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Toll-like Receptor 2 (TLR2), Transforming Growth Factor-�,Hyaluronan (HA), and Receptor for HA-mediated Motility(RHAMM) Are Required for Surfactant Protein A-stimulatedMacrophage Chemotaxis*□S

Received for publication, March 10, 2012, and in revised form, August 20, 2012 Published, JBC Papers in Press, September 4, 2012, DOI 10.1074/jbc.M112.360982

Joseph P. Foley‡§1, David Lam¶, Hongmei Jiang¶, Jie Liao¶, Naeun Cheong¶, Theresa M. McDevitt§, Aisha Zaman§,Jo Rae Wright�, and Rashmin C. Savani‡§¶2

From the ‡Division of Pharmacology and Toxicology, Department of Pharmaceutical Sciences, University of the Sciences inPhiladelphia, Philadelphia, Pennsylvania 19104, the §Division of Neonatology, Department of Pediatrics, Children’s Hospital ofPhiladelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the �Department of Cell Biology,Duke University Medical Center , Durham, North Carolina 27710, and the ¶Divisions of Pulmonary and Vascular Biology andNeonatal-Perinatal Medicine, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Background: Surfactant protein A stimulates macrophage chemotaxis.Results: SPA interaction with TLR2 stimulates JNK- and ERK-dependent TGF� production, which in turn stimulatesRHAMM- and hyaluronan-dependent macrophage chemotaxis.Conclusion: SPA activates a novel and specific pathway to stimulate macrophage chemotaxis.Significance:Uncovering the mechanisms that regulate innate immunity in the lung is crucial for understanding the responsesto infection and injury.

The innate immune system protects the host from bacterialand viral invasion. Surfactant protein A (SPA), a lung-specificcollectin, stimulates macrophage chemotaxis. However, themechanisms regulating this function are unknown. Hyaluronan(HA) and its receptors RHAMM (receptor for HA- mediatedmotility, CD168) and CD44 also regulate cell migration andinflammation.We therefore examined the role ofHA,RHAMM,and CD44 in SPA-stimulated macrophage chemotaxis. Usingantibody blockade and murine macrophages, SPA-stimulatedmacrophage chemotaxis was dependent on TLR2 but not theother SPA receptors examined. Anti-TLR2 blocked SPA-in-duced production of TGF�. In turn, TGF�1-stimulated che-motaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophagesfrom TLR2�/� mice failed to migrate in response to SPA butresponded normally to TGF�1 and HA, effects that wereblocked by anti-RHAMMantibody.Macrophages fromWTandCD44�/� mice had similar responses to SPA, whereas thosefrom RHAMM�/� mice had decreased chemotaxis to SPA,

TGF�1, and HA. In primary macrophages, SPA-stimulatedTGF� production was dependent on TLR2, JNK, and ERK butnotp38.Pam3Cys,aspecificTLR2agonist,stimulatedphosphor-ylation of JNK, ERK, and p38, but only JNK and ERK inhibitionblockedPam3Cys-stimulated chemotaxis.Wehave uncovered anovel pathway for SPA-stimulated macrophage chemotaxiswhere SPA stimulation via TLR2 drives JNK- and ERK-depen-dent TGF� production. TGF�1, in turn, stimulatesmacrophagechemotaxis in a RHAMM and HA-dependent manner. Thesefindings are highly relevant to the regulation of innate immuneresponses by SPA with key roles for specific components ofthe extracellular matrix.

Lung infections andmany noninfectious lung disorders, suchas bronchopulmonary dysplasia, acute respiratory distress syn-drome, cystic fibrosis, and occupational lung diseases, involvean inflammatory response with elaboration of growth factorsand cytokines and an abnormal remodeling of the extracellularmatrix. Toll-like receptors (TLRs)3 and a family of proteinscalled collectins play central roles in regulation of host defensevia recognition of specific pathogen-associated molecular pat-terns (PAMPs) on various microorganisms (1). Although theseinnate immune receptors are widely expressed, they are of par-ticular importance to dendritic cells and macrophages.

* This work was supported, in whole or in part, by National Institutes of HealthGrants HL62472 and HL073896 (to R. C. S.) and HL068072 (to J. R. W.). Thiswork was also supported by a Sponsored Research Agreement from Seika-gaku Corp., Japan (to R. C. S.).

We dedicate this manuscript to Jo Rae Wright, who passed away on January11, 2012. Her inspiration, encouragement, and enthusiasm, together withscientific rigor and insight, helped guide and shape this project to itsfruition.

□S This article contains supplemental Figs. 1–3.1 Submitted part of this work for a Master’s degree in Pharmacology and

Toxicology at the Department of Pharmaceutical Sciences, University ofthe Sciences, Philadelphia, PA.

2 To whom correspondence should be addressed: Divisions of Pulmonary andVascular Biology and Neonatal-Perinatal Medicine, Dept. of Pediatrics,5323 Harry Hines Blvd., Dallas, TX 75390-9063. Tel.: 214-648-2145; Fax: 214-648-2096; E-mail: [email protected].

3 The abbreviations used are: TLR, Toll-like receptor; BMDM, bone marrow-derived macrophage; Pam3Cys, tripalmitoyl-S-glycero-Cys-(Lys)4; HA, hy-aluronan (hyaluronic acid); HABP, hyaluronan-binding protein; HABPep,synthetic HA-binding peptide; HA6, 6-mer HA oligosaccharide; HA900,900-kDa HA; RHAMM, receptor for HA-mediated motility (CD168); SPA, sur-factant protein A; PAMP, pathogen-associated molecular pattern; HMW,high molecular weight; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hy-droxymethyl)propane-1,3-diol; SIRP�, signal inhibitory regulatory protein�; PGN, peptidoglycan.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 44, pp. 37406 –37419, October 26, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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The pulmonary collectins, surfactant proteins A (SPA) andD, are critical regulators of macrophage activity (2). They haveimportant roles in aggregation, opsonization, and phagocytosisof pulmonary pathogens, as well as production of reactive oxy-gen species and cytokines (3, 4). In particular, SPA stimulatesmacrophage chemotaxis (5) and phagocytosis of apoptotic neu-trophils, mechanisms that result in production of TGF� (6). Anumber of cell surface proteins bind to SPA. Key among theseare TLR2, TLR4, SIRP�, and calreticulin/CD91, proteins thatintimately regulate the innate immune response (7–9).Transforming growth factor-� (TGF�) is a multifunctional

regulator of cell growth and differentiation that induces theexpression of various extracellular matrix components, includ-ing hyaluronan, collagen, and fibronectin (10). Additionally,TGF� is also a strong chemoattractant for macrophages, lym-phocytes, mast cells, and fibroblasts (11). Interestingly, higherconcentrations of TGF� are anti-inflammatory and contributeto the abrogation of the inflammatory response after injury (6).Hyaluronan (HA), a nonsulfated glycosaminoglycan polymer

of repeating disaccharide units of N-acetylglucosamine andglucuronic acid, is regulated byTGF� and alsomodifies inflam-matory cell behavior, in particular, chemotaxis (reviewed inRef. 12). An increased recovery ofHA in bronchoalveolar lavagehas been found in a variety of human lung disorders, such asacute respiratory distress syndrome and occupational lung dis-orders, and in animal models, such as bleomycin-induced lunginjury (13, 14). In this rodent model, elevated low molecularweight HA content in bronchoalveolar lavage temporally cor-relates with an influx of macrophages into the lung. Further-more, administration of an HA-binding peptide (HABPep)inhibits macrophage accumulation after bleomycin injury, sug-gesting that HA is critical in macrophage recruitment to thelung (14).HA exerts its biologic effects via specific cell surface recep-

tors. Although a number of HA-binding proteins have beenisolated, two distinct cell surface receptors, CD44 andRHAMM (Hmmr, CD168), have been studied in some detail.Binding of HA to CD44 has been implicated in lymphocytehoming, tumorigenesis, and activation of monocytes (15).Binding of HA to RHAMM promotes cell migration in associ-ation with tyrosine phosphorylation and focal adhesion turn-over (16). Furthermore, blockade of either HA or RHAMMinhibits TGF�1-stimulated fibroblast motility (17), as well asmacrophage motility after bleomycin lung injury (18). Interest-ingly, TLR2/4 double knock-out macrophages do not respondto HA stimulation (19), and CD44 associates with TLR4, sug-gesting that TLRs andHA receptors likely cooperate tomediateintracellular signaling by HA (20).Because HA has been implicated in macrophage migration

