2+/+) mice were obtained by breed-ing heterozygote pairs. C57BL/6 mice were purchased from Harlan Lab-oratories. Thioglycolate peritonitis was induced in 8–12-wk-old mice byinoculation of sterile Brewer’s thioglycolate (1 ml 10% p/v; DIFCO).Peritoneal lavage with ice-cold PBS was carried out 4 d later. The differentimmune cell populations were analyzed by flow cytometry. Peritonealexudates were cultured at 37˚C and 5% CO2 in RPMI 1640 medium(Invitrogen) supplemented with 5% FCS (BioWhittaker-Lonza) and 100 U/ml penicillin, 100 mg/ml streptomycin, 1000 U/ml gentamicin, 2 mML-glutamine, and 0.1 mM nonessential amino acids. Nonadherent cellswere washed out with PBS. Ninety percent of adherent cells in culture wereCD11b+ F4/80+ macrophages.

MCP-1, RANTES, MIP-1a, and MIP-1b chemokines were purchasedfrom R&D Systems. Pan-specific PI3K inhibitor LY294002 and thep110g-specific inhibitor AS252424 were from Sigma-Aldrich. The se-lective Cox-2 inhibitors celecoxib and NS398 were from Alexis Bio-chemicals. LPS from Escherichia coli (026:B6) was purchased fromSigma-Aldrich. The doses of the above inhibitors and chemicals wereselected based on previous studies (43) or after dose-response tests. Thedose of LY294002 and AS252424 inhibitors was selected based on theliterature and after testing cell viability (Supplemental Fig. 1).

FACS analysis

Immune cell populations from the peritoneal cavity of cox-2+/+ or cox-22/2

mice were stained for flow cytometry using mAbs against F4/80, CCR7,CD29, ICAM-1 (CD54), and Gr1 (eBioscience). mAbs against CD45R/B220, CD11a, CD11b, CD11c, NK1.1, CD3, and CD18 were from BDBiosciences. CCR2 and CCR5 polyclonal Abs were a gift of Dr. MatthiasMack (Institute for Technical Microbiology Biotechnology, Mannheim,Germany). F-actin was visualized using phalloidin (Invitrogen). All sam-ples were acquired in a FACSCalibur cytometer (BD Biosciences) andanalyzed using FlowJo 4.1 software (TreeStar).

In vivo adhesion and migration assays

Thioglycolate-elicited peritoneal macrophages from cox-2+/+ or cox-22/2

donor mice were stained with a PKH26 Red Fluorescent Cell Linker kitfrom Sigma-Aldrich. A total of 53 106 cells in 1 ml PBS was injected intothe peritoneal cavity of C57BL/6 recipient mice that had been inoculatedwith Brewer’s thioglycolate 4 d earlier. One microgram of LPS (E. coli026:B6; Sigma-Aldrich) in 300 ml PBS was injected i.p. 30 min later.Control animals were injected only with PBS. Animals were killed after5 min to analyze macrophage adhesion to the peritoneal membrane or after4 h to study macrophage migration to inguinal lymph nodes. Peritonealcavity was washed with ice-cold PBS to determine the number of PKH26+

cells. To study the adhesion of PKH26-stained macrophages in vivo, the

peritoneal membrane was fixed with 4% PFA, and images were analyzed.Inguinal lymph nodes were frozen in OCT freezing medium. Five-microncryosections were collected for fluorescence microscopy. Image acquisi-tion was carried out with a CCD Leica camera using a 403 objective.

In vitro migration assays

Peritoneal macrophages were cultured in the top chamber of 5- or 8-mmtranswell plates (Corning) for 3 h using complete RPMI 1640 medium with5% FCS. Nonadherent cells were removed by washing the chambers with2% FCS, RPMI 1640 medium. The chemokine was placed in the bottomchamber, and cell migration was analyzed after 4 or 24 h at 37˚C. Cellswere treated with the pan-specific PI3K inhibitor LY294002 or with thep110g-specific inhibitor AS252424 for 30 min prior to induction of cellmigration by RANTES. Nonmigrated cells in the top chamber were re-moved by rubbing with a cotton stick. Migrated cells in the bottom of thetranswell filter were fixed with 1% paraformaldehyde (PFA) and stainedwith Violet Crystal (Sigma-Aldrich). Images of eight fields of the transwellfilter were taken using a 203 objective to quantify the number of migratedcells. In vitro migration of peritoneal macrophages from individual animals(n . 3 per group) was tested in duplicate. Data are shown as relativemigration (number of cells in the presence of chemokine/number of cellsin the absence of any chemokine) 6 SD.

In vitro cell-adhesion assays

Cell adhesion to culture plates precoated with fibronectin (2 mg/ml; Sigma-Aldrich) and ICAM-1 (1 mg/ml; R&D Systems) was tested after labelingperitoneal macrophages with BCECF AM cell tracker (0.1 mg; Invitrogen)for 30 min at 37˚C. Macrophage adhesion was performed at 37˚ for 20min. Fluorescence intensity from the total number of cells was measuredusing FLUOstar Optima (BMG LABTECH). Then, nonadherent cells wereremoved by washing several times with PBS, and fluorescence of remainingcells (adherent cells) was measured. Cell adhesion to fibronectin or ICAM-1was calculated as the mean fluorescence of adherent cells divided by themean fluorescence of total cells and is represented as a percentage.

Fluorescence microscopy

To study the structure of the actin cytoskeleton, peritoneal macrophageswere cultured on cover slips for 20 min, at an early stage, or for 18 h toevaluate podosome and focal adhesion formation. Cox-2 enzymatic activitywas blocked in these experiments by adding celecoxib 1 h before treatingthe cells with LPS or PGE2. Then, cells were washed with PBS and fixedwith 4% PFA for 15 min. Cover slips were blocked with 1% BSA in PBSand incubated with an anti-vinculin mAb (Sigma-Aldrich) and phalloidin–Alexa Fluor 488 (Invitrogen). Vinculin was detected using a donkey anti-mouse Ab coupled to Alexa Fluor 555 (Invitrogen). Cover slips werewashed three times with PBS and then once with H2O and 70% ethanol,dried for 5 min, and mounted using Mowiol (Calbiochem). Images fromthree independent experiments were captured with a Radiance 2000Confocal System (Bio-Rad) or a Confocal LSM510 Meta (Zeiss) coupledto an inverted microscope Axiovert S100 TV (Zeiss). Abs against CD11b(biotin conjugated) and focal adhesion kinase (FAK; pTyr397) were pur-chased from eBioscience and Invitrogen, respectively.

