11 LysoPtdOH enhances CXCL16 production stimulated by LPS from

ORIGINAL ARTICLE

LysoPtdOH Enhances CXCL16Production Stimulated by LPS from Macrophages and Regulates T cell Migration

Shijun Kang ?Chunlan Yang ?Rongcheng Luo

Received:10February 2008/Accepted:30August 2008/Published online:2October 2008óAOCS 2008

Abstract As a transmembrane chemokine,CXCL16has been detected in various tissues and organs under normal and pathological conditions,it also plays an important role in macrophages/dendritic cells (DC)and T cell interactions and traf?cking during in?ammation and immune respon-ses.LysoPtdOH,a bioactive lipid mediator has been indicated to regulate DC and epithelial functions during wound healing and in?ammation responses.However,the direct link of CXCL16expression with lysoPtdOH has not been https://www.360docs.net/doc/127759917.html,ing monocyte-derived macrophages/DC (MoDC),we investigated the roles of lysoPtdOH in CXCL16production and cell surface presentation.We found that macrophages/MoDC constitutively express and secrete CXCL16,lysoPtdOH signi?cantly enhanced CXCL16protein production stimulated with lipopolysac-charide (LPS)by more than twofold,which was re?ected by increased mRNA transcription by 64-fold.Production of CXCL16increased by lysoPtdOH and LPS from macro-phages was inhibited around 70%by Pertussis toxin (G i/o speci?c inhibitor),exoC3(Rho speci?c inhibitor),and pyrrolidine dithiocarbamate (the NF-j B-dependent path-way inhibitor)separately.LysoPtdOH treatment increased macrophages’chemotactic activity to activated T cells.The soluble form of CXCL16produced by macrophages/MoDC was functionally chemoattractive to T cells.

Keywords LysoPtdOH áMacrophages áCXCL16áProduction Abbreviations DC

Dendritic cells

LysoPtdOH

Lysophosphatidic acid LPA1,LPA2,LPA3LysoPtdOH receptors LPS Lipopolysaccharide

MFI Mean ?uorescence intensity MoDC

Monocyte-derived DC

Introduction

Chemokines are structurally related,small (8–14kDa)polypeptide signaling molecules that bind to the chemokine receptors,a family of seven transmembrane G protein-coupled receptors.The chemokines and their respective receptors are divided into the CXC,CC,C,and CX 3C families,based upon the positions of their conserved two N-terminal cys residues.The chemokines genes are clus-tered on genomic loci,including chromosome 4q12–q13(CXC acting mainly on neutrophils),4q21,and 17q11.2(CC chemokines acting mainly on monocytes).The che-mokine receptor gene clusters exist on chromosomes 2and 3.Many chemokines share common receptors and bind to multiple receptors [1].Chemokines were originally char-acterized by their ability to induce chemotaxis of leukocytes.They have since been shown to act on multiple cell types,including T lymphocytes.Chemokines lead to the directed migration of leukocytes especially T lym-phocytes to the in?ammation and tumor sites [2].

CXCL16,originally found in dendritic cells (DC)in lymphoid organ T cell zones,contains a transmembrane

S.Kang (&)áR.Luo (&)

Department of Oncology,Nanfang Hospital,

Southern Medical University,510515Guangzhou,China e-mail:DrKang5@https://www.360docs.net/doc/127759917.html, R.Luo

e-mail:rcluorc@https://www.360docs.net/doc/127759917.html,

C.Yang

Department of Medicine,Changning County Hospital,644300Sichuan,China

Lipids (2008)43:1075–1083DOI 10.1007/s11745-008-3238-6

domain and sheds a soluble form[3,4].CXCL16promotes interactions between macrophages/DC and CD8T cells and guides effector T cell traf?cking in lymphoid organs and some nonlymphoid tissues[3,5,6].The membrane bound CXCL16also mediates the?rm adhesion of CXCR6-expressing T cells[6].Recent reports indicated that the elevated CXCL16mediated a unique pathogenic role in chronic in?ammatory diseases such as rheumatoid arthritis,cardiac vascular atherosclerosis,and cancers[7–9].CXCL16is not exclusively expressed on the cell sur-face.It is also found as a soluble molecule that is constitutively generated by proteolytic cleavage of its transmembrane variant,a process called shedding[10].