through interactions with RHAMM and CD44, and both SPAand HA effects are influenced by TLR2/4, we examinedwhether SPA stimulation of macrophage chemotaxis isdependent uponHA/receptor interactions.Wehere report thatSPA-stimulated chemotaxis results fromTLR2- and JNK/ERK-dependent TGF� production, which in turn requires RHAMMand HA, but not CD44, to promote macrophage migration.These studies are highly relevant to inflammatory responses inthe lung, suggest that interactions of the extracellular matrix

with inflammatory cells are key in this novel pathway, andreveal a new role for SPA in innate immune responses.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—SPA was purified from broncho-alveolar lavage of human alveolar proteinosis patients asdescribed previously (21). Purified SPAcontainednodetectableendotoxin as determined by theLimulus assay (data not shown)and no TGF� as determined by the mink lung epithelial cellassay (supplemental Fig. 1). Highly purified and defined HAoligosaccharides, including HA, a six sugar oligosaccharide ofHA, 8-, 14-, and 34-mer and a 900-kDaHA (HA900,HMWHA)that were free of endotoxin, protein, or nucleic acid, were thekind gifts of Seikagaku Corp. (Tokyo, Japan). Anti-RHAMMantibody (R36), generated in rabbits against amino acids 585–605 encoded in the full-length RHAMM cDNA (22, 23), hasbeen described previously (24). CD44 antibodies includedKM81 (generously provided by Ellen Puré, Wistar Institute,University of Pennsylvania), CD44v3 (Calbiochem), and IM-7(BD Biosciences). Other antibodies used in the study includedanti-SIRP� (Upstate, Charlottesville, VA), anti-calreticulin(Affinity Bioreagents, Golden, CO), anti-TLR2 (Zymed Labora-tories Inc.), and anti-TLR4 (e-Bioscience, San Diego). All sig-naling antibodies were obtained from Cell Signaling Technol-ogy (Danvers, MA) and included rabbit monoclonal antibodiesto total ERK1/2 (p42 MAPK, catalog no. 4695) and phospho-p38 (pMAPKAPK-2-T222, catalog no. 3316), and rabbit poly-clonal antibodies to phospho-ERK1/2 (p-p44/42MAPK-T202/Y204, catalog no. 9101), total p38 (p38 MAPK, catalog no.9212), total JNK (SAPK/JNK, catalog no. 9252), phospho-JNK(pSAPK/JNK, catalog no. 9251), and �-actin (catalog no. 4967).Pan-specific TGF� antibody was purchased from R&D (Min-neapolis, MN), and TGF�1 was purchased from Sigma (catalogno. T 7039). The synthetic TLR2 ligands, tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys), and FSL1, are endotoxin-freeand were obtained from Invivogen. Pharmacologic blockers ofthe MAPKs ERK (PD98059, 10 �M), JNK (SP600125, 10 �M),and p38 (SB202190, 10 �M) were obtained from Calbiochem.Peptides—HA-binding peptide (HABPep), mimicking a

charged sequence previously shown to bind HA (RGG-GRGRRR) (25) and scrambled peptide containing the sameamino acids but in a random, non-HA binding order(RGRRGRGRG), have been described previously (14). Peptideswere commercially produced to �95% purity (Invitrogen).Cell Culture—RAW264.7 murine macrophages were

obtained from ATCC (catalog no. TIB-71, Manassas, VA) andwere maintained in DMEM supplemented with 10% heat-inac-tivated FBS, 2 mM L-glutamine, 100 �g/ml streptomycin, and100 units/ml penicillin at 37 °C under 5% CO2. Macrophagesolder than 10 passages were not used. The optimal dose ofTGF�1 to stimulateHAproduction in RAW264.7 cells was firstdetermined as a dose response, and 3–10 ng/ml TGF�1 stimu-lated maximal HA production (supplemental Fig. 2).Mouse Lines and Bone Marrow-derived Macrophages

(BMDM)—All mice were housed in the Animal Care Facilitiesof either The Children’s Hospital of Philadelphia or Universityof Texas Southwestern Medical Center, Dallas. All animalexperiments were reviewed and authorized by the Institutional

Mechanisms of SPA-stimulated Macrophage Chemotaxis

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Animal Care and Utilization Committees of both institutions,and experiments were conducted in accordance with NationalInstitutes of Health guidelines. All mice were on the C57/Bl6background, and wild type littermates were used as controls.TLR2�/� mice were the kind gift of Dr. S. Akira (OsakaUniver-sity, Osaka, Japan); mice expressing a TGF�-responsive con-struct (12 repeats of the CAGA sequence of the plasminogenactivator inhibitor 1 (PAI1) promoter) driving GFP (CAGA-GFP mice) were the kind gift of Dr. Hal Dietz (Johns HopkinsUniversity, Baltimore, MD); CD44�/� mice were the kind giftof Dr. Tak Mak (Amgen, Toronto, Canada), and RHAMM�/�

mice were the kind gift of Dr. Eva A. Turley (London RegionalCancer Centre, London, Ontario, Canada).Bone marrow-derived macrophages were obtained by sacri-

ficing mice and flushing the bone marrow from the tibia andfemur of each mouse with macrophage medium (RPMI 1640with 10% FCS, 20% L929 conditioned medium, 1% penicillin/streptomycin/glutamine) under sterile conditions. Marrowcells weremade into a single cell suspension by gentle pipettingand strained. Cells were centrifuged and resuspended; redblood cells were lysed by flash exposure to hypotonic solutionand then cultured in macrophage medium. Nonadherent cellswere replated and provided fresh medium. Cells were allowedto further differentiate for 3 days and were then used for assays.Chemotaxis Assay—Macrophage chemotaxis was deter-

mined using a modified Boyden chemotaxis chamber contain-ing a 96-well microchemotaxis plate (MBA-96, Neuro Probe,Cabin John, MD) as described previously (26). Briefly, the bot-tomwells of the chamber contained 40�l of various concentra-tions of chemoattractants (SPA, TGF�, HA, and Pam3Cys) indefined medium (DMEM or DM), without fetal calf serum(FCS). Positive and negative controls included 10% FCS orDMEM, respectively. The upper wells were filled with 5 � 105cells/ml suspended in 100 �l of DMEM in the presence orabsence of blocking reagents (antibodies to RHAMM, CD44,SIRP�, calreticulin, TLR2, TLR4, HABPep, and JNK/ERK/p38and inhibitors) with, as appropriate, nonimmune rabbit,mouse, chicken IgG, HABP preincubated with an excess of HA,and DMSO serving as controls. A 5-�m pore polycarbonatemembrane filter was placed between the bottom and top cham-ber. The chamber was incubated for 6 h at 37 °C. Adherent cellson the upper surface of themembrane were treated with 200 �lof 1 mM EDTA for 15–20 min and wiped off. Cells that hadmigrated into the membrane were stained with Diff-QuickTMand counted in five randomly selected high power fields in eachwell. Each chemoattractant solution was tested in 6 wells, andeach experiment was repeated at least three times. Data wereexpressed as the number ofmacrophages thatmigrated into themembrane for each test solution, converted to a percentage ofcontrol (DM), and combined for at least three separateexperiments.To confirm that the migration of macrophages was indeed

chemotaxis, a checkerboard analysis was performed accordingto the method established by Zigmond and Hirsch (27). Che-motaxis ofmacrophages to the chemoattractant was confirmedif directional migration toward the chemoattractant in thelower chamber was negated by inclusion of the same concen-tration of the chemoattractant in the top chamber.