SDS-PAGE and Western blotting

Total protein extracts were obtained, and cell lysates were subjected toWestern blot analysis using conventional SDS-PAGE, followed by proteintransfer to nitrocellulose membranes, as described (43). Abs against pax-illin, pTyr118 Paxillin (BD Bioscience), vinculin (Sigma-Aldrich), FAK(Invitrogen), ERK1/2, p-ERK1/2, p-JNK, JNK, Akt, p-Akt Ser473, PI3Kp85, PI3K p101 (Cell Signaling), PI3K p110g, PI3K p110d, and b-actin(Santa Cruz Biotechnology) were used.

Rac and Cdc42 activation

Rac- and Cdc42-activation assays were carried out by pull-down assaysusing a GST fusion protein containing the p21-binding domain (PBD) ofp21-activated kinase (PAK) for Rac and Cdc42 (10 mg; Millipore). Briefly,a subconfluent monolayer of peritoneal macrophages was lysed with 1 mlice-cold cell lysis buffer (25 mM HEPES, 150 mM NaCl, 1% IGEPALCA-630, 10% glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, andprotease and phosphatase inhibitor mixture; Roche). Cell lysates wereimmediately centrifuged at 10,000 3 g for 20 min at 4˚C. Protein con-centration was determined by the bicinchoninic acid method (ThermoScientific). The same concentration of protein was incubated for 1 h at 4˚Cwith PAK-1 PBD (Upstate) with mild agitation. Beads were washed threetimes with lysis buffer. Bound proteins were eluted with Laemmli samplebuffer and separated by SDS-PAGE. Rac and Cdc42 were detected by

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Western blot with specific Abs (anti-Cdc42 Ab from Santa Cruz Bio-technology; anti-Rac1 Ab [clone 23A8] from Millipore).

Statistics

Statistical analysis of the experimental data was carried out usingGraphPad 5. The Student t test (unpaired) and the Mann–Whitney non-parametric test were performed to compare different assay groups. Thestatistical test done for each particular experiment is described in the figurelegends. The p values ,0.05 were considered statistically significant.

ResultsCox-2 deficiency affects cell migration to the peritoneal cavity

Thioglycolate-induced peritonitis was used as experimental modelto evaluate the role of Cox-2 expression in cell migration to theperitoneum. Interestingly, we found a significant reduction in thetotal number of cells migrating to the peritoneal cavity in cox-22/2

mice compared with cox-2+/+ mice (Fig. 1A). FACS analysis ofperitoneal exudates showed that CD11b+ CD11c+ F4/80+ macro-phages (44) are the main cell population in the peritoneal cavity atday 4 after peritonitis induction with sodium thioglycolate (Fig.1B). The decrease in the total number of cells in cox-22/2 micewas almost exclusively due to a reduction in the migration ofF4/80+ CD11c+ CD11b+ macrophages (∼50% reduction in totalcell number, p , 0.05). In contrast, the number of CD3+ cells(T lymphocytes), CD45R/B220+ cells (mainly B lymphocytes),NK1.1+ cells (NK cells), and Gr1+ cells (granulocytes) recruited tothe peritoneal cavity was similar in cox-2+/+ and cox-22/2 mice(Fig. 1C). Cell survival was not compromised in the peritonealcavity of cox-22/2 mice (Supplemental Fig. 1A). Moreover, thiseffect cannot be ascribed to significant alterations in circulatingmonocytes in cox-22/2 mice (data not shown).

In vitro cell migration of cox-22/2 macrophages in response tochemokines is defective

The reduced number of cox-22/2 cells recruited into the peritonealcavity may indicate a defect in the migratory capacity of macro-phages. Thus, we tested in vitro migration of thioglycolate-elicitedperitoneal macrophages from cox-2+/+ or cox-22/2 mice in re-sponse to CCR2- and CCR5-dependent chemokines (MCP-1,RANTES, MIP-1a, and MIP-1b). The absence of Cox-2 in peri-toneal macrophages significantly reduced in vitro cell migration in

response to these chemokines (Fig. 2A), whereas cox-22/2 bonemarrow–derived DCs migrated normally in response to MIP-3b,one of the ligands of CCR7 that mediates the migration of DCs(Supplemental Fig. 2). Then, we looked for alterations in theexpression of different chemokine receptors by FACS. Elicitedperitoneal macrophages express CCR2 and CCR5 but lack cellsurface expression of CCR7 and CXCR4, in agreement withprevious reports (30, 34) (Fig. 2B, data not shown). We observedsimilar levels of CCR2 and CCR5 in thioglycolate-elicited peri-toneal macrophages from cox-2+/+ and cox-22/2 mice, suggestingthat the cell migration defect cannot be ascribed to differences inchemokine receptor expression.To determine whether effects on the migration of cox-22/2-null

macrophages are related to the absence of Cox-2 activity, weanalyzed the effect of the inhibition of Cox-2 enzymatic activityon cell migration of wild type macrophages. Cox-2 is expressedat low levels on freshly isolated thioglycolate-elicited peritonealmacrophages, and it is rapidly induced after LPS or PGE2 stim-ulation (43). NS-398 is a Cox-2–specific inhibitor that blockedPGE2 production by macrophages after LPS stimulation at a doseof 0.1 mM (Fig. 1C, Supplemental Fig. 1C). In vitro migration ofcox-2+/+ macrophages treated with NS-398 1 h before inducingcell migration in response to RANTES was impaired, suggestingthat Cox-2 enzymatic activity and PG synthesis are required formacrophage migration (Fig. 2D).