LysoPtdOH is a serum-borne lysophospholipid that signals through the cognate G protein-coupled receptors to evoke a great variety of responses in numerous cell types. In addition to stimulating cell proliferation and survival, lysoPtdOH might regulate the traf?cking,cytokine pro-duction,and T cell-activating functions of DC[11–13]. Reports have indicated that lysoPtdOH receptors might also appear on the human macrophage surface,however, in?uences of lysoPtdOH on cytokines/chemokines pro-duction in macrophages/DC have been controversial[14–16].The effect of lysoPtdOH on CXCL16expression in macrophages/DC has never been https://www.360docs.net/doc/127759917.html,ing mono-cyte-derived macrophages/DC(MoDC),we investigated how lysoPtdOH regulate CXCL16production and shed-ding.The increased production of CXCL16stimulated by lysoPtdOH in macrophages/MoDC may involve multiple signaling transduction pathways including G i/o,Rho,and NF-j B,which would affect the chemotactic activity of macrophages/MoDC as well.

Materials and Methods

Reagents

The endotoxin content of the sodium salt of18:1lys-oPtdOH(1-oleoyl-2-hydroxy-sn-glycero-3-phosphate; Avanti Polar Lipids)in working solution was\50pg/ml. Pertussis toxin(PTx),lipopolysaccharide(LPS),and pyr-rolidine dithiocarbamate(PDTC)were purchased from Sigma(St Louis,MO).The C3exoenzyme(exoC3)was purchased from Cytoskeleton(Denver,CO).Anti-lys-oPtdOH receptor antibodies,anti-CXCL16antibody,and isotype-matched control were from BD Pharmingen(San Diego,CA).

Cell Source and Culture

CD14?monocytes were puri?ed from heparinized peripheral blood mononuclear cells(PBMC),which were either purchased from BRT Laboratory(Baltimore,MD)or healthy donors using immunomagnetic selection according to the manufacturer’s instructions(Miltenyi Biotec, Auburn,CA).All donors(both male and female,20–50year old)provided written informed consent according to federal,state and institutional guidelines.Monocyte-derived macrophages were obtained by culturing CD14? monocytes(0.59106cells/ml)in complete RPMI-1640 (Invitrogen,Carlsbad,CA)with10%human serum (Sigma,MO)in the presence of GM-CSF(50ng/ml,R&D Systems,Minneapolis,MN)for6days.MoDC were gen-erated by supplementing with GM-CSF and IL-4(50ng/ ml,each)for over6days.Macrophages/MoDC were washed and incubated with LPS(100ng/ml,Sigma Chemicals)for24h to induce cytokine secretion.To determine whether lysoPtdOH affect macrophages/MoDC activation or modulate the effects of LPS,macrophages/ MoDC were incubated with and without lysoPtdOH or LPS.

Flow Cytometry Analysis

Cells were counted(29106)and stained with?uores-cence-labeled antibodies against lysoPtdOH receptors and CXCL16for30min,washed and analyzed by?ow cytometry(FACS,Becton Dickenson Immunocytometry Systems,San Jose,CA).Gated on live cells,expression of LPA1,LPA2,LPA3,and CXCL16was analyzed in com-parison with isotype-match controls.Mean?uorescence intensities(MFI)were analyzed with CellQuest software (Becton Dickenson Immunocytometry Systems).

Qualitative RT-PCR

Cells were lysed using TRIzol Reagents.Total RNA was precipitated from the aqueous phase using isopropyl alcohol after separation by chloroform.After DNase I treatment,cDNA was synthesized from1l g total RNA using MLV-Reverse Transcriptase(Invitrogen,Carlsbad, CA)according to the manufacturer’s directions.RT-PCR pro?led as followed:48°C for45min,94°C for2min,30 cycles(94°C for30s,60°C for1min,68°C for2min), 68°C for5min.Primer sequences used in RT-PCR were 50-CGGAGACTGACTGTCAGCAC-30and50-GGTCCAG AACTATGCCGAGA-30for LPA1mRNA;50-TGGCCTA CCTCTTCCTCATGTTCCA-30,50-GGGTCCAGCACAC CACAAATGCC-30for LPA2mRNA;50-GGACACCCAT GAAGCTAAT-30and50-TCTGGGTTCTCCTGAGAG AA-30for LPA3mRNA[17,18].b-actin primer sequences were described previously[19].To analyze the reactions, 10l l of each reaction was subjected to electrophoresis on a1%agarose gel and DNA was detected by ethidium bromide staining.