Actin-based Cytoskeletal Rearrangements—Macrophageswere plated at a concentration of 1 � 106 cells/ml in DMEMwith 10% FCS in chamber slides (catalog no. 154526, Lab-Trek�� Chamber Slide System, Fisher). After cells were allowed toadhere for 24 h, the medium was changed to DMEM with 1%FCS overnight. The next day, cells were exposed to SPA (100�g/ml), TGF�1 (10 ng/ml), HA6 oligosaccharide (4 mM), orPam3Cys (5 �M) in defined medium for 1 h. Cells were thenwashed twice with PBS, fixedwith 3% paraformaldehyde in PBSfor 10 min, and washed twice again with PBS. Cytoskeletalstaining was achieved by using FITC-phalloidin (MolecularProbes, Eugene, OR) treatment for 10 min at RT in a dark envi-ronment. After washing in double distilled H2O, chamberswere removed, and slides were mounted with Fluoromount(28). The cells were examined under oil-immersion at �600magnification.TGF� Mink Lung Bioassay and TGF� ELISA—SPA prepara-

tions and conditionedmediawere assayed for activeTGF� con-tent using the mink lung epithelial cell line according to theprotocol described by Abe et al. (29). This TGF-�-responsivecell line was stably transfected with the human plasminogenactivator inhibitor (PAI-1) promoter linked to a luciferasereporter gene. Briefly, 1.8 � 105 mink lung epithelial cellline/ml were allowed to attach for 3 h and then cultured over-night with 30 �l of SPA, medium, or 40–1200 pg/ml TGF�standard (Sigma). Mink lung epithelial cell line extracts werecollected the next day and assayed for luciferase activity usingthe luciferase assay system per the manufacturer’s instructions(Promega). Data were expressed as picograms/ml of TGF� pre-sented as a percent of control (PBS).For the active TGF� ELISA, 5 � 106 macrophages were

plated onto 6-well dishes in DMEM supplemented with 10%FBS and maintained at 37 °C. The medium was replaced withDMEMwithout FBS overnight tomake cells quiescent. Macro-phages were then exposed to SPA (100 �g/ml) or Pam3Cys atdiffering concentrations from 0.5 to 10 �M for 24 h. Active andtotal TGF� was measured using an ELISA kit from R&D Sys-tems (catalog no. DY1679; Minneapolis, MN) as per the manu-facturer’s instructions. TGF� was measured in both the cellpellets and supernatants. Activation of TGF� to obtain totalTGF� content was achieved by acidification as per the manu-facturer’s instructions. To determine their contribution toSPA-stimulated TGF� production, macrophages exposed toPam3Cys (5 �M) were also incubated with JNK, ERK, or p38inhibitors (each 10 �M) for 24 h, and TGF� content was againdetermined in the supernatant.ELISA-like Assay for HA—Supernatants collected from 1 �

106 cells/ml were assayed for HA content by an ELISA-likeassay as described previously (30) with several modifications.This ELISA measures the competition of HA present in thesample versus HA coated on a 96-well plate for binding to abiotinylated HA-binding protein (Seikagaku, Japan). Briefly, 60�l of cellular supernatant or Healon standard (GE Healthcare)were loaded onto nonfat dry milk-blocked Covalink-NH96-Microwell plates (Nunc, Fisher Corp.) after overnight pro-tease digestion.After addition of 60�l of biotinylatedHA-bind-ing protein to eachwell and incubation at 37 °C for 1 h, 100�l ofthe sample biotinylated HA-binding protein incubation solu-




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tion were transferred to a HA-coated Covalink plate and incu-bated for 1 h at 37 °C to allow to competitive binding (0.2mg/mlHA, ICN Inc.). HA binding was detected by an avidin-biotincomplex reagent (Vectastain) and o-phenylenediamine(Sigma). The change in absorbance at 450 nm after a 15-min

incubation was measured. HA concentrations of the samplewere normalized to cellular protein and expressed as nano-grams of HA/mg of protein.Western Blot Analysis—Western blot analysis was performed

using the NOVEX NuPAGE system from Invitrogen with1-mm of 4–15% BisTris gels according to standard protocol asdirected by the manufacturer and as described previously (18).Briefly, 10�g of cell lysatewas loaded to eachwell; gelswere runat 100 V at 4 °C for 1 h in NuPAGEMOPS/SDS running bufferunder reducing conditions. Protein was transferred to nitrocel-lulose membrane and run at 30 V for 60 min at room tempera-ture. The membrane was blocked using 5% nonfat dry milk inTween/Tris-buffered saline (TTBS) (100 mM Tris base, 1.5 M

NaCl adjusted to a pH of 7.4 with 0.1% Tween 20). Primaryantibodies were applied overnight at 4 °C. The next day, themembrane was washed using TTBS four times for 10 min eachtime. A horseradish peroxidase-conjugated goat anti-rabbitsecondary antibody was applied for 1 h at room temperature.Following this, the membrane was washed with TTBS followedby two 15-min washes with TBS. The blots were devel-oped using a chemiluminescence system from AmershamBiosciences.Statistical Analysis—All experiments were repeated at least

three times, and chemotaxis was assessed with 5–6 wells percondition in each experiment. Data from triplicate experimentswere combined using percent of control for each experiment. Alldata are presented asmeans� S.E. Analysis of variancewithNeu-man-Keuls post hoc testingwasused to assess differencesbetweenthreeormoregroups. Significancewas acceptedat the0.05 level ofprobability. Comparisons between two groups of observationsweredeterminedbyStudent’s t test assumingunequal variances.Ap value of less than 0.05 was considered significant.

RESULTS

SPA-stimulated Chemotaxis Is Dependent on TLR2 but Inde-pendent of TLR4, SIRP�, and Calreticulin—Among a numberof its innate immune functions, the pulmonary collectin SPAstimulates macrophage migration. To determine the mecha-nisms regulating this function, we first determined the optimalSPA dose for macrophage chemotaxis. SPA stimulated macro-phage chemotaxis in a dose-dependent manner, with maxi-mum 5–7-fold stimulation at a dose of 100 �g/ml (Fig. 1A). Toconfirm the chemotactic nature of this response, a checker-board analysis was performed with SPA either in the bottom,top, or in both chambers of a modified Boyden chamber (Table

FIGURE 1. SPA-stimulated macrophage chemotaxis requires TLR-2,RHAMM, and HA. A, as assessed in a modified Boyden chemotaxis chamberassay, SPA stimulated macrophage chemotaxis in a dose-dependent mannerwith maximum stimulation at 100 �g/ml (*, p � 0.05 versus DM). Definedmedium (DMEM) was used as a negative control. B, SPA (100 �g/ml)-stimu-lated chemotaxis was tested in the presence of blocking antibodies againstSIRP�, calreticulin/CD91, TLR4, and TLR2 with nonimmune rabbit, rat, andchicken IgGs as species-specific controls. SPA alone significantly stimulatedmacrophage chemotaxis (*, p � 0.05 versus DMEM). SPA-stimulated che-motaxis was only inhibited by anti-TLR2 antibody (#, p � 0.05 versus SPAalone, SPA � IgG). C, SPA-stimulated chemotaxis (100 �g/ml) in the presenceof HABPep, anti-RHAMM, and three CD44 antibodies, IM-7, KM81, or CD44v3,with nonimmune IgG and HABPep incubated with an excess of HA used ascontrols. SPA alone significantly stimulated macrophage chemotaxis (*, p �0.05 versus DMEM). SPA-stimulated chemotaxis was significantly inhibited tobase line in the presence of HABPep or anti-RHAMM antibody (#, p � 0.05versus SPA alone/IgG/HA � HABPep), but not with any CD44 antibody tested.All data are normalized as percent of control (DM) presented as mean � S.E.for three independent separate experiments, each with samples run at leastin triplicate.

TABLE 1Checkerboard analysis of SPA-stimulated chemotaxisChemotaxis was assessed in a modified Boyden chamber assay as described under“Experimental Procedures.” Both 10% FCS or SPA (100 �g/ml), when added in thebottom chamber, produced a significant increase in migration (*, p � 0.05 versusDM/DM). Abrogation of the gradient by adding SPA to both the bottom and topchambers completely inhibited the chemotactic response (#, p � 0.05 versusDM/SPA). Data are presented as means � S.E. normalized to percent of control(DM) for three separate experiments. These results confirm that SPA-stimulatedmigration is a chemotactic response. ND means not determined.

Top chamberBottom chamber DM 10% FCS SPA 100 �g/ml

DM 100 � 34 113 � 14 91 � 2210% FCS 394 � 63* 127 � 15# NDSPA 100 �g/ml 594 � 168* ND 94 � 44#




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1). As expected, 10% FCS or SPA in the bottom chamber signif-icantly stimulated directed migration from the top to the bot-tom chamber (Table 1). However, when the concentration gra-dient was abolished by adding SPA or 10% FCS to the top andbottom chambers, no chemotaxis was observed, and migrationwas similar to the negative control (Table 1). These results con-firm that SPA-stimulated migration is a chemotactic response,and we chose to use 100 �g/ml as the dose of SPA for all sub-sequent experiments.