In vivo macrophage migration is reduced in the absence ofCox-2

Macrophages are able to emigrate from the peritoneal cavity toadjacent lymph nodes in response to LPS (11). To address whetheremigration of cox-22/2 peritoneal macrophages to lymph nodeswas also affected, we transferred peritoneal-elicited macrophagesfreshly isolated from cox-2+/+ and cox-22/2 mice into the peri-toneal cavity of C57BL/6 mice. To track macrophages after in-jection, we prestained them using the red fluorescent membranelinker PKH26. As shown in Fig. 3A, the number of PKH26+ cellsremaining in the peritoneal cavity of mice 4 h after i.p. injection ofLPS was significantly lower than in mice injected with PBS, in-dicating macrophage emigration. Interestingly, the number of cox-22/2 macrophages (PHK26+ cells) remaining in the peritoneal

FIGURE 1. Macrophage recruitment to the peri-

toneal cavity is diminished in cox-22/2 mice. (A)

Quantification of the total number of cells migrated

to the peritoneal cavity of cox-2+/+ or cox-22/2 mice

4 d after i.p. injection of sodium thioglycolate (10%

p/v). Data were collected from three independent

experiments, in which three mice/group were ana-

lyzed. Each symbol represent an individual mouse,

and the horizontal lines represent the mean. (B and C)

Analysis of cell populations in the peritoneal cavity

of cox-2+/+ and cox-22/2 mice by flow cytometry.

Percentage of cells (B) or total number of cells re-

covered (C) is shown as mean 6 SEM (n = 9 mice/

group from data collected in three independent ex-

periments). Mann–Whitney nonparametric test was

performed with data shown in (A) and (C); statistical

significance is indicated. CD3, T cells; CD11b, macro-

phages; CD11c, macrophages/DCs; CD45R/B220, B

cells; F4/80 and CD11b, macrophages; Gr-1, gran-

ulocytes; NK1.1, NKT cells.

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cavity of C57/BL6 mice after LPS challenge was significantlyhigher than in those who received cox-2+/+ macrophages, sug-gesting a reduced ability of cox-22/2 macrophages to migrate outof the peritoneum. Cell survival was not compromised in the peri-toneal cavity of these mice after LPS treatment (SupplementalFig. 1B). To corroborate that emigration of Cox-2–deficient macro-phages was affected, we isolated inguinal lymph nodes from thoseanimals. We detected PKH26+ cells in inguinal lymph nodes thatwere F4/80+ CD11b+ CD11c+ macrophages (Supplemental Fig.3). Detection of PKH26+ macrophages by fluorescent microscopyshowed a significant reduction in the number of cox-22/2 macro-

phages in lymph nodes compared with the number of cox-2+/+

macrophages (Fig. 3B). Taken together, these results suggest thatCox-2 is important for in vivo migration of peritoneal macro-phages to lymph nodes in response to LPS.

FIGURE 2. Impaired in vitro migration of Cox-2–deficient macrophages

in response to chemokines. (A) Macrophage migration in response to

chemokines. Thioglycolate-elicited peritoneal macrophages from cox-2+/+

or cox-22/2 mice were cultured over transwell plates in the presence of the

indicated concentrations of MCP-1, RANTES, MIP-1a, or MIP-1b in the

bottom chamber. Cell migration was quantified after 4 h and is presented as

relative migration (number of cells in the presence of chemokine/number

of cells in the absence of any chemokine) 6 SD. Data pooled from two

independent experiments are shown. Thioglycolate-elicited macrophages

from two or three mice were used separately in each independent exper-

iment. *p, 0.05, unpaired t test with a total of four or five mice/group. (B)

Cell surface expression of CCR2 and CCR5 chemokine receptors in cox-

2+/+ or cox-22/2 macrophages was analyzed by FACS. A control isotype

Ab was used as a negative control. Results shown are representative of

three independent experiments. (C) The Cox-2–specific inhibitor NS-398

blocks PGE2 production by peritoneal macrophages. NS-398 (0.1 mM) was

added to the cells 1 h prior to stimulation with LPS (1 mg/ml). PGE2

production was measured by ELISA 18 h later. (D) Cox-2 enzymatic in-

hibition impairs macrophage migration in response to RANTES (10 ng/

ml). Results shown are representative of three independent experiments.

**p , 0.005, unpaired t test.

FIGURE 3. In vivo migration and adhesion of macrophages to the

peritoneal membrane are reduced in the absence of Cox-2. Peritoneal

macrophages from cox-2+/+ or cox-22/2 mice were isolated after 4 d of

thioglycolate injection. Cells were labeled with PKH26 before transfer into

the peritoneal cavity of C57BL/6 mice to track cell emigration from the

peritoneal cavity to lymph nodes. (A) Analysis of peritoneal exudates from

C57BL/6 mice after LPS challenge. Cox-2+/+ and cox-22/2 macrophages

were quantified by FACS 4 h after LPS injection (1 mg in PBS, i.p.). Data

from two independent experiments (n = 2–3 mice/group) were pooled and

are shown as percentage (6 SD) of PKH26+ cells (n = 5–6 mice/group).

*p = 0.0147, unpaired t test. (B) Fluorescence detection of PKH26+

macrophages in inguinal lymph nodes. The number of PKH26+ macro-

phages in 5-mm cryosections was quantified. Four fields (403 objective

lens) were analyzed per tissue section. Data are shown as mean 6 SEM

(n = 5–6/group). *p = 0.0484, unpaired t test. (C) FACS analysis of peri-

toneal macrophages (CD11b+/ PKH26+) from cox-2+/+ or cox-22/2 mice

that remained in peritoneal exudates of C57BL/6 mice. Cox-2+/+ and cox-

22/2 macrophages were stained with PKH26 prior to cell transfer. Cell

adhesion was induced by i.p. injection of LPS or PBS (negative control) 5

min before cell recovery. Bar graph shows the mean percentage (6 SEM)

of PKH26+ cells that remained in the peritoneal exudates. Data are from

three independent experiments (n = 12–13 mice/group). *p = 0.0056,

unpaired t test. (D) Immunofluorescence detection of PKH26+ cells at-

tached to the peritoneal membrane. Four fields (403 objective lens) were

analyzed for each tissue sample to quantify the number of PKH26+ cells.

