of May 12, 2018.This information is current as

Axl/Gas6 Pathway-InducibleαCells Are Regulated by an IFN-

Survival and Migration of Human Dendritic

Paus, Silvia Bulfone-Paus and Mirella GiovarelliRalfCappello, Silvia Rossi, Daniele Pierobon, Zane Orinska,

Sara Scutera, Tiziana Fraone, Tiziana Musso, Paola

http://www.jimmunol.org/content/183/5/3004doi: 10.4049/jimmunol.0804384August 2009;

2009; 183:3004-3013; Prepublished online 5J Immunol 

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4.DC1http://www.jimmunol.org/content/suppl/2009/08/05/jimmunol.080438

Referenceshttp://www.jimmunol.org/content/183/5/3004.full#ref-list-1

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Survival and Migration of Human Dendritic Cells AreRegulated by an IFN-�-Inducible Axl/Gas6 Pathway1

Sara Scutera,2* Tiziana Fraone,2† Tiziana Musso,* Paola Cappello,† Silvia Rossi,*Daniele Pierobon,† Zane Orinska,‡ Ralf Paus,§¶ Silvia Bulfone-Paus,2,3‡ and Mirella Giovarelli2†

Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In thisstudy, we show that Axl expression is induced by IFN-� during human dendritic cell (DC) differentiation from monocytes(IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-�. Thiseffect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axldown-regulation on the surface of human IFN-�-treated DC correlates with an increased proteolytic cleavage of Axl and withelevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axlexpression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced che-motaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidencethat Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. Thisencourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunitycharacterized by high levels of IFN-�. The Journal of Immunology, 2009, 183: 3004–3013.

A xl is an important member of the receptor tyrosine ki-nase family constituted by Tyro3, Axl, and Mer (TAM4

family) (1–3). Each member shares a similar extracellu-lar domain structure, and a conserved catalytic kinase domain inthe cytoplasmic portion. Both Axl and Mer undergo proteolyticprocessing to yield a soluble form (soluble Axl (sAxl) and solubleMER) (4–6). sAxl, generated by ADAM10-mediated cleavage, ispresent in cell-conditioned medium of primary and transformed

cell lines and in human and mouse serum (7). The TAM receptorligands are two closely related vitamin K-dependent proteins:Gas6, the product of the growth arrest-specific gene, and proteinS, a negative regulator of blood coagulation (8–12). Gas6 bindsthe three receptors with different affinity (Axl � Tyro3 � Mer),whereas protein S seems to be a specific agonist for Tyro3 and Meronly (8).

TAM receptors are broadly expressed by cells of the immune,nervous, reproductive, and vascular systems, and by different tu-mor cell lines (2, 3). Several studies indicate that the Gas6/Axlsystem plays an important role in cell adhesion and migration (13,14). Furthermore, Gas6/Axl signaling modulates cell growth andinhibits apoptosis. For example, Axl promotes survival of endo-thelial and neuronal cells (15, 16), and protects murine fibroblastsand human endothelial cells from apoptosis induced by TNF orother stimuli (17–19). Both Gas6 and protein S mediate the rec-ognition of apoptotic cells and their subsequent phagocytosis bymacrophages through the recognition of phosphatidylserine ex-posed on apoptotic cell membranes (1, 3, 20). Mutant mice lackingthe three TAM receptors show defective clearance of apoptoticcells and develop severe lymphoproliferative disorders accompa-nied by broad-spectrum autoimmunity (21).

Recently, Rothlin et al. (22) reported that, in murine macro-phages and dendritic cells (DC), TAM receptor signaling limits theTLR-induced production of proinflammatory cytokines throughthe induction of the inhibitory proteins suppressor of cytokine sig-naling (SOCS) 1 and SOCS3. Intriguingly, Axl was found to as-sociate with the type I IFN receptor (IFNAR1). Also, IFNAR1 aswell as its transcription factor STAT1 were shown to be essentialfor SOCS induction (22). In this murine model, type I IFNs, whichare induced downstream of TLR activation, also up-regulate Axlexpression via IFNAR-STAT1 signaling (22).