SPA binds a number of cell surface proteins, including TLR2,where it competitively inhibits the binding of peptidoglycan(31), zymosan (32), and TLR4 (33). In addition, Gardai et al. (7)developed a model to explain the pro- and anti-inflammatoryactions of SPA. During quiescent conditions, in the absence ofbacterial invasion, the globular head of SPA binds to SIRP�(signal inhibitory regulator protein �) on resident macro-phages, an anti-inflammatory signal, whereas in the presence ofpathogens and/or their products, the globular head of SPA is

FIGURE 2. SPA-stimulated increase in HA and chemotaxis is dependent on TGF�, and SPA-stimulated TGF� production is dependent on TLR2. A, mediawere assayed for HA content by an ELISA-like assay after incubation with SPA (100 �g/ml) or TGF�1 (10 ng/ml) for 1, 6, 12, and 24 h and normalized to theprotein content of the cells in each culture. Data are presented as nanograms of HA per mg of protein � S.E. for three independent experiments with samplesrun in triplicate within each experiment. Macrophages cultured in DMEM had no increase in HA content of the medium over time. SPA or TGF�1 treatmentproduced similar time-dependent increases in HA production that were significant at 12 and 24 h (*, p � 0.05 versus DMEM). B, SPA stimulated HA productionin the presence of TGF� antibody or rabbit IgG (each 50 �g/ml). SPA alone or with IgG resulted in a significant increase in media HA concentration (*, p � 0.05versus DMEM). SPA stimulation of HA was significantly inhibited to base line in the presence of a pan-specific TGF� antibody (#, p � 0.05 versus SPA/SPA�IgG).C, SPA stimulated chemotaxis in the presence of anti-TGF�, anti-RHAMM, and anti-TLR2 antibodies. SPA alone significantly stimulated macrophage chemotaxis(*, p � 0.05 versus DMEM). Anti-TGF� antibody inhibited SPA-stimulated chemotaxis to levels similar to that achieved by anti-RHAMM and anti-TLR-2 antibod-ies (#, p � 0.05 versus SPA/SPA � IgG). Data are representative of three independent experiments with at least four replicates within each experiment. D, mediawere assayed for TGF� content after incubation with SPA in the presence of either anti-TLR2 or anti-RHAMM antibodies. Data are presented as mean � S.E. ofpercent of DM control. SPA � IgG produced significant induction of TGF� (*, p � 0.05 versus DMEM). SPA induction of TGF� was significantly inhibited to baseline in the presence of anti-TLR2 antibody (#, p � 0.05 versus SPA/SPA � IgG). However, anti-RHAMM antibody failed to block SPA stimulation of TGF� secretion.Data are representative of three experiments with at least three replicates within each experiment. Collectively, these data show that SPA stimulates TGF�production via interaction with TLR2, and TGF� stimulates macrophage chemotaxis in a RHAMM-dependent manner. E, genetic confirmation of TLR2 require-ment for SPA-stimulated TGF� production. BMDM from WT, RHAMM�/�, and TLR2�/� mice were exposed to SPA, and active TGF� content of the media wasmeasured by ELISA. SPA stimulated TGF� production in both WT and RHAMM�/� mice, a response that was significantly blunted in TLR2�/� macrophages.Data are presented as mean � S.E. and are representative of two independent experiments with samples run in triplicate within each experiment.




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occupied, thereby allowing the binding of the collagenous tailsof SPA to calreticulin/CD91, a pro-inflammatory signal. Todetermine the specific SPA/receptor interaction mediatingmacrophage chemotaxis, we examined whether this responsecould be inhibited by using the blocking antibodies to SIRP�,calreticulin, TLR2, or TLR4 as used in the study of Gardai et al.(7). Anti-TLR2 antibody significantly inhibited SPA-stimulatedchemotaxis, whereas anti-SIRP�, anti-calreticulin/CD91, andanti-TLR4 had no effect (Fig. 1B). Interestingly, blockingSIRP�, normally an anti-inflammatory signal, did not result inincreased chemotaxis, suggesting that SPA-stimulated che-motaxis is an active signal and not just removal of repressiondue to SIRP�. Additionally, nonimmune rabbit, rat, andchicken IgG used as controls also had no effect. These datasuggested that TLR2 is necessary for SPA-stimulated che-motaxis, whereas SIRP�, calreticulin, and TLR4 are not.SPA-stimulated chemotaxis IsMediated by RHAMMandHA

but Not CD44—Because cell motility is regulated by HA and itsreceptors RHAMM and CD44, we next examined whetherSPA-mediated chemotaxis was dependent onHAand its recep-tors by using either HA-binding peptide (HABPep) or anti-HAreceptor antibodies in the top chamber. HABPep preincubatedwith an excess of HA and rabbit IgG served as controls. SPA-stimulated macrophage chemotaxis was inhibited to base linein the presence of either HABPep or anti-RHAMM antibody(Fig. 1C), but three CD44 antibodies, IM-7, KM81, andCD44v3, previously shown to blockmurine cell migration (34–36), failed to block SPA-stimulated chemotaxis (Fig. 1C). Theseresults indicated that HA interaction with RHAMM, and notCD44, mediates SPA-stimulated macrophage chemotaxis.SPA Causes a Time-dependent Increase in HA Production

That Is Dependent on TGF�—Because our data suggested thatHA acting throughRHAMMwas necessary for SPA-stimulatedmacrophage chemotaxis, we next determined whether SPAcould stimulate HA synthesis. TGF�1, which is known to stim-ulate HA synthesis (37), was used as a positive control. Macro-phages were treated with SPA, TGF�1, or DMEM for 1, 6, 12,and 24 h. HA content of the media was determined using anELISA-like assay for HA, and data were normalized to totalprotein of the cell pellet. Untreated macrophages did not accu-mulate HA in the medium (Fig. 2A). Interestingly, treatmentwith SPA or TGF�1 produced similar 3–4-fold time-depen-dent increases in HA production that reached statistical signif-icance at 12 and 24 h (Fig. 2A). Because the SPA that we wereusing was not contaminated with TGF� (supplemental Fig. 1),we next examined whether SPA-stimulated HA productioncould be blocked by a pan-specific TGF� antibody using rabbitIgG as a control. SPA alone or with IgG produced a significant3-fold increase in HA production as compared with DMEM(Fig. 2B). This responsewas completely inhibited by anti-TGF�antibody (Fig. 2B), suggesting that SPA-stimulated HA synthe-sis was an indirect process and as result of TGF� production.

Because SPA-stimulated HA production was dependent onTGF�, SPA-stimulated chemotaxis was examined in the pres-ence of anti-TGF� and anti-RHAMM antibodies, with anti-TLR2 antibody used as a positive control for inhibition. Con-sistent with results for HA synthesis, TGF� antibodysignificantly inhibited SPA-stimulated chemotaxis to levels

similar to those achieved by anti-RHAMMand anti-TLR2 anti-bodies (Fig. 2C). These data indicated that TLR2, TGF�, andRHAMM are all necessary for SPA induction of macrophagechemotaxis.SPA Stimulation of TGF� Synthesis Is Dependent on TLR2

but Not RHAMM—Because SPA stimulates TGF� secretion inmacrophages (38), we next attempted to block this effect usinganti-TLR2 or anti-RHAMM antibody. RAW264.7 macro-phages were treated with SPA in the presence of either anti-TLR2 or anti-RHAMM antibody or rabbit IgG used as a con-trol. Although anti-TLR2 antibody inhibited SPA induction ofTGF� to base line, anti-RHAMMantibody had no effect on thisresponse (Fig. 2D). To confirm these effects using a geneticapproach, we obtained primary BMDM from wild type,TLR2�/�, and RHAMM�/� mice, stimulated them with SPA,and measured TGF� accumulation in the medium. There waslittle to no TGF� secretion in the absence of SPA stimulation(Fig. 2E, RPMI). SPA significantly stimulated TGF� productionin WT and RHAMM�/� macrophages, but this response wassignificantly inhibited in TLR2�/� macrophages (Fig. 2E).To further confirm the role of TLR2 in TGF� production

with SPA treatment, we obtained bonemarrow-derivedmacro-phages from mice harboring a transgene in which the TGF�-responsive PAI1 promoter was fused to a GFP reporter (39).Quiescent cells were treated with either TGF� as a positivecontrol or SPA. In the absence of stimulation, there was little tono GFP staining. SPA treatment caused a robust expression ofGFP, equivalent to that stimulated by TGF�. Both anti-TLR2and anti-TGF� antibodies blocked the SPA-stimulated effecton the expression of GFP (Fig. 3). Collectively, these data sug-gested that SPA-stimulated TGF� production requires TLR2but not RHAMM. Furthermore, data showing that anti-RHAMM antibody blocked SPA-stimulated chemotaxis (Fig.2C) suggested that RHAMM is a more downstream player inthe pathway.