Data from three independent experiments are shown as mean 6 SEM (n =

12 mice/group). *p = 0.0006, unpaired t test.

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Cox-2 expression is required for macrophage adhesion to themesothelium

Cell migration is closely linked to cell adhesion. Thus, we testedthe effect of Cox-2 deficiency on macrophage adhesion to theperitoneal membrane in our in vivo experimental model. Wetransferred PKH26-labeled cox-2+/+ and cox-22/2 thioglycolate-elicited peritoneal macrophages into C57/BL6 mice before in-jecting them with LPS. After 5 min, the peritoneal cavity waswashed with ice-cold PBS. LPS treatment allows macrophageadhesion to the mesothelium, as shown by the significant reductionin the number of cox-2+/+ macrophages (PKH26+ cells) recovered inthe peritoneal exudates of LPS-treated mice compared with PBS-injected control mice. Interestingly, this reduction was not observedin mice transferred with cox-22/2 macrophages (Fig. 3C). Next,we analyzed cell adhesion to the peritoneal mesothelium by fluo-rescent microscopy. Quantification of PKH26+ macrophages fromseveral fields of view showed increased adhesion of cox-2+/+

macrophages to the peritoneal membrane upon LPS injection. Incontrast, cox-22/2 macrophages failed to attach to the mesothelium,remaining instead in the peritoneal cavity (Fig. 3D). Thus, a reducedability of cells to adhere to the mesothelium would explain thedecrease in migration of cox-22/2 macrophages in response to LPS.

Macrophage adhesion is reduced in the absence of Cox-2

To study in vitro changes in cell adhesion of thioglycolate-elicitedperitoneal macrophages from cox-22/2 mice, we performed cell-adhesion assays in plates coated with ICAM-1 or fibronectin.

Compared with cox-2+/+ macrophages, there was a significantdecrease in the number of cox-22/2 macrophages that adhered toeither ICAM-1 or fibronectin (Fig. 4A, 4B). Furthermore, celladhesion to fibronectin was significantly reduced when cox-2+/+

macrophages were pretreated with a specific Cox-2 inhibitor(Celecoxib), showing that Cox-2–dependent PG production playsan important role in cell adhesion (Fig. 4C).b1 and b2 integrins are essential for firm adhesion of macro-

phages to the mesothelium (45). Thus, we analyzed cell surfaceexpression of several integrins in thioglycolate-elicited macro-phages from cox-2+/+ and cox-22/2 mice. We did not find anydifferences in the cell surface expression of integrins CD11a,CD11b, CD18, or CD29 or the adhesion molecule ICAM-1 be-tween cox-2+/+ and cox-22/2 cells (Fig. 4D).

Cox-2–deficient macrophages show an altered actincytoskeleton organization

Upon integrin-mediated anchoring, firm cell adhesion requirespolarization of the actin cytoskeleton, which is key for cell mi-gration (33). Actin cytoskeleton is organized as filaments gen-erating two well-distinguished structures: podosomes and focaladhesions. Podosomes are involved in cell diapedesis, whereasfocal adhesions are related more to cell anchoring and interstitialmigration (46). To understand the role of Cox-2 in actin cytoskel-eton reorganization, we performed experiments with thioglycolate-elicited peritoneal macrophages from cox-2+/+ and cox-22/2 mice.We examined actin cytoskeleton remodeling during short-term(20 min) and long-term (18 h) cell adhesion by staining actin

FIGURE 4. Cell surface expression of integrins

is normal in Cox-2–deficient macrophages. Adhe-

sion of thioglycolate-elicited peritoneal macro-

phages from cox-2+/+ and cox-22/2 mice to plates

coated with ICAM-1 (A) or fibronectin (B). *p ,0.05, **p , 0.005, unpaired t test. (C) Effect of

Cox-2 inhibition on cell adhesion to fibronectin.

Macrophages were pretreated for 1 h with a specific

inhibitor of Cox-2 (celecoxib, 100 nM). *p , 0.05.

(D) Cell surface expression of F4/80, CD11a,

CD11b, CD18, CD29, and ICAM-1 in peritoneal

macrophages from cox-2+/+ and cox-22/2 mice.

Black graphs represent data from cox-2+/+ macro-

phages. Gray line represents data from cox-22/2

macrophages. The isotype Ab control is shown as

a gray graph. Bar plots show mean fluorescence

intensity (MFI) 6 SEM. All results are represen-

tative of three independent experiments.

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filaments with phalloidin conjugated to Alexa Fluor 488. Detec-tion of vinculin by immunofluorescence, one of several proteinsinvolved in anchoring F-actin to the membrane, was performed inparallel to further characterize podosomes and focal adhesions.Cox-2+/+ macrophages cultured on fibronectin-coated cover-

slips for 20 min showed a peripheral distribution of F-actin as-sociated with vinculin. On the contrary, cox-22/2 peritoneal mac-rophages failed to reorganize the actin cytoskeleton. Neitherpodosomes nor focal adhesions were clearly seen at this earlystage of cell adhesion (Fig. 5A). Further study of some of theproteins involved in focal adhesion, including vinculin, paxillin,FAK, and actin, showed no difference in the total protein ex-pression levels between cox-2+/+ and cox-22/2 peritoneal macro-phages (Fig. 5B). F-actin levels were equivalent in both types ofmacrophages, indicating that altered reorganization of the cyto-skeleton was not due to changes in actin expression or polymer-ization at this early stage of cell adhesion (Fig. 5C). Tyrosinephosphorylation of paxillin is involved in the regulation of dy-namics of actin cytoskeletal organization in motile cells (47).Analysis of the levels of phospho-paxillin (Tyr118) in cox-2+/+ andcox-22/2 macrophages by Western blot showed decreased levelsof phospho-paxillin in Cox-2–deficient cells (Fig. 5D). This resultprompted us to study cell adhesion of cox-2+/+ and cox-22/2

peritoneal macrophages after long-term cell culture.After 18 h of in vitro culture of thioglycolate-elicited peritoneal

macrophages on coverslips coated with fibronectin, podosomeswere detected as a dense core of actin surrounded by vinculin andintegrins distributed in a ring shape (Fig. 6A, Supplemental Fig. 4).The number of cox-22/2 macrophages containing podosomes wassignificantly diminished compared with cox-2+/+ macrophages.