Type I IFNs are pleiotropic cytokines that play an important rolein direct antiviral defense, and link the innate and adaptive immuneresponse (23). Although many different cell types of both hema-topoietic and nonhematopoietic origin produce type I IFNs in re-sponse to infectious agents or inflammatory stimuli, it is well

*Department of Public Health and Microbiology, †Center for Experimental Researchand Medical Studies, San Giovanni Battista Hospital, and Department of Medicineand Experimental Oncology, University of Torino, Torino, Italy; ‡Department of Im-munology and Cell Biology, Research Center Borstel, Borstel, Germany; §Depart-ment of Dermatology, University of Lubeck, Lubeck, Germany; and ¶School ofTranslational Medicine, University of Manchester, Manchester, United Kingdom

Received for publication January 2, 2009. Accepted for publication June 30, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Ministero dell’Istruzione Universita e Ricerca, Pro-gramma di Ricerca Scientifica di Interesse Nazionale, and Regione Piemonte-Progettidi Ricerca Sanitaria Finalizzata e Applicata, and by grants from the FondazioneCasse di Risparmio di Torino (Progetto Alfieri) and Deutsche Forschungsgemein-schaft (to S.B.-P.). P.C. is a recipient of Fondazione Italiana per la Ricerca SulCancro, fellowship; T.F. is a recipient of Fondazione Angela Bossolascofellowship.

S.S. and T.F. performed research, analyzed and interpreted data, and wrote the paper;T.M. designed research, and analyzed and interpreted data; P.C. and Z.O. analyzedand interpreted data; D.P. and S.R. performed research and collected data; R.P. in-terpreted data and wrote the paper; and S.B.-P. and M.G. designed research, analyzedand interpreted data, and wrote the paper.2 S.S., T.F., S.B.-P., and M.G. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Silvia Bulfone-Paus, Depart-ment of Immunology and Cell Biology, Research Center Borstel, Parkallee 22,D-23845 Borstel, Germany. E-mail address: sbulfone@fz-borstel.de4 Abbreviations used in this paper: TAM, Tyro3, Axl, and Mer; BMDC, bone mar-row-derived dendritic cell; DC, dendritic cell; HPRT, hypoxanthine phosphoribosyl-transferase; MFI, mean fluorescence intensity; pAb, polyclonal Ab; PI, propidiumiodide; sAxl, soluble Axl; SOCS, suppressor of cytokine signaling; TACE, TNF-�converting enzyme; ADAM, a disintegrin and metalloproteinase; MMP, matrixmetalloproteinase.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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established that plasmacytoid DC are the most potent IFN-�-pro-ducing cells after microbial challenge (24, 25). Mounting evidenceshows that type I IFNs modulate DC biology at different levels. Forexample, IFN-� (in combination with GM-CSF), rapidly induces thedifferentiation of monocytes into potent, functional DC (IFN/DC)(26–34). These IFN/DC show increased expression of costimulatorymolecules and exhibit more potent Ag-presenting activities comparedwith DC generated in the presence of IL-4 (IL-4/DC) (26–34).IFN/DC undergo complete maturation upon LPS stimulation and mi-grate in response to �-chemokines, as a consequence of CCR7 up-regulation (30). Functionally, IFN/DC exhibit potent allostimulatoryactivity in an MLR assay and induce humoral and Th1-polarized cel-lular responses in SCID mice reconstituted with human PBMCs (26).IFN/DC also exhibit a cytotoxic activity that is mainly mediated by asubset expressing CD56 (33, 34).