FIGURE 3. Effect of SPA on macrophages obtained from CAGA-GFP mice.BMDM were obtained from CAGA-GFP mice expressing a transgene consist-ing of 12 repeats of the PAI1 promoter linked to GFP as a reporter (39). ThePAI1 promoter is highly TGF�-sensitive, and GFP expression correlates withactive TGF� expression and signaling. Cells were plated and made quiescentovernight in 1% FCS. Three hours after stimulation, they were observed underdirect fluorescence microscopy and imaged at �400 magnification. Quies-cent cells (Control) showed little to no GFP. Stimulation with active TGF�1, apositive control, showed robust GFP staining. Similar increases in GFP stain-ing were observed with SPA, and both anti-TLR2 and anti-TGF� antibodiesblocked this response. Nonimmune IgG had little to no effect on the GFPsignal stimulated by SPA. Data shown are representative of two independentexperiments with samples examined in triplicate within each experiment.




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TGF�1 Stimulation of Chemotaxis IsDependent onRHAMM—Because TGF� was the likely mediator of SPA-stimulated che-motaxis, and RHAMM and TLR2 were critical for thisresponse, we examined whether TGF�1-stimulated che-motaxis was also dependent upon these receptors. TGF�1stimulated a 3–4-fold increase inmacrophage chemotaxis (Fig.4A), an effect that was blocked by anti-RHAMM but not byanti-TLR2 antibody (Fig. 4A). As expected, TGF� antibody,used as a positive inhibitory control, blocked TGF�-stimulatedchemotaxis. These results suggested that RHAMM/HA inter-actions were downstream of TGF�, whereas TLR2 wasupstream of TGF�.HA Stimulation of Chemotaxis Is Dependent on RHAMM

andNot CD44—The data thus far suggested that RHAMM/HAinteractions were necessary and were downstream of TGF�-stimulated chemotaxis. To examine this further, we obtainedendotoxin-, protein-, and nucleic acid-free HA oligosaccharide(HA6) and used it as a chemoattractant. Preliminary dose-re-sponse experiments defined 4mM as the optimal concentrationfor this response (data not shown). HA6 stimulated a 4–5-foldincrease in chemotaxis (Fig. 4B). This effect was blocked to baseline by anti-RHAMM antibody and HABPep but not by anti-bodies to TLR2, TLR4, TGF�, or three separate antibodies toCD44 (IM7, KM81, and CD44v3) (Fig. 4B). Preincubation ofHABPep with HMW HA (900 kDa, HA900) abrogated theinhibitory effect of HABPep, thereby confirming the specificityof the inhibition. The results of the various blockers used aresummarized in Table 2.To further define the effects of molecular size on HA-stimu-

lated macrophage motility, we used HA6 versus HA900 in thechemotaxis assay. HABPep alone had no effect on macrophagechemotaxis (Fig. 4C). A 6-fold increase in chemotaxis was seenwith HA6 stimulation, whereas HA900 did not stimulate che-motaxis (Fig. 4C). Interestingly, when bothHA6 andHMWHAwere added as chemoattractants together, no chemotaxis wasobserved suggesting thatHMWHAacts in a dominant negativemanner. Finally, we used HA oligosaccharides of varying sizesto determine the effect of HA size on macrophage chemotaxis.All HA oligosaccharides (6-, 8-, 14-, and 34-mer) were used at 4mM,whereasHA900was used at the highest dose possiblewith-out prohibitive viscosity of the material (0.01 mM). IncreasingHA oligosaccharide size was associated with increased stimu-lation of chemotaxis, whereas HA900 had no effect (Fig. 4D).These data demonstrated that HA-stimulated macrophagechemotaxis is both dose- and molecular size-dependent,requires RHAMM, and HMWHA acts in a dominant negativemanner to inhibit HA6-stimulated chemotaxis.

FIGURE 4. TGF�1 stimulation of chemotaxis is dependent on RHAMM andHA. A, TGF�1-stimulated macrophage chemotaxis was examined in the pres-ence of anti-TGF�, anti-TLR2, and anti-RHAMM antibodies. TGF�1 alone sig-nificantly stimulated macrophage chemotaxis (*, p � 0.05 versus DMEM). Thiseffect was significantly inhibited to base line in the presence of anti-TGF� andanti-RHAMM antibodies (#, p � 0.05 versus TGF�1 or TGF�1 � �gG) but not byanti-TLR2 antibody. B, we next determined the chemotactic response to HA6and the receptors involved in this response. HA6 (4 mM) stimulated a 4 –5-foldresponse in macrophage chemotaxis (*, p � 0.05 versus DMEM). Only anti-RHAMM antibody and HABPep blocked this response to base line (#, p � 0.05versus HA6 alone). Anti-TLR2, -TLR4, and -TGF� and three antibodies to CD44(IM7, KM81, and CD44v3) had no effect. C, to compare the effects of HMW andlow molecular weight HA, we used HA6 (6-mer HA, 100 �g/ml) and HA900(900-kDa HA, 100 �g/ml) as chemoattractants. HA6 stimulated macrophage

chemotaxis 6-fold (*, p � 0.05 versus DMEM and HA900), whereas HA900 hadno effect. Interestingly, HA900 inhibited HA6-stimulated chemotaxis whenthe two HA products were combined (#, p � 0.05 versus HA6). D, HA of variousmolecular sizes, each at 4 mM except HA900 (100 �g/ml), were tested in themodified Boyden chamber assay. A molecular size-dependent increase in HA-stimulated chemotaxis was observed with maximal chemotaxis observedwith 34-mer HA (*, p � 0.05 versus DMEM; #, p � 0.01 versus DMEM; n.s. meansnot significant versus DMEM). HA900 had no effect on chemotaxis. All che-motaxis data are presented as mean � S.E. normalized as percent of control(DM) for three separate experiments, with at least four replicates within eachexperiment.




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Chemotactic Responses to SPA, TGF�, and HA in TLR2�/�,CD44�/�, and RHAMM�/� Macrophages—To geneticallyconfirm the antibody blocking experiments, we obtainedBMDM from WT and TLR2�/� mice and examined theirresponses to SPA, TGF�, and HA as chemoattractants. Asexpected,WTmacrophages had a 5–6-fold response to each ofthe three chemoattractants (Fig. 5A, top panel), and each of theresponses was blocked to base line by anti-RHAMM antibody.TLR2�/�macrophages did not respond to SPA (Fig. 5A, bottompanel). Interestingly, however, macrophages from TLR2�/�

mice had robust chemotactic responses to bothTGF� andHA6(Fig. 5A, bottom panel), and each of these responses wasblocked to base line by anti-RHAMMantibody (Fig. 5A). Thesedata confirm that SPA interaction with TLR2 mediates thepathway upstream from TGF�.Next, we examined BMDM from littermate WT and