Inhibition of Cox-2 enzymatic activity on cox-2+/+ macrophagesby celecoxib showed a similar reduction in the number of cellscontaining podosomes. Cox-22/2macrophages treated with celecoxibshowed no changes in the number of cells with podosomes com-pared with nontreated cox-22/2 cells (Fig. 6B).In contrast, focal adhesions were detected as dot-like accumu-

lations of actin that are often found at the end of actin stressfibers. These focal adhesions also contained vinculin and phospho-FAK (pTyr397), as reported previously (48) (Supplemental Fig. 4).Comparison of cox-2+/+ and cox-22/2 macrophages showed nodifference in the number of cells that had any focal adhesion.Furthermore, macrophage treatment with celecoxib had no effecton focal adhesion formation (Fig. 6C).

PGE2 modulates actin cytoskeleton organization and restoresmacrophage adhesion in the absence of Cox-2

PGE2 produced by DCs in response to LPS and TNF-a modulatesactin cytoskeleton organization. PGE2 dissembles podosomes, pro-moting migration of DCs (36). Less clear is the role of PGE2 infocal adhesion formation. Different reports suggest that PGE2 caneither promote or inhibit focal adhesion formation, depending onthe cell type (37, 49). To better understand the role of PGE2 in theorganization of actin cytoskeleton in macrophages, we examinedcells, using confocal microscopy, that were treated or not withPGE2 (2 mM) for 18 h. Visualization of actin filaments (stainedwith phalloidin) and vinculin showed a significant reduction in thenumber of cells containing podosomes after treatment with PGE2

(Fig. 7A, 7B). On the contrary, a higher number of cells containingactin stress fibers and focal adhesions was observed in macro-phages treated with PGE2 compared with nontreated cells (Fig.

FIGURE 5. Cox-22/2 macrophages show abnormal cytoskeleton remodeling at early stages of cell adhesion. (A) Visualization of actin cytoskeleton by

confocal fluorescent microscopy (603 objective lens) at early stages of cell adhesion. Adhesion of thioglycolate-elicited peritoneal macrophages from cox-2+/+

or cox-22/2 mice was performed at 37˚C for 20 min. Actin cytoskeleton remodeling was analyzed using phalloidin coupled to Alexa Fluor 488 and

a monoclonal anti-vinculin Ab (secondary Ab coupled to Alexa Fluor 555). Representative images from three biological replicates/genotype are shown. (B)

Analysis of paxillin, vinculin, FAK, and actin protein levels by Western blot. Cell extracts were isolated from thioglycolate-elicited peritoneal macrophages

from cox-2+/+ or cox-22/2 mice. Quantification of protein levels relative to actin is indicated below each Western blot panel. (C) FACS analysis of actin

polymerization. F-actin was stained using phalloidin. A representative plot of three biological replicates/genotype is shown. (D) Western blot analysis of the

levels of phospho-paxillin (Tyr118) in cox-2+/+ or cox-22/2 macrophages. Cells were adhered to plates for 90 min before preparation of cell protein lysates. The

amount of phospho-paxillin (Tyr118) was quantified relative to the total amount of paxillin and is indicated below each Western blot panel. A representative

experiment of three is shown.

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7A, 7C). Previously, we showed that inhibition of Cox-2 enzy-matic activity with celecoxib diminished macrophage adhesion toICAM-1. To test whether exogenous PGE2 rescues cell adhesionof thioglycolate-elicited cox-22/2 macrophages, we treated themwith 2 mM PGE2 1 h before assaying macrophage adhesion toplates coated with ICAM-1. PGE2 treatment of cox-2+/+ macro-phages significantly increased cell adhesion to ICAM-1. Adhesionof cox-22/2 macrophages to ICAM-1 was significantly reduced inthe absence of exogenous PGE2, as expected. In contrast, thetreatment of cox-22/2 macrophages with PGE2 partially recoveredcell adhesion to ICAM-1 (Fig. 7D).Next, we tested whether LPS affected focal adhesion formation

in macrophages. Production of endogenous PGE2 by macrophagesis Cox-2 dependent and increases after LPS activation (Fig. 2C,

Supplemental Fig. 1). Analysis of actin and vinculin distributionin thioglycolate-elicited macrophages adhered to fibronectin afterLPS stimulation showed a significant increase in the formation ofactin stress fibers (Fig. 8A). The number of macrophages in whichfocal adhesions were detected increased significantly after LPSactivation (Fig. 8B). Inhibition of Cox-2 enzymatic activity bycelecoxib did not change the number of macrophages in whichfocal adhesions were detected compared with macrophages thatwere not activated by LPS. On the contrary, celecoxib blockedfocal adhesion formation in response to LPS. Finally, we evaluatedcell adhesion of thioglycolate-elicited macrophages to fibronectin(Fig. 8C). Celecoxib significantly decreased macrophage adhesionto fibronectin, whereas macrophage stimulation with LPS increasedit. Cox-2 inhibition by celecoxib significantly reduced the adhesion

FIGURE 6. Formation of podosomes

during long-term cell adhesion is re-

duced in the absence of Cox-2 enzy-

matic activity. (A) Visualization of actin

cytoskeleton by confocal fluorescent

microscopy (603 objective lens) at late

stages of cell adhesion. Macrophage

adhesion to fibronectin was performed

at 37˚C for 18 h. Where indicated, cox-

2+/+ and cox-22/2 macrophages were

treated with celecoxib (100 nM) 1 h

earlier. Actin cytoskeleton was stained

using phalloidin coupled to Alexa Fluor

488. Podosomes and focal adhesions

were identified with a monoclonal anti-

vinculin Ab. A donkey anti-mouse Ab

coupled to Alexa Fluor 555 was used

for detection. Representative images

are shown. Podosomes are denoted by

arrows. (B) Genetic or chemical inhi-

bition of Cox-2 activity decreases the

number of cells with podosomes. (C)

Quantification of the number of cells

with focal adhesions. Each symbol in-

dicates the percentage of cells with

podosomes (B) or focal adhesions (C)

in one field of view. Two independent

immunofluorescence stainings were ana-

lyzed per condition for each of the at

least three biological replicates/genotype.