ELISA

CXCL16concentrations in the spent culture supernatant were measured by ELISA kits purchased from R&D Systems(Minneapolis,MN).Brie?y,the capture anti-body in the working concentration was diluted and coated in a96-well microplate overnight at4°C.After washing and blocking,100l l of the sample or standard in appropriate diluents were added and incubated for2h at room temperature following the manufacturer’s pro-tocol.The detection antibody conjugated with biotin in the working concentration was added after washing for 2h,then streptavidin conjugated with HRP was incu-bated for1h.Color was developed by adding substrates for20min and the optical density of each well was determined using a microplate reader set to650nm. CXCL16concentrations were calculated according to the standard controls.

Real-time PCR

Cells were lysed,total RNA was puri?ed and cDNA was synthesized as above.RT-PCR was set up in a96-well plate using a TaqManòUniversal PCR Master Mix kit from Applied Biosystems(Foster City,CA).The reac-tions were run on an ABI PRISMò7300Sequence Detection System and compared to the actin control.Pre-validated primers and probes speci?c for CXCL16 (Hs00222859_m1)and b-actin(Hs00194811_m1)were purchased from Applied Biosystems.After normalization RNA ampli?ed from different cells was exported as TIFF ?les.

T cells Activation and Migration Assay

CD3?T cells were puri?ed from normal donor PBMC as above by positive selection(Miltenyi Biotec,Auburn,CA). Puri?ed CD3?T cells were stimulated with anti-CD3/ CD28coated beads for5days before analysis or migration assay[20].Activated CD3?cells were loaded into the top chamber of a trans-well(5.0l m pore size,Costar,New York,NY).In the bottom well,a total of500l l spent culture supernatant or fresh media as control were plated. Anti-CXCL16blocking antibody was purchased from R&D Systems.Migration was allowed to proceed for2h. Then cells that had migrated to the bottom wells were counted and the absolute numbers of migrated cells were calculated.Some macrophages were pretreated with dif-ferent inhibitors PTx(1l g/ml),exoC3(1l g/ml),or PDTC (200l M),then treated with lysoPtdOH/LPS,spent culture supernatant were collected and plated in the bottom wells [13,21].Statistics

One-way ANOVA with Dunnett’s post test was performed using GraphPad InStat version3.05to compare the aver-ages of different experiment groups.P\0.05was accepted as being signi?cant.

Results

Macrophages and MoDC Express

LysoPtdOH Receptors

Macrophages and MoDC were derived from CD14? monocytes from normal donor PBMC without age or gender discrimination.FACS con?rmed the cell surface expression of LPA1and LPA2,but not LPA3on macro-phages/MoDC,as well as their precursors CD14? monocytes(Fig.1a).Similar results were observed when analyzing mRNA expression of lysoPtdOH receptors by qualitative RT-PCR,where expression of both LPA1and LPA2mRNA was readily detectable(Fig.1b).

Macrophages and MoDC Express and Secrete CXCL16

FACS con?rmed macrophages and MoDC expressed CXCL16on the cell surface.A few monocytes expressed CXCL16on their cell surface,almost all macrophages acquired CXCL16after differentiation,MoDC expressed CXCL16with the highest intensity on the cell surface (Fig.2a).ELISA showed both macrophages and MoDC secreted soluble CXCL16,while macrophages released more CXCL16into their spent culture supernatants (Fig.2b).