Because IFN-� is a crucial regulator of DC functions, and be-cause Axl expression is regulated by IFN-�, we addressed thequestion as to whether there is direct evidence of a regulation ofAxl expression by IFN-� in human DC. We focused on humanIFN/DC, because they are generated in the presence of IFN-�, andthere is accumulating evidence that IFN/DC differentiate frommonocytes in systemic lupus erythematosus, an autoimmune con-dition characterized by relatively large amounts of circulatingIFN-� (35). In addition, IFN/DC are considered promising candi-dates for immunotherapeutic studies (36). For comparison, we in-vestigated conventional IL-4/DC (37). IFN/DC and IL-4/DC weregenerated from human monocytes, and their expression of Axl,Tyro3, Mer, and the ligand Gas6 was investigated in immature andmature DC. In addition, the activity of Gas6 on DC survival andchemotaxis was assessed. These studies revealed that IFN/DC, butnot IL-4/DC, acquire cell surface Axl during their differentiation.TLR-dependent maturation stimuli (LPS, poly(I:C), TLR7/8 li-gand) significantly down-regulate the Axl expression on IFN/DCthrough increased proteolytic cleavage. Gas6 protects DC fromserum deprivation-induced apoptosis, and promotes chemotaxis ofDC in an Axl-dependent manner. These results suggest that IFN-�can regulate DC survival and migration through the up-regulationof Gas6/Axl-mediated signaling.

Materials and MethodsDC generation

Human PBMC were obtained from the venous blood of voluntary healthydonors by Histopaque density gradient centrifugation (Sigma-Aldrich) andenriched in monocytes with an isolation kit (Miltenyi Biotec). The resultingpreparations were consistently �95% CD14� as determined by FACS-Calibur (BD Biosciences). Monocytes were incubated in six-well cultureplates (1 � 106 cells/ml) for the indicated times in RPMI 1640 mediumwith 10% heat-inactivated FBS (Life Technologies) supplemented with100 ng/ml GM-CSF and 50 ng/ml IL-4 (both PeproTech) to generate IL-4/DC, or with 100 ng/ml GM-CSF and 1000 IU/ml IFN-� or IFN-� (Pepro-Tech) to generate IFN/DC. In some experiments, after 3 days of culture,IFN/DC, previously labeled with anti-Axl primary mAb (R&D Systems)and with a PE anti-mouse Ig (DakoCytomation), were purified with anti-PEmicrobeads (Miltenyi Biotec) to a purity of �95% Axl� cells. Axl� andAxl� IFN/DC were then used for further analyses.

Culture conditions

On day 3, IFN/DC were stimulated for 24 h with different stimuli: LPSderived from Escherichia coli O55:B5 (100 ng/ml) and poly(I:C) (15 �g/ml) (both from Sigma-Aldrich); TLR7/8 ligand (5 �g/ml; InvivoGen); andCD40L-transfected cells J558 (1:4 ratio; provided by S. Sozzani, Univer-sity of Brescia, Brescia, Italy). In the same manner, IL-4/DC were treatedwith IFN-� (1000 IU/ml) and LPS (100 ng/ml) alone or in combination.Where indicated, IFN/DC were cultured with a broad-spectrum hydrox-amic acid-based metalloproteinase inhibitor GM6001 or the TNF-� pro-tease inhibitor TAPI-1 (Calbiochem).

Real-time PCR

RNA was extracted from cells with TRIzol reagent, and cDNA was syn-thesized from 2 �g of total RNA by using random oligonucleotides asprimers and a SuperScriptII kit (all reagents were from Invitrogen). ThecDNA was then amplified for the housekeeping gene hypoxanthine phos-phoribosyltransferase (HPRT), Axl, and Gas6 using the following primers:HPRT (sense, 5�-TGA CCT TGA TTT ATT TTG CAT ACC-3�; antisense,5�-CGA GCA AGA CGT TCA GTC CT-3�); Axl (sense, 5�-CGT AACCTC CAC CTG GTC TC-3�; antisense, 5�-TCC CAT CGT CTG ACAGCA-3�); and Gas6 (sense, 5�-AAC TCC CCA GGG AGC TAC A-3�;antisense, 5�-GCA CGG CAA GAT GTC CTC-3�). The Universal ProbeLibrary system (Roche) was used to select specific probes. Real-time RT-PCR analysis was performed on Light Cycler (Roche) using TaqMan assay(Invitrogen) with the following thermal steps: initial denaturation at 95°Cfor 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, an-nealing at 56°C for 30 s, and extension at 72°C for 60 s.