The unpaired t test was used to compare

two groups.

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capacity of LPS-activated macrophages, which was now compara-ble to the cell adhesion capacity of nontreated macrophages.Altogether, the data showed that Cox-2 enzymatic activity and

endogenous production of PGE2 were required for actin cyto-skeleton organization and macrophage adhesion.

Cox-2 expression is required for PI3K-dependent cell signaling

Macrophage activation through TLRs is intimately related to celladhesion. LPS stimulation of monocytes and macrophages acti-vates PI3K, which regulates tyrosine phosphorylation of paxillin(50, 51). Lower levels of phospho-paxillin in cox-22/2 macro-phages suggested that cell signaling activation may be altered inthe absence of Cox-2. b2 integrins are known to activate multiplesignaling cascades required for macrophage adhesion and mi-gration, including ERK1/2, JNK, and PI3K (30, 34). We nextanalyzed the status of some key molecules after adhesion ofthioglycolate-elicited macrophages. Cell adhesion for 90 min in-duced phosphorylation of JNK, ERK1/2, and Akt. The levels of p-ERK1/2 and p-JNK were similar in Cox-2–deficient macrophagescompared with wild type ones. Interestingly, lower levels of p-Aktwere observed in cox-22/2 peritoneal macrophages (Fig. 9A). Thefact that p-Akt levels were reduced suggested a defect in PI3K-mediated signaling. Thus, we analyzed the levels of the PI3K reg-ulatory subunits p85 and p101, along with the PI3K catalytic sub-units p110g and p110d, which were described previously as beingkey molecules for leukocyte migration (40, 41). p85, p101, andp110d proteins were expressed at similar levels in cox-2+/+ andcox-22/2 macrophages. Notably, p110g protein expression wassignificantly reduced in cox-22/2 peritoneal macrophages (Fig. 9B).Treatment of thioglycolate-elicited macrophages with PGE2 for90 min increased p110g protein expression (Fig. 9C). Short-terminhibition of Cox-2 enzymatic activity by celecoxib decreased

p110g protein levels in macrophages, which were restored byadding PGE2 (Fig. 9D).To determine whether lower levels of p-Akt in cox-22/2 macro-

phages resulted in deficient signaling downstream of this kinase, westudied the activation of GTPases involved in cell adhesion andmigration. Rac-1 and cdc42 were pulled down, after cell adhesionmediated by ICAM-1, using the PBD of PAK. The presence ofactive Rac-1–GTP and Cdc42-GTP was severely reduced in cox-22/2

macrophages compared with cox-2+/+ macrophages (Fig. 9E, 9F).PI3K signaling was proved to be fundamental for cell migra-

tion. Class I PI3K p110g and p110d are key for regulating themigration of immune cells. In particular, p110g2/2 mice havea defective accumulation of macrophages in a septic peritonitismodel due to reduced macrophage migration toward severalchemokines, although less is known about its affect on cell ad-hesion (41). We performed cell-adhesion assays to ICAM-1 in thepresence of a nonspecific PI3K inhibitor, LY294002, or a p110g-specific inhibitor, AS252424. Pretreatment of peritoneal macro-phages with these inhibitors diminished cell adhesion to ICAM-1(Fig. 9G) without affecting cell survival (Supplemental Fig. 1D).Furthermore, PI3K and p110g inhibitors severely decreasedmacrophage migration in response to RANTES, thus highlightingthe importance of PI3K p110g catalytic activity in macrophagecell adhesion and migration (Fig. 9H).

DiscussionMigration of leukocytes to the inflamed tissues is a hallmark ofinflammation (2). Previous studies reported that Cox-derived PGscan modulate leukocyte migration to the inflamed tissue (52).Mice deficient in Cox-2 or mPGES-1, a downstream PG synthase,have a reduced recruitment of leukocytes to the inflammatory foci,as shown in different mouse models, including thioglycolate-

FIGURE 7. PGE2 promotes macro-

phage adhesion. (A) Analysis of podo-

somes and focal adhesion formation after

treatment with PGE2. PGE2 (2 mM) was

added before triggering cell adhesion

of thioglycolate-elicited peritoneal mac-

rophages to fibronectin for 18 h. Images

were acquired using 603 objective lens.

Arrows denote podosomes or focal adhe-

sions (green = phalloidin, red = vinculin).

(B) PGE2 decreases the number of cells

with podosomes. (C) PGE2 increases focal

adhesion formation. Quantification of the

percentage of cells with podosomes (B)

and focal adhesions (C) was done by an-

alyzing several fields of view from two

independent experiments with cells from

three individual mice stained indepen-

dently. Each symbol represents the per-

centage of cells in one field of view. All

data were pooled, and the unpaired t test

was used to compare the two groups. (D)

Exogenous PGE2 rescues cell adhesion

of cox-22/2 macrophages. Thioglycolate-

elicited peritoneal macrophages from cox-

2+/+ and cox-22/2 mice were treated or

not with PGE2 1 h before assaying mac-

rophage adhesion to plates coated with

ICAM-1. Cells from four biological rep-

licates were assayed per genotype in

triplicate. The unpaired t test was used to

compare the different groups, as indicated.