CD44�/� mice. BothWT andCD44�/� macrophages had sim-ilar 4-fold responses to SPA and TGF� (Fig. 5B). In both geno-types, SPA responses were inhibited by anti-TLR2 antibody(Fig. 5B), and TGF� responses were inhibited to base line byanti-RHAMM antibody (Fig. 5B). These data confirmed thatCD44�/� is completely dispensable in the chemotacticresponses to SPA and TGF�. We next determined whetherCD44 was involved with HA6-stimulated chemotaxis. BMDMfromWTandCD44�/�mice had similar 5–6-fold chemotacticresponses to HA6 (Fig. 5C), suggesting that CD44 was notrequired for HA6-stimulated chemotaxis.To definitively confirm the role of RHAMM in these chemo-

tactic responses, we obtainedBMDMfrom littermateRHAMMWT and KOmice. SPA, TGF�, and HA6 all stimulated macro-phage chemotaxis in WTmice (Fig. 5D). RHAMMKOmacro-phages had a significantly lower chemotactic response withSPA, TGF�, and HA6 (Fig. 5D). Collectively, these data genet-ically confirm that SPA stimulates chemotaxis via TLR2 andthat RHAMM, but not CD44, is necessary for chemotaxis stim-ulated by SPA, TGF�, and HA.SPAStimulation of Cytoskeletal Rearrangement Is Dependent

upon TLR2 and RHAMM—The assembly of actin-based exten-sions, namely filopodia and lamellipodia, are integral compo-nents of cell migration. In previous studies, Tino and Wright(28) demonstrated filopodial extensions with SPA stimulationof macrophages. Because anti-RHAMM antibody blockedSPA-stimulated migration, we examined the effect of thisblockade on actin organization. RAW264.7 cells were stimu-lated with SPA, and cytoskeletal rearrangements were deter-mined using FITC-phalloidin (green) and nuclei stained using

TABLE 2Summary of antibody blockade resultsBecause there is a distinct specificity of antibody blockade of chemotaxis to SPA,TGF�, and HA, a hierarchy is established where SPA-stimulated chemotaxisrequires TLR2, TGF�, HA, and RHAMM and RHAMM/HA interactions univer-sally required for all chemoattractants tested.

ChemoattractantsBlockers SPA TGF� HA

�TLR2 � � ��TGF� � � �HABPep � � ��RHAMM � � ��CD44 � � �

FIGURE 5. Chemotactic responses in TLR2�/�, CD44�/�, and RHAMM�/�

macrophages. A, BMDM were obtained from WT and TLR2�/� mice, and che-motaxis to SPA, TGF�, and HA6 was determined. Chemotaxis is represented bycells per high power field in three independent experiments with four replicatesper group in each experiment. Data are presented as mean�S.E. In concordancewith the data obtained with the RAW264.7 murine macrophage cell line, WTmacrophages had 5–6-fold chemotactic responses to SPA (100 �g/ml), TGF� (10ng/ml), and HA6 (4 mM) (*, p � 0.05 versus DM). Each of these responses wasblocked to base line by anti-RHAMM antibody (#, p � 0.05 versus SPA, TGF�, orHA6) but not by normal IgG. Complete medium (CM, DMEM � 10% FCS) wasused as a positive control. TLR2�/� macrophages did not respond to SPA (ˆ, p �0.05 versus CM, TGF�, and HA) but had robust 4-fold responses to TGF� and HA6,both of which were inhibited to base line by anti-RHAMM antibody (#, p � 0.05versus TGF� or HA alone or with normal IgG). B, BMDM were obtained from WTand CD44�/� mice, and chemotactic responses to SPA and TGF�were examined.Both sets of macrophages showed equivalent chemotactic responses to SPA andTGF� (*, p � 0.05 versus DM), and these responses were inhibited to base line byanti-RHAMM antibody (#, p � 0.05 versus SPA or TGF� alone or with IgG). C, WTand CD44�/� macrophages also had similar 4–6-fold chemotactic responses toHA6 (*, p � 0.05 versus RPMI). D, BMDM were obtained from WT and RHAMM�/�

mice, and chemotaxis to SPA, TGF� and HA6 was determined. RHAMM WTmacrophages demonstrated significant chemotactic responses to all three stim-ulants. RHAMM�/� macrophages showed significantly lower chemotacticresponses to SPA and no response to TGF�and HA6 (#, p�0.05 versus WT macro-phages). Collectively, these data confirm that CD44 is dispensable, and RHAMM isa key mediator in the chemotactic responses to SPA, TGF�, and HA.




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DAPI (blue) (supplemental Fig. 3). Quiescent cells in DM hadfew filopodia or cytoplasmic extensions. SPA treatment ofmacrophages, with or without normal IgG, showed increasedspike-like filopodia. However, these cytoskeletal rearrange-ments were completely blocked by R36, and cell morphologywas similar to quiescent cells.BMDM from WT and TLR2�/� mice were made quiescent

overnight and examined after SPA, TGF�, or HA6 stimulation.WT macrophages responded to all three conditions, with orwithout normal IgG, and showed extensive filopodia and lamel-lipodia formation (Fig. 6, A and B). TLR2�/� macrophagesfailed to respond to SPA but had robust filopodial and lamelli-podial formation with TGF� and HA6 treatment, and both ofthese responses were blocked by anti-RHAMM antibody (Fig.6, A and B). Collectively, these data confirm the critical roles ofTLR2 and RHAMM in SPA-mediated cytoskeletal rearrange-ments relevant to cell motility.TLR2-mediated TGF� Production and Chemotaxis Require

theMAPK JNK and ERK—Because we had demonstrated TLR2as the principal receptor that mediated SPA-stimulated TGF�production, we tested whether Pam3Cys, a TLR2-specificligand, could stimulate macrophage chemotaxis and whetherblockade of TGF�, RHAMM, and HA could inhibit thisresponse. First, we conducted a dose response study thatshowed that 5 �M Pam3Cys stimulated a 5–6-fold increase inmacrophage chemotaxis (Fig. 7A). Using this concentration ofPam3Cys, we next tested whether HABPep and antibodies toTGF� and RHAMM could block the chemotaxis stimulated byTLR2 ligation. Pam3Cys stimulated chemotaxis to the samedegree as 10 ng/ml TGF� used as positive control (Fig. 7B). Allthree blockers, HABPep, anti-TGF�, and anti-RHAMM anti-bodies blocked Pam3Cys-stimulated chemotaxis to base line,

whereas scrambled peptide and nonimmune rabbit IgG had noeffect (Fig. 7B). We next determined if Pam3Cys stimulatedlatent and active TGF� production in macrophages. Concen-trations of Pam3Cys as low as 0.1 �M stimulated a 5-foldincrease in production of both latent and active TGF� com-pared with control cells maintained in DMEM alone (Fig. 7C).MAPKs play an important role for TLR2-induced inflamma-

tion (40, 41). We therefore examined if MAPK signaling wasalso important in TLR2-stimulated macrophage chemotaxis.We first determined the phosphorylation of MAPKs afterPam3Cys treatment in both RAW264.7 cells (Fig. 8A) and pri-mary BMDM obtained from WT mice (Fig. 8B). RAW264.7macrophages were exposed to Pam3Cys (a TLR2/1 agonist),FSL1 (a TLR2/6 agonist), LPS (a TLR4 agonist), HA6, orTGF�1, and cells were harvested in orthovanadate-containinglysis buffer at 10 min. Immunoblot analyses for phospho-ERK,phospho-JNK, and phospho-p38, aswell as total ERK, JNK, p38,and �-actin as loading controls, were performed. Macrophagesexposed to Pam3Cys showed robust phosphorylation of JNK,ERK, and p38, and LPS treatment was associated with ERKphosphorylation, but none of the other treatments were asso-ciated with significant effects on phosphorylation (Fig. 8A). Inprimary BMDMfromWTmice, we also examined the effects ofthe signaling inhibitors on the phosphorylation pattern withPam3Cys treatment (Fig. 8B). Similar to the findings inRAW264.7 cells (Fig. 8A), WT BMDM had robust phosphory-lation of ERK, JNK, and p38 with Pam3Cys treatment, and eachpharmacologic inhibitor blocked the phosphorylation of therelevant signaling component (Fig. 8B), thereby confirming thespecificity of the inhibitors in these cells.We then tested the effects of pharmacologic inhibition of

JNK, ERK, and p38 on SPA-stimulated TGF� production in

FIGURE 6. Cytoskeletal changes in response to SPA, TGF�, and HA6 in WT and TLR2�/� mice. BMDM from WT and TLR2�/� mice were plated and madequiescent overnight. Three hours after stimulation with SPA, TGF�, or HA6, the cytoskeleton was stained using FITC-phalloidin (green) and nuclei with DAPI(blue). A, WT macrophages that were mostly round in shape at base line showed increased formation of filopodia and lamellipodia when treated with SPA withor without normal IgG. Treatment with anti-RHAMM antibody (R36) inhibited SPA-stimulated cytoskeletal changes. B, both WT and TLR2�/� macrophagesexposed to TGF� or HA6 showed similar robust cytoskeletal changes with formation of lamellipodia and filopodia as seen in WT macrophages exposed to SPA,and these responses were completely inhibited when cells were also exposed to R36.