LysoPtdOH Stimulates CXCL16Production

from Macrophages

To examine the effect of lysoPtdOH on CXCL16expres-sion,we cultured macrophages,stimulated them with LPS to trigger chemokine release.We observed that lysoPtdOH at10l M signi?cantly increased the production and release of CXCL16from macrophages by more than twofold as detected by ELISA(Fig.3a).LysoPtdOH did not affect CXCL16cell surface presentation shown by FACS (Fig.3b).Quantitative real-time PCR showed lysoPtdOH upregulated CXCL16mRNA expression in macrophages also(Fig.3c).Control macrophages had basal CXCL16 mRNA(detectable at cycle35),lysoPtdOH increased CXCL16mRNA(detectable at cycle32),LPS induced CXCL16mRNA(detectable at cycle28),however, lysoPtdOH and LPS synergized in CXCL16mRNA tran-scription(detectable at cycle22).By calculating the cycle

threshold (CT)values of each sample and D CT,we found that lysoPtdOH enhanced CXCL16mRNA transcription by 64-fold.

Signaling Pathways Regulating CXCL16Production Enhanced by LysoPtdOH

By binding to speci?c LPA receptors,lysoPtdOH activates complex cell signaling including PTx sensitive and insen-sitive G proteins depending on cell types,triggering tyrosine phosphorylation and regulating rho-dependent actin reor-ganization [19,22].PTx,a speci?c inhibitor of G i/o proteins,which has been shown to inhibit G i/o -dependent lysoPtdOH effects in different cell systems.Pretreatment of macro-phages with 10ng/ml of PTx for 16h before lysoPtdOH

suppressed CXCL16protein production stimulated by LPS.In addition,pretreatment with 1l g/ml of exoC3,a speci?c inhibitor of Rho and 200l M of PDTC,an inhibitor of the NF-j B-dependent pathway signi?cantly decreased lys-oPtdOH enhanced CXCL16protein production from macrophages,suggesting that the enhancement of lys-oPtdOH on CXCL16production are G i/o ,Rho,and NF-j B dependent (Fig.4).Similar results were also observed at the RNA level as detected by RT-PCR.LysoPtdOH Enhances Chemotactic Activity of Macrophages

Macrophages play an important role in immune cell traf-?cking into in?ammation and tumor sites.To

investigate

Fig.1a Expression of

lysoPtdOH receptors on the cell surface of macrophages and MoDC.Macrophages and MoDC were derived from

CD14?monocytes by culturing with GM-CSF or GM-CSF ?IL-4for 6days.Cells were

harvested and stained with PE labeled anti-LPA antibodies.Cells were washed and analyzed by FACS.Filled curves show results from cells stained with isotype-matched control mAbs.Curves with just lines show results from cells stained with speci?c antibodies.The numbers shown in each histogram represent MFI of LPA ,LPA 2or LPA 3over control.Graphs shown are representative of three

independent experiments.b RT-PCR analysis of mRNAs

encoding lysoPtdOH receptors in macrophages and MoDC

CXCL16production stimulated by lysoPtdOH would functionally attract more T lymphocyte migration,che-motactic activity of spent culture supernatant collected from lysoPtdOH/LPS treated macrophages was tested in a trans-well system.Results showed that spent culture supernatant from lysoPtdOH/LPS stimulated macrophages signi?cantly increased CD3?T cells migration,and pre-incubation the spent culture supernatant with 10l g/ml blocking antibody against human CXCL16signi?cantly blocked CD3?cells migration.In contrast,normal mouse IgG (10l g/ml)had no effects on CD3?cell migration,implying the enhanced chemotactic activity of macro-phages stimulated by lysoPtdOH was mediated through the increased production of soluble CXCL16(Fig.5a).Con-sistent with the ELISA results of CXCL16production regulated by lysoPtdOH through several signaling trans-duction pathways above,the chemotactic activity of macrophages was also mediated through G i/o -,Rho-,and NF-j B-dependent pathways,since CD3?cells migration toward to spent culture supernatant collected from macro-phages were blocked by pretreatment macrophages with

PTx,exoC3,or PDTC (Fig.5b).LysoPtdOH and LPS alone did no signi?cantly enhance CD3?cells migration.

Discussion

Chemokines play an important role as paracrine and autocrine mediators in healthy and diseased skin.They have been implicated in wound healing,in?ammatory processes and tumors [23,24].CXCL16has been linked with different biological activities.As a surface-expressed molecule CXCL16functions as a scavenger receptor for oxidized low-density lipoprotein [25],binds to CXCR6expressed on leukocytes and establishes ?rm cell to cell contact [5].The constitutively proteolytic soluble form CXCL16functions as a chemoattractant for CXCR6-expressing cells such as T cells [26–28].CXCL16has been detected in wound ?uids,acute and chronic in?am-mation sites and tumor tissues,implicating its important roles in broad physiological and pathological situations [10,24

].