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induced peritonitis (53, 54). Nevertheless, the underlying mech-anisms remain unclear. Our results point to the importance of Cox-2 in macrophage adhesion and migration, which are markedlyimpaired in cox-22/2 mice. Moreover, analysis of the signalingpathways involved in these effects indicates that Cox-2 is requiredto maintain normal protein levels of p110g in these cells. Thepaxillin/p110g-PI3K/Cdc42/Rac1 axis is defective in cox-22/2

macrophages, resulting in altered cell adhesion and migration.In recent years, the view of macrophages merely as phagocytes

has been expanded with the description of new macrophagepopulations with differentiated effector functions, including themas APCs to activate the adaptive immune response (55, 56). Newdata show that monocyte–macrophage turnover involves cell mi-gration in and out of the inflammatory focus with importantphysiological consequences (57, 58). However, macrophage emi-gration from the inflammatory focus is not completely understood.We found that, in the thioglycolate-elicited peritonitis model,

Cox-2 deficiency leads to a significant selective reduction in themigration of CD11b+ F4/80+ CD11c+ myeloid cells to the peri-toneal cavity, in line with previous data obtained from mPGES1-null mice (53, 54). More than 90% of CD11b+ cells were positivefor the macrophage-specific Ag F4/80, which is expressed bymature macrophages (44), indicating a defect in the migration ofthose cells to the peritoneum. Using a model of thioglycolate-elicited peritonitis to study macrophage emigration describedpreviously (10, 11), we show in this study that macrophage emi-gration to lymph nodes is reduced after LPS challenge in theabsence of Cox-2. Cox-22/2 macrophages were defective withregard to migration toward CCR2- and CCR5-binding chemo-

kines, as were cox-2+/+ macrophages treated with Cox-2–specificinhibitors, whereas cox-22/2 bone marrow–derived DCs migratednormally in response to MIP-3b (a ligand of the CCR7 receptor).The migration defect was not due to alterations in CCR2 andCCR5 surface expression on cox-22/2 macrophages. Neverthe-less, taking into account the results showing altered signaling incox-22/2 macrophages, it must be considered that chemokinereceptor signaling could be also impaired.Impaired macrophage migration can be explained by the sig-

nificant reduction in cell adhesion to the mesothelium (in vivoassays) or to ICAM-1 or fibronectin (in vitro assays). Integrinsare known to activate multiple signaling cascades required formacrophage migration (2, 30, 34). The “integrin adhesome”involves.100 proteins linked together by .500 interactions (59).Recently, we found that integrin-mediated signaling induces Cox-2 upregulation, which is associated with FAK activation inmesangial cells (60). In this study, we found that cox-22/2 mac-rophages have a defect in cell adhesion that is due to defectiveactin cytoskeleton reorganization. A reduced number of cox-22/2

macrophages present podosomes after adhesion. Podosomes weredescribed as short-lived adhesion organelles with a core of actinsurrounded by adhesion molecules, including integrins and met-alloproteinases, which are able to digest extracellular matrix and,thus, are important for interstitial migration (61). F-actin poly-merization was not affected in cox-22/2 macrophages, suggestingthat these cells may have a defect in cell-signaling transduction.Activation of FAK and Rho-family GTPases is required for cy-toskeleton remodeling and cell adhesion (62). FAK phosphor-ylates downstream-signaling molecules, such as paxillin (Tyr118),

FIGURE 8. PGE2 production by macrophages

after LPS stimulation mediates focal adhesion

formation. (A) Analysis of focal adhesions by

confocal microscopy in thioglycolate-elicited

macrophages treated with LPS (green = phal-

loidin, red = vinculin). Celecoxib (100 nM) was

added, where indicated, to block Cox-2 enzy-

matic activity. Macrophage adhesion to fibro-

nectin was assayed after 18 h. Images were

acquired using 603 objective lens. (B) Quanti-

fication of the percentage of cells with focal

adhesions. Each symbol represents the percent-

age of cells in one field of view. Data are from

two independent experiments. The unpaired t

test was used to compare the different groups, as

indicated. (C) Analysis of macrophage adhesion

to fibronectin. Macrophages were treated with

celecoxib and/or LPS, as indicated. Cells from

three independent experiments were assayed in

triplicate. All data were pooled together, and the

unpaired t test was used to compare the different

groups.

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which is required for migration of cancer cells (63). The role ofCox-2–derived PGs in FAK activation and cell migration wassuggested previously. HCT-116 cells overexpressing Cox-2 showan enhanced phosphorylation of FAK, resulting in elevated kinaseactivity (64). Moreover, PGE2 induces FAK and paxillin phos-phorylation and subsequent migration in cancer cells (65). P-paxillin regulates adhesion dynamics associated with focal adhe-sions (47). We show in this study that exogenous PGE2 increasesfocal adhesion formation in macrophages. LPS induces Cox-2expression and PGE2 synthesis in macrophages, which are alsoassociated with actin cytoskeleton remodeling. LPS dissociatespodosomes in DCs, as shown previously (36). We observed asimilar effect on focal adhesion assembly in macrophages. Thiseffect is Cox-2 dependent, and it can be blocked by pretreatingmacrophages with celecoxib.Phosphorylation of paxillin on Tyr118 is reduced significantly in

cox-22/2 macrophages. Although some reports showed the abilityof different PGs to modulate cell adhesion, the molecular mech-anism is not well understood. A remarkable finding of our study isthe link established between Cox-2 expression and PI3K signal-ing. PI3K inhibition blocks tyrosine phosphorylation of Pyk2and paxillin, affects actin cytoskeleton remodeling, and decreasesmonocyte and macrophage adhesion (50, 51). Analysis of some ofthe main cell-signaling cascades (ERK1/2, JNK, and PI3K) in cox-22/2 macrophages after cell adhesion showed that phosphoryla-tion of Akt is reduced in the absence of Cox-2. The role of PI3Kin cell adhesion and migration has been studied extensively inthe last few decades. PI3K activation is required for macrophagespreading and migration (66). PI3K is activated through chemo-kine receptors, as well as LPS and PGE2 (67, 68). The PI3Kfamily of proteins is classified into three groups: class I, class II,and class III. Class I PI3Ks are subdivided into two groups: cat-alytic and regulatory PI3Ks, with four and six isoforms, respec-tively. Class I PI3K p110g and p110d have been described as keyfor regulating the migration of leukocytes (40). Cox-22/2 mac-rophages synthesize lower levels of p110g compared with wildtype macrophages. Interestingly, treatment of macrophages withexogenous PGE2 for a short period of time increases p110g pro-tein levels, whereas inhibition of Cox-2 enzymatic activity bycelecoxib reduces p110g expression. p110g protein levels arerestored by exogenous PGE2.p110g2/2 macrophages show a reduced capacity to migrate