The cDNA was also amplified for the genes ADAM10 and ADAM17/TNF-� converting enzyme (TACE) using the following primers: ADAM10(sense, 5�-ACC TGG GAA ACA GTG CAG TCC-3�; antisense, 5�-GGTCAG ATG CTG GGC AGA GAG-3�) and ADAM17 (sense, 3�-ATG AGGACC AGG GAG GGA AAT ATG-5�; antisense, 5�-CAC TCC TGG GCCTTA CTT TCA ATG-3�).

The iQ SYBR Green Supermix (Bio-Rad) was used to run relative quan-titative real-time PCR of the samples, according to the manufacturer’s in-structions. Reactions were run in triplicate on an iCycler (Bio-Rad), andgenerated products were analyzed with the iCycler iQ Optical System Soft-ware (version 3.0a; Bio-Rad). Gene expression was normalized based on18S rRNA contents with overlapping results, and the data are evaluated as2��Ct (where Ct represents cycle threshold) values.

Immunophenotypic analysis

Cells were washed and resuspended in PBS (Sigma-Aldrich) supplementedwith 0.2% BSA and 0.01% sodium azide, and incubated with fluorochrome-conjugated mAbs and isotype-matched negative controls (DakoCytomation)after blocking nonspecific sites with rabbit IgG (Sigma-Aldrich) for 30 min at4°C. The following mAbs were used: anti-Axl, anti-Mer, and anti-Tyro3(R&D Systems); anti-CD80, anti-CD83, and anti-CD86 (BD Biosciences);anti-MHCII (Ancell); and anti-CCR5, anti-CD11c, and anti-CD123 (BD Bio-sciences). To evaluate the NK phenotype, we also used Abs against CD56(Southern Biotechnology Associates), CD49b, NKG2D, TRAIL, IFN-� (BDBiosciences), IL-15 (R&D Systems), and granzyme B (Holzel Diagnostica).Type I IFN receptor expression was monitored by staining cells with anti-IFNAR1 or anti-IFNAR2 (PBL InterferonSource). For intracellular staining,cells were fixed with cold 2% formaldehyde solution and permeabilized with5% saponin solution before staining. For granzyme B, IFN-�, and IL-15 anal-ysis, the cells were also previously treated for 4 h with brefeldin A. Sampleswere then collected and analyzed using a FACSCalibur CellQuest (BD Bio-sciences). Cells were electronically gated according to their light-scatter prop-erties to exclude cell debris.

Protein detection

Concentrations of sAxl, TNF-�, and IL-6 in cell supernatants were eval-uated by DuoSet ELISA kits (R&D Systems), according to the manufac-turer’s recommendations. Values are given as the mean concentration �SEM of three independent experiments.

Apoptosis assay

IFN/DC were cultured in RPMI 1640 medium/1% FBS in presence orabsence of Gas6 to evaluate its capacity of protection. Human or mouserGas6 (R&D Systems) was used in this study with identical results. Thecells were treated for 15 and 30 h and then evaluated for apoptosis with theannexin V-FITC apoptosis detection kit (Calbiochem).

Migration assay

Migration of IL-4/DC and IL-4/DC treated with IFN-� for 24 h was mea-sured in duplicate with a Transwell system (24-well plates; 8.0 �m poresize; Corning Costar). RPMI 1640 medium alone or 5, 25, 50 nM Gas6(R&D Systems) or 250 ng/ml rCCL4 (PeproTech) was added to the lowerchamber. Wells with medium only were used as a control for spontaneousmigration. A total of 2.5 � 105 cells in 100 �l was added to the upperchamber and incubated at 37°C for 4 h. In some migration experiments,IL-4/DC treated with IFN-� were pretreated with a polyclonal Ab (pAb)against Axl (10 �g/ml) or isotype control pAb (10 �g/ml) (both from R&DSystems) for 30 min at 4°C. For receptor desensitization, IL-4/DC treatedwith IFN-� were first stimulated with 250 nM Gas6 for 30 min at 4°C, thenallowed to migrate toward 25 nM Gas6. Cells migrated into the lower

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chamber were harvested, concentrated to a volume of 200 �l, and countedby flow cytometry. Events were acquired for a fixed time of 60 s. Thecounts fell within a linear range of the control titration curves obtained bytesting increasing cell concentrations. Values are given as the mean numberof migrated cells � SEM.