Fig.2a Macrophages and MoDC express CXCL16on the cell surface.Macrophages and MoDC were cultured as above and stained with anti-CXCL16-PE and analyzed by FACS.Filled curves repre-sent cells stained with isotype-matched control mAb.Curves consisting of lines alone were from cells stained with anti-CXCL16.The numbers shown in each histogram were MFI of CXCL16over control.The data shown are representative of ?ve repeated experi-ments.b Macrophages and MoDC secrete soluble CXCL16.Cells

were washed on day 5,resuspended at 59105/ml and cultured.The spent culture supernatants were harvested after 24h.The concentra-tions of CXCL16were measured by ELISA.Data shown are means ±SE of three experiments with https://www.360docs.net/doc/127759917.html,pare to the media control,monocytes secrete basal level CXCL16(*P \0.06);macrophages give the highest level of CXCL16(**P \0.01);while MoDC shed the median level of CXCL16( P \0.05)

Lipids have long since been recognized as signaling molecules that have the capacity to control important cel-lular processes,including cell proliferation,apoptosis,metabolism and migration and trigger profound physio-logical responses [29].The same signaling lipid can provoke different cellular responses,depending on the precise cell type and underlying signaling network present in the target cell.LysoPtdOH,a natural occurring water-soluble phospholipid was originally identi?ed as a key molecule in lipid biosynthesis.It is stored within the cell at concentrations up to 60l M and may accumulate in extracellular ?uids such as in the in?ammatory exudate at concentrations as high as 10l M [22].Both lysoPtdOH and CXCL16have been detected in the wound ?uid in multiple tissues upon injury with the similar kinetics [24,30–32],which may allow the coordinated skin repair with sub-sequent cell in?ltrations [33,34].The instrumental role of disintegrin-like metalloproteinase ADAM10on CXCL16’s proteolytic shedding has been introduced recently [10,24],and lysoPtdOH has been reported to affect the production and shedding of several transmembrane proteins [35,36],here we have shown that lysoPtdOH treatment signi?cantly increased the production and shedding of CXCL16stimu-lated by LPS as well,supporting the important roles of lysoPtdOH in multiple cellular systems by regulating macrophages/MoDC’s chemotactic activity toward to

T

Fig.3a LysoPtdOH increases CXCL16production and shedding in macrophages.Macrophages were prepared as above.Cells were treated with LPS,lysoPtdOH or lysoPtdOH plus LPS for 16h,levels of CXCL16in spent culture supernatant were measured by ELISA.Data presented as the mean ±SE of ?ve experiments with similar https://www.360docs.net/doc/127759917.html,pare to macrophage control supernatant,LPS induced CXCL16release from macrophage (*P \0.01);LysoPtdOH increased CXCL16production and shedding from macrophage stimulated by LPS (**P \0.05);while lysoPtdOH alone had no effect on CXCL16secretion ( P [0.05).b LysoPtdOH lead to undetectable change on CXCL16cell surface presentation in macro-phages.Above macrophages were stained with anti-CXCL16-PE and

analyzed by FACS.Black ?lled curve represent cells stained with isotype-matched control mAb.Gray ?lled curve was for cells stained with anti-CXCL16after lysoPtdOH stimulation.Broken line curve was for cells stained with anti-CXCL16after LPS activation.Solid line curve was for cells stained with anti-CXCL16after lysoPtdOH and LPS treatment.Data shown are representative of three repeatable experiments.c LysoPtdOH upregulated CXCL16mRNA expressions in macrophages shown by real-time PCR.The same number of cells treated as above were lysed,RNA was puri?ed,cDNA was synthesized and the same amount of cDNA was used for RT-PCR

cells.The correlated expression of CXCL16and its pro-tease ADAM-10within the synovium and cultured macrophages has been reported recently [37],which may explain why the change of cell surface presentation of CXCL16affected by lysoPtdOH was undetectable by FACS in our culture system.