toward several chemokines (41, 42), an observation similar towhat we described in cox-22/2 macrophages. Reduced expressionof p110g and reduced phosphorylation of Akt likely leads to theobserved impaired activation of Rac and Cdc42 in cox-22/2

macrophages upon adhesion. PI3K regulates the activation of Racand Cdc42 (69-71). The Rho family of GTPases transduces signalsgenerated from chemokine receptors and adhesion molecules,serving as cross-talk points between different signaling pathwaysinvolved in migration (72). Our results point out that Cox-2 ex-pression is required to maintain the PI3K–GTPase axis for celladhesion and migration. PI3K isoforms have redundant functionsin leukocytes, but Cox-2–deficient macrophages have a selectivedefective expression of p110g but not p110d. The differentialexpression of the PI3K isoforms was shown to be important ina cell- and time-dependent manner, being a tightly regulatedprocess (40, 73). Our data reinforce this idea. PI3K isoforms canhave unique functions, and the relevance of Cox-2 in regulatingcell migration by selectively affecting p110g could be cell de-pendent.Interestingly, we also found that PGE2 increases the adhesion

of peritoneal cox-2+/+ macrophages and restores the cell adhesiondefect of cox-22/2 macrophages. This is associated with a change

FIGURE 9. Cox-2 deficiency impairs PI3K-dependent signaling. (A)

Protein phosphorylation of ERK1/2, JNK, and Akt in cox-2+/+ or cox-22/2

macrophages. Cells were adhered to cell culture plates for 90 min before

preparation of cell protein lysates. Protein levels of total or phosphorylated

proteins were detected with specific Abs. Protein quantification of each

phospho isoform is shown as a number below each panel. Phospho protein

abundance was quantified after correcting against the total amount of each

protein. Then, it was calculated relative to the amount in wild type cells. (B)

Protein expression of p110g is reduced in cox-22/2 macrophages. Protein

levels of PI3K p85, p101, p110d, and p110g were analyzed by Western blot.

Relative quantification of p110g protein in cox-22/2 macrophages compared

with wild type is shown below the panel. (C) PGE2 increases p110g protein

expression. Thioglycolate-elicited macrophages were treated with PGE2

(100 nM) during cell adhesion to ICAM-1. Cell lysates were prepared after

90 min. Hsp90 was used as a loading control and for relative protein

quantification (number is shown below the panel). (D) Inhibition of Cox-2

enzymatic activity decreases p110g protein expression in macrophages.

Thioglycolate-elicited macrophages were incubated with celecoxib for 1 h

before setting up a cell adhesion assay to ICAM-1. Exogenous PGE2 was

added right before setting up a cell adhesion to ICAM-1. Cell lysates were

prepared after 90 min. Hsp90 was used as a loading control and for relative

protein quantification (number is shown below the panel). (E and F) Rac-1

and Cdc42 activation is impaired in cox-22/2 macrophages. Rac and Cdc42

proteins were pulled down using PAK-1 PBD. The presence of the activated

GTP-Rac and GTP-Cdc42 was analyzed by Western blot. Actin was used as

a loading control and for relative protein quantification (number is shown

below each panel). (G) The nonspecific PI3K inhibitor LY294002 and the

p110g-selective inhibitor AS252424 reduce macrophage adhesion to ICAM-

1. (H) Migration of macrophages is regulated by p110g. Wild type macro-

phages were incubated with 2 mM of LY294002 or AS252424 for 30 min

prior to induction of cell migration toward RANTES (50 ng/ml). At least

three independent experiments were performed for each experiment. *p ,0.05 versus cells not treated with inhibitor, **p , 0.005 versus migrated

cells to RANTES not treated with inhibitors, unpaired t test.

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in cell morphology and a normalized actin cytoskeleton organi-zation. Our hypothesis is that autocrine PGE2 secretion by acti-vated macrophages is required to maintain macrophages in apromigratory state by maintaining high levels of p110g, which isrequired for adhesion/migration. How this takes place is currentlyunder study in our laboratory. Previous results from our laboratoryindicate that macrophages’ autocrine PGE2 secretion is likelyrequired to maintain gene expression of cytokines, Cox-2, andmPGES-1 (74). This might be equally true for signaling kinaseslike p110g. EP4 deficiency decreases macrophage survival, com-promising PI3K/Akt and NF-kB pathways (75). These pathwaysare key in the development of the inflammatory response in earlyatherosclerosis; thus, EP4 deficiency significantly reduced theearly development of atheroma plaques in the aortic valves.Missing Cox-2 during any stage of macrophage activation willaffect the final outcome of the inflammatory response. Low pro-duction of PGE2 in Cox-22/2 macrophages, in response to an in-flammatory stimulus, results in less mature/active macrophageswith a differential ability to migrate. This hypothesis is in linewith the proposed role for PGE2 in the migration of monocytes asa booster of chemokine-induced migration (26).In summary, our results suggest that the paxillin/p110g PI3K/

Cdc42/Rac1 axis is defective in cox-22/2 macrophages. Likely,this leads to defective actin polymerization and focal adhesionformation in those cells, as well as impaired cell adhesion andmigration. Experiments in progress will help to identify whichPGs produced by Cox-2 are required for the observed effect onmacrophage migration, although previous in vitro studies sug-gested that Cox-2–mediated production of both PGE2 and PGD2 isresponsible for macrophage migration in response to LPS (76).Together, our results may be important to further understand therole of Cox-2 in inflammatory diseases, especially those in whichan essential role for macrophage emigration is emerging, such asatherosclerosis (57, 58, 77, 78).

AcknowledgmentsWe thank B. Barrocal, H. Salgado, M. Chorro, and M. Maza for excellent

technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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