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primary WT BMDM (Fig. 8C). SPA stimulated a 10-foldincrease in TGF� as compared with unstimulated controls, andthis response was completely inhibited using JNK and ERK, butnot p38 inhibitor (Fig. 8C). Because inhibition of JNK and ERKdecreased Pam3Cys-induced TGF� expression, we sought todetermine whether JNK and ERK blockade would also inhibitPam3Cys-stimulated macrophage chemotaxis. Chemotaxis toPam3Cys was determined in WT BMDM in the presence orabsence of JNK, ERK, or p38 inhibitors. Pam3Cys-stimulated a10–12-fold increase in macrophage chemotaxis, with no effect

noted with DMSO, the vehicle for the inhibitors (Fig. 8D).Interestingly, and in line with the effects on TGF� production,JNK and ERK, but not p38 blocker, inhibited Pam3Cys-stimu-lated chemotaxis to base line (Fig. 8D), suggesting thatPam3Cys stimulation of TLR2 results in the activation of JNKand ERK, which are critical steps in TGF� expression and sub-sequent macrophage chemotaxis.

DISCUSSION

We describe a novel pathway regulating SPA-stimulatedmacrophage chemotaxis. Interaction of SPA with TLR2 resultsin JNK- and ERK-dependent TGF� production. This growthfactor then stimulates macrophage chemotaxis, a process thatrequiresHAand its receptor RHAMM.Ligation of RHAMMbyHA promotes actin cytoskeletal rearrangements that arerequired for cell motility. These data suggest the pulmonarycollectin SPA regulates macrophage recruitment via TLR2 andthat the extracellular matrix components HA and RHAMMregulate the inflammatory response in concert with the innatehost defense system of the lung.SPA, the most abundant surfactant protein in the lung, is an

integral part of the innate immune system. It binds to andenhances the uptake of Staphylococcus aureus, Escherichia coli,Pseudomonas aeruginosa, and various other bacteria andviruses, as well as stimulates macrophage chemotaxis (42). SPAcan modulate the inflammatory response to a variety of stimuli(43). For instance, SPA abrogates inflammation caused by LPSexposure. Intratracheal instillation of LPS increases lung SPAprotein andmRNA content, changes that correlate with dimin-ished inflammation and secretion of cytokines. In addition,administration of LPS to SPA�/� mice results in an increasedproduction of inflammatory cytokines and nitric oxide as com-pared with wild type mice. It is likely that SPA competes withLPS for TLR4 binding. However, SPA directly enhances nitricoxide and TNF� production by macrophages in response tovarious stimuli (44), with this response likely beingmediated byinteraction with calreticulin/CD91.A number of cell-associated binding proteins and receptors

have been identified for SPA. A 210-kDa SPA receptor exists onBMDM, alveolar macrophages, and the U937 macrophage cellline (45), and increased SPA binding to this receptor occurswith LPS and IFN� treatment. Additionally, expression of thisreceptor is dependent upon the activation state ofmacrophageswith highest affinity binding occurring on monocytes andreduced binding on GM-CSF-activated macrophages (46).Another potential receptor for SPA is the receptor for the com-plement protein C1q implicated in SPA-stimulated phagocyto-sis in monocytes (47). Additionally, the scavenger protein gly-coprotein-340 binds SPA in a carbohydrate recognitiondomain-independent manner, but it does not affect binding ofSPA to alveolar macrophages or SPA stimulation of macro-phage chemotaxis. However, glycoprotein-340 directly stimu-lates random migration (chemokinesis) of alveolar macro-phages (48). SPA can also bind to CD14, a known patternrecognition co-receptor for LPS and PGN, that mediatesrelease of pro-inflammatory cytokines (49).Gardai et al. (7) showed that SPA acts as a dual function

surveillancemolecule to enhance or suppress cytokine produc-

FIGURE 7. Pam3Cys, a TLR2 ligand, stimulates TGF� production and che-motaxis that requires RHAMM. A, dose response to Pam3Cys-stimulatedchemotaxis. RAW264.7 cells, examined in the chemotaxis assay, showed adose-dependent increase in chemotaxis to Pam3Cys with the optimal dosebeing 5 �M. This dose of Pam3Cys was used for all subsequent experiments.B, Pam3Cys stimulated chemotaxis to the same degree as 10 ng/ml TGF�1.Chemotaxis to Pam3Cys was inhibited by antibodies to TGF� and RHAMM(R36), as well as HABPep, but not by scrambled peptide or normal rabbit IgGused as controls. (*, p � 0.05 versus Pam3Cys � TGF� and Pam3Cys � R36; #,p � 0.05 versus Pam3Cys alone.) C, Pam3Cys stimulated the production ofboth latent and active TGF� with maximal effect at even the lowest concen-trations studied (*, p � 0.05 versus DMEM without Pam3Cys).




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tion depending on the binding orientation of the carbohydraterecognition domain of SPA. SPAmaintains normal lung home-ostasis by binding to SIRP� that blocks pro-inflammatory sig-naling by activating SHP-1, a phosphatase that in turn dephos-phorylates p38. However, upon recognition of PAMPs onforeign organisms, the collagenous tail of SPA binds to calreti-culin/CD91 and elicits the phosphorylation of p38 and thedownstream activation of the NF�B pro-inflammatory signalpathway (7). Thus, intraperitoneal injection of mice with SPAhalf an hour before LPS exposure suppresses pro-inflammatorycytokine production. However, simultaneous injection of thetwo results in enhanced inflammation.Our studies define a novel pathway where SPA binds TLR2

and activates JNKandERK to increaseTGF�production. In thecontext of the whole animal model, we predict different phasesof the response, with early lower concentrations of TGF� stim-ulating macrophage chemotaxis to recruit these cells, and laterhigher concentrations of TGF� being inhibitory and anti-in-flammatory to stop macrophage motility and limit the inflam-

matory response. In support of the pathway proposed in thereport, Kramer et al. (50) instilled SPA into the lungs of venti-lated preterm lambs and observed increased accumulation ofinflammatory cells confirming that SPA promotes the recruit-ment of inflammatory cells to the lung.As noted above, TLRs, primary receptors of the innate

immune system, provide immediate recognition of PAMPs onforeign pathogens to allow for clearance and phagocytosis min-utes to hours after infectious challenge (51). TLR4 is responsi-ble for LPS recognition and signaling (52), whereas TLR2 isresponsible for recognition and binding toGram-positive PGN,which also elicits proinflammatory cytokine release (31).Indeed, macrophages from TLR2�/� mice are unable to pro-duce pro-inflammatory cytokines in response to PGN (53). SPAinhibits PGN-induced NF�B activation and TNF� secretionthrough a specific competitive binding of SPA toTLR2 onU937cells and alveolar macrophages. In addition, SPA can also alterthe interaction of TLR2 with zymosan, a yeast cell wall compo-nent, also causing down-regulation of NF�B activation and

FIGURE 8. Pam3Cys-stimulated TGF� production and chemotaxis requires ERK and JNK but not p38. We examined the intracellular signaling events,specifically the MAPKs ERK, JNK, and p38, stimulated by TLR2 ligation, TLR4 ligation, HA6, and TGF�1, and we used pharmacologic inhibitors to test thecontribution of each pathway to Pam3Cys-stimulated TGF� production and chemotaxis. A, in RAW264.7 cells, Pam3Cys, a TLR2/1 ligand (2nd lane), and FSL1,a TLR2/6 ligand (3rd lane), stimulated the phosphorylation of ERK, JNK, and p38. LPS (25 �g/ml, 4th lane), a TLR4 ligand, only stimulated ERK phosphorylation.HA6 (4 mM, 5th lane) and TGF�1 (10 ng/ml, 6th lane) had no significant effects on any MAPK and remained similar to that of DMEM control cells (No treat, 1stlane). Data are presented as mean � S.E. of densitometry from three independent experiments. B, using BMDM from WT mice, we examined the effects ofPam3Cys and blockers of ERK, JNK, and p38 on the phosphorylation of these proteins. Pam3Cys treatment resulted in a robust phosphorylation of ERK, JNK, andp38 and all three blockers (ERK, PD98059; JNK, SP600125; and p38, SB202190; each at 10 �M concentration) blocked the relevant pathway confirming thespecificity of each blocker in these primary cells. C, in WT BMDM, SPA (100 �g/ml) stimulated a 10-fold increase in TGF� production (*, p � 0.05 DMSO versusRPMI). Pharmacologic inhibition of JNK and ERK, but not p38, inhibited Pam3Cys-stimulated TGF� production to base line (#, p � 0.05 versus SPA � DMSO).D, also in WT BMDM, pharmacologic inhibition of JNK and ERK, but not p38, inhibited Pam3Cys-stimulated chemotaxis (*, p � 0.05 versus control; #, p � 0.05versus Pam3Cys � DMSO). Data are presented as mean � S.E. from three experiments with samples run in triplicate within each experiment. Collectively, thesedata confirm that TLR2 stimulation by SPA or Pam3Cys results in JNK- and ERK-dependent TGF� production and subsequent chemotaxis and confirm thatuniversal ligation of TLR2 has the same effects as SPA.