Statistical analysis

Statistical analysis was performed by Student’s t test (GraphPad Prism 4;GraphPad). Values of p � 0.05 were considered significant. Values areexpressed as the mean � SEM.

ResultsIFN-� specifically induces Axl expression during DCdifferentiation

Axl has been reported to be modulated by IFN-� treatment(22, 38). Therefore, we investigated whether the expression of Axland/or Gas6 is regulated during the IFN-�-mediated differentiationof DC from human monocytes. IFN/DC were generated by cul-turing CD14� monocytes with GM-CSF and IFN-�, and Axl ex-pression was analyzed by flow cytometry. Conventional IL-4/DCwere used for comparison.

As shown in Fig. 1A, cell surface Axl was expressed by �50% ofIFN/DC, whereas it was undetectable in IL-4/DC. A clear-cut induc-

tion of Axl expression (33 � 5% of positive cells) was detected asearly as 1 day after GM-CSF and IFN-� treatment. At day 3, 50% ofthe cells were Axl positive, and Axl expression remained stable until5 days posttreatment (Fig. 1A). Both IFN/DC and IL-4/DC showedtypical phenotypes, as previously reported (28 –31). IFN/DCexpressed higher levels of the costimulatory molecules CD80,CD86, and MHCII, compared with IL-4/DC (data not shown).In accordance with the Axl cell surface expression, Axl mRNAlevels, measured by real-time RT-PCR, increased from day 1 today 3 upon IFN-� treatment; Gas6 was also up-regulated, but ina delayed manner compared with its receptor (Fig. 1B).

To confirm that IFN-� is the factor responsible for Axl up-regulation during DC differentiation, monocytes were treated withGM-CSF, IFN-�, or GM-CSF and IFN-�, and Axl expression wasassessed after 48 h. GM-CSF-treated monocytes did not expressAxl. IFN-� induced Axl expression in 15% � 4 of monocytes;however, the combination of both cytokines increased Axl expres-sion in a higher proportion of monocytes (33 � 7%) (Fig. 1C).

Axl expression induced by IFN-� was found to be dose depen-dent, with a maximal induction in the dose range of 1,000–10,000IU/ml, and some induction with concentrations as low as 50 IU/ml(Fig. 1D). Comparable results were obtained using IFN-� (data not

FIGURE 1. Axl expression in DC generated in the presence of IFN-� and GM-CSF. A, CD14� monocytes were cultured with GM-CSF (100 ng/ml)and either IFN-� (1000 U/ml) (IFN/DC) or IL-4 (50 ng/ml) (IL-4/DC) for 1, 3, and 5 days. Cell surface Axl expression (filled histograms) was evaluatedby flow cytometry. Empty histograms show staining with isotype-matched control Abs. Positive cell percentage is reported in each panel. B, Total RNAextracted from IFN/DC and IL-4/DC was subjected to real-time RT-PCR with primers specific for Axl and Gas6. mRNA level was normalized accordingto the expression of the housekeeping gene HPRT. Relative mRNA levels were adjusted to IL-4/DC (equal to 1) and are the means � SEM of threeexperiments. C, Monocytes were cultured in the presence of GM-CSF (100 ng/ml) only, IFN-� (1000 U/ml) only, or both for 48 h. Cells were stained forAxl and analyzed by flow cytometry. D, Monocytes were cultured in the presence of GM-CSF (100 ng/ml) and IFN-� at the indicated concentrations for3 days. Axl expression was evaluated by flow cytometry. The results shown are representative of three independent experiments.