Playing an important role in innate immunity,macro-phages detect the invading pathogens through Toll-like receptors (TLR).LPS,a major cell wall component of gram-negative bacteria,binds to mainly TLR4and other cell surface molecules such as HSP70,HSP90,chemokine receptor 4(CXCR4),and growth differentiation factor 5,as well as some cytoplasm molecules then lead to mainly activation of NF-j B [38–42].Therefore,whether the involvement of NF-j B in lysoPtdOH-enhanced CXCL16production stimulated by LPS was solely induced by lys-oPtdOH from LPS cannot be decided in this experimental setting.Meanwhile activation of G i/o -,Rho-in lysoPtdOH and LPS stimulated macrophages is more likely through LPA receptors binding and signaling [19,43–45].

LysoPtdOH has been indicated as promoting early T cell migration to tissue sites of immune responses and regu-lating T cell proliferation and secretion of numerous cytokines [46,47].LysoPtdOH has also been reported to regulate the differentiation and activation of monocytes/macrophages and their further interactions with endothelial cells [48,49].However,this is the ?rst demonstration that lysoPtdOH increases CXCL16production and shedding in addition to other in?ammatory cytokines [21,50,51

],

Fig.4Signaling transductions regulating CXCL16shedding stim-ulated by LysoPtdOH.Macrophages prepared as above were pretreated with different inhibitors for 16h before lysoPtdOH and/or LPS stimulations,concentrations of CXCL16in spent culture supernatant were measured by ELISA.Data were presented as the mean ±SE from three experiments with similar https://www.360docs.net/doc/127759917.html,pare to spent supernatant from lysoPtdOH/LPS treated macrophages,CXCL16secretion in supernatant collected from PTx,exoC3,or PDTC pretreated macrophages were signi?cantly inhibited (*P \0.01;**P \0.05; P \

0.01)

Fig.5a LysoPtdOH increased chemotactic activity of macrophages.Spent culture supernatant was put in the bottom well of a trans-well system and activated CD3?T cells were loaded into the top chambers of the trans-well system.Migration was allowed for 2h,and cells that had migrated to the bottom wells were collected and counted.Data are presented as the mean ±SE of ?ve experiments with similar https://www.360docs.net/doc/127759917.html,pared to the supernatant from control macrophages,supernatant collected from LPS treated cells slightly induced CD3?T cells migration (*P \0.05);supernatant collected from LPA/LPS treated cells macrophages attracted more CD3?T cells migration (**P \0.01),which was signi?cantly blocked by preincubation supernatant with anti-CXCL16blocking antibody ( P \0.01);non-speci?c mIgG antibody had no effect on the chemotactic activity of supernatant collected from LPA/LPS treated macrophages (àP [0.05).b Signaling pathways regulating chemotactic activity of macrophages.Spent culture supernatant collected from macro-phages treated with different inhibitors was also placed in the bottom wells of a trans-well system and migration of CD3?T cells was calculated.Data presented as the mean ±SE of three experiments with similar https://www.360docs.net/doc/127759917.html,pare to spent supernatant from LPA/LPS treated macrophages,migration of CD3?T cells toward supernatant collected from PTx,exoC3,or PDTC pretreated macrophages were signi?cantly blocked (*P \0.01;**P \0.01; P \0.01)

therefore directing more activated T cells to the in?am-mation and tumor sites to develop functional immune responses[13,52].Since most of CXCL16binding CXCR6expressing cells in blood are IFN-secreting helper T cells,cytotoxic T cells,or CD56?T cells that are enri-ched for effector functions,thus upregulation of CXCL16 by lysoPtdOH in macrophages and MoDC most likely leads to the(T helper1)Th1and cytotoxic effector func-tion in in?ammation and tumor sites.The direct effect of lysoPtdOH on MoDC maturation and the ability of DC to in?uence Th1cell polarization has been investigated[16], this study elucidated its role in CXCL16production in macrophages as a focus.We hope this mechanic link between lysoPtdOH and chemokines advances our knowledge of lipid mediated immune responses. Acknowledgments We thank all the healthy blood donors provided written informed consent according to federal,state and institutional guidelines.The authors declare no con?icting and commercial interest.

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