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TNF� secretion (53). Collectively, these data strongly supportan anti-inflammatory role for SPA in controlling host defensein response to foreign challenge through TLR2 interactions.However, our data show that blockade of TLR-2 abolishes SPAstimulation of chemotaxis and that SPA/TLR2 interactionsresult in the production and release of TGF�, an anti-inflam-matory growth factor. These effects likely promote the recruit-ment of activated macrophages to affected areas of the lungand, when the concentration of TGF� is high enough, inhibitsfurther macrophage migration.TGF�1 is a key regulator of inflammatory cell motility and is

a strong chemoattractant for a variety of cell types, includingmacrophages, neutrophils, T-cells, and fibroblasts (17, 54).TGF�1 increases HA synthesis (37) and the expression ofRHAMM in a dose- and time-dependentmanner (55). Further-more, TGF�1-stimulated fibroblast migration can be blockedby RHAMM antibody and by HABPep, suggesting thatRHAMMandHA regulation of TGF�1-stimulated cell motilityis likely a universal mechanism (17). Our data show that SPA/TLR2 interaction stimulates TGF� release and that TLR2blockade does not inhibit TGF�-mediated chemotaxis; TGF�is therefore a downstream component of the chemotacticresponse to SPA.CD44 is a type 1 transmembrane HA receptor that was ini-

tially implicated in lymphocyte homing and tumorigenesis.More recently, CD44 is required for the clearance of lowmolec-ular weight HA from injured sites and is crucial to resolvinglung inflammation (56). Indeed,Gallo and co-workers (57) haverecently reported mechanisms by which CD44 mediates inter-nalization of HA. Furthermore, co-immunoprecipitation ofCD44 and TLR4 suggests a close relationship between HAreceptors and TLR signaling (20). However, in our studies ofSP-A-stimulated chemotaxis, CD44�/� cells responded toSP-A in a manner similar to that of WTmacrophages, indicat-ing that CD44 is not involved in TLR2-stimulated TGF� pro-duction and cell motility.The cell-associated receptor RHAMM (Hmmr, CD168) reg-

ulates the motility of various cell types such as fibroblasts, lym-phocytes, smooth muscle cells, endothelial cells, and macro-phages (58). Binding of HA to RHAMM results in tyrosinephosphorylation, focal adhesion turnover processes, and acti-vation of various signaling molecules, including Src, and Ras,through the ERK kinase cascade (16, 59, 60). Several injurymodels have demonstrated an increased expression ofRHAMM and HA in macrophages, fibroblasts, and smoothmuscle cells migrating in response to tissue injury (24, 60, 61).Furthermore, we have previously shown that blockingRHAMM inhibits immune cell chemotaxis and randommigra-tion in vitro (58) and inflammation in vivo (18). The roles ofCD44 and RHAMM in inflammatory cell responses have beenstudied using knock-out mice and blocking antibody experi-ments. CD44, acting through HA, has been implicated ininflammatory cell activation as well as rolling and adhesion tothe endothelium (62). However, bleomycin lung injury in CD44knock-out animals results in an unresolved exaggerated inflam-mation primarily consisting ofmacrophages in associationwithelevated HA concentrations (56). Furthermore, blockade ofRHAMM inhibits macrophage motility in vitro and recruit-

ment to the lung after bleomycin injury (18). These previousreports suggest differential roles for these two receptors withrespect to inflammation after lung injury, with RHAMMmedi-ating recruitment and CD44 required for resolution of inflam-mation. The data in this report clearly demonstrate that SPA,TGF�, and HA-stimulated migration are dependent onRHAMM and not CD44. Additionally, our data demonstratethat HA-stimulated macrophage chemotaxis is dose- andmolecular size-dependent, with lowmolecularweightHAmax-imally stimulating and HMWHA inhibiting cell motility.A number of previous reports have implicated intracellu-

lar signaling pathways in TLR-stimulated inflammatoryfunctions. These include activation of NF�B, JNK, ERK, andp38 (63). Adhikary et al. (40) studied TLR2-specific signalingpathways and showed that Pam3Cys stimulation of cornealepithelial cells resulted in phosphorylation of JNK, ERK, andp38 but that only blockade of JNK had an inhibitory effect onactivation of NF�B. Indeed, JNK�/� mice had decreasedresponses to S. aureus-stimulated inflammation. Our resultsdiffer somewhat from those of Adhikary et al. (40) in thatboth JNK and ERK were required for the expression ofTGF�. These differences are possibly due to cell type speci-ficity in responses.In summary, our data confirm an important role for TLR2-

stimulated and JNK/ERK-mediated TGF� production andmacrophage recruitment, as shown in the schematic modelin Fig. 9. Our data are consistent with the model that TLR2recognition of PAMPs differs from that of collectins such asSPA, in that SPA competitively inhibits the binding of pep-tidoglycan to TLR2, thereby decreasing NF�B activation(31), but it leads to activation of a signaling pathway thatresults in TGF� production. TGF�-stimulated macrophagemotility, in turn, is dependent on HA and RHAMM, indicat-ing that these components of the extracellular matrix per-form vital tasks in the recruitment of macrophages to the siteof infection, but they also serve to dampen the inflammatoryprocess so as to protect the host from unrelenting inflamma-tion and tissue damage. Further understanding of the mech-anisms involved in TLR2-stimulated chemotaxis may allow

FIGURE 9. Schematic describing the pathway for TLR2-mediated TGF�production and chemotaxis. The studies presented here show that SPA orPam3Cys ligation of TLR2 increases TGF� production in a JNK/ERK-depen-dent manner. In turn, TGF� acts to recruit macrophages to the site of infec-tion/injury, an event that is mediated by TGF� receptors, but requiresRHAMM and HA. When TGF� concentrations are high enough at the site ofinfection/injury, there is a general suppression of inflammation. Modulationof RHAMM and HA may provide novel therapeutic targets to modulate innateimmune responses.




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the development of novel agents to attenuate unmitigatedinflammation after lung injury.

Acknowledgments—We thank the following for their kind gifts ofreagents: Dr. Hal Dietz (The Johns Hopkins University, Baltimore,MD) for the CAGA-GFP mice; Dr. Tak Mak (Amgen, Toronto,Ontario, Canada) for the CD44�/�mice; andDr. EvaA. Turley (Lon-don Regional Cancer Centre, London, Ontario, Canada) for theRHAMM�/� mice. The assistance of Masaki Kosemura and Taka-hiro Masa (Seikagaku Corp.) in preparation of hyaluronan oligosac-charides is greatly appreciated. We thank Drs. Ralph DeBerardinis,Felix Yarovinsky, and Carole Mendelson (University of Texas South-western) for critical review of the manuscript.

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McDevitt, Aisha Zaman, Jo Rae Wright and Rashmin C. SavaniJoseph P. Foley, David Lam, Hongmei Jiang, Jie Liao, Naeun Cheong, Theresa M.

Protein A-stimulated Macrophage Chemotaxisand Receptor for HA-mediated Motility (RHAMM) Are Required for Surfactant

, Hyaluronan (HA),βToll-like Receptor 2 (TLR2), Transforming Growth Factor-

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