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shown). IFN-� specifically induced Axl, but did not affect the expres-sion of Tyro3 and Mer, the other two members of the TAM family,whose level was comparable in both IFN/DC and IL-4/DC at day 3 ofdifferentiation (Fig. 2A). These results indicate that IFN-� specificallyinduces Axl expression during IFN/DC differentiation.

Because only about half of the IFN/DC population was positive forAxl (Figs. 1A and 2A), we next investigated whether Axl expressioncorrelated with the expression of selected lineage and activation mark-ers. Axl� and Axl� IFN/DC were sorted by microbeads and pheno-typically characterized by FACS analysis. Both populations showedan almost identical pattern of the DC lineage markers CD11c, CD123,and CCR5, and of the cytotoxicity markers that are highly expressedby and shared between IFN/DC and NK cells (CD56, CD49B, gran-zyme B, TRAIL, NKG2D) (33, 34) (Fig. 2B). Intracellular immuno-reactivity for IFN-� and IL-15, two cytokines shown to bepresent in IFN/DC (32, 34), was also expressed at comparablelevels by both Axl� and Axl� IFN/DC. Interestingly, however,these two populations differed in their expression of IFNAR1(mean fluorescence intensity (MFI) 190 for Alx�; MFI 36for Axl�) (Fig. 2C), a cytokine receptor that is physically as-sociated with Axl (22). Therefore, we can postulate that, be-cause they share DC lineage markers, Axl� and Axl� IFN/DCmost likely originate from the same precursor cells, and laterdifferentiate into two functionally distinct populations.

Axl expression is regulated during DC maturation

Given that Axl is regulated during IFN/DC differentiation, next weasked whether DC maturation influences Axl expression. To thisend, IFN/DC were induced to mature with LPS, poly(I:C), TLR7/8ligand, and CD40L, and Axl expression was measured by FACSanalysis. Mer and CD83 were used for comparison (Fig. 3A). Asshown in Fig. 3A, unexpectedly, Axl expression was signifi-cantly down-regulated by maturation stimuli, whereas the DCmaturity indicator CD83 was highly up-regulated and Mer wasnot affected.

Because IFN-� regulates IL-4/DC maturation (39–42), we ana-lyzed Axl expression in IL-4/DC stimulated for 24 h with eitherIFN-� or LPS. As shown in Fig. 3B, IFN-�-mediated activation ofIL-4/DC resulted in Axl up-regulation comparable to the levels ob-served in IFN/DC. LPS did not affect basal Axl and Mer surface levelson IL-4/DC; however, it strongly inhibited Axl expression induced byIFN-� (Fig. 3B). IFN-�, therefore, can induce Axl expression inIFN/DC as well as in IL-4/DC, and its effect is inhibited in bothmodels by maturation stimuli. Therefore, only IFN-�, independentlyof whether it is used as a maturation or differentiation stimulus, spe-cifically induces Axl expression in both DC cell types. In contrast, inboth IFN/DC and IL-4/DC, maturation-inducing stimuli have the op-posite effect, down-regulating Axl expression.

FIGURE 2. Expression of Axl family members on DC generated in the presence of IFN-� and GM-CSF and immunophenotypic pattern of Axl�/Axl�

IFN/DC. A, Representative flow cytometric analysis of TAM on monocytes after 3-day culture in the presence of GM-CSF and either IFN-� or IL-4.Percentage of positive cells is indicated. B, IFN/DC were purified with Axl mAb-conjugated microbeads, leading to a purity of the Axl� cells of �95%.Thereafter, Axl� (f) and Axl� (�) IFN/DC were stained for the indicated markers and were analyzed by flow cytometry. Marker expression is given asmean (�SEM) of at least five DC preparations. C, Analysis of IFNAR1 and IFNAR2 subunit surface expression on Axl� and Axl� IFN/DC. Emptyhistograms show staining with isotype-matched control Ab. Percentage of positive cells and MFI is reported in each panel.

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LPS-induced reduction of surface Axl coincides with increasedproduction of sAxl

Rothlin et al. (22) reported an increase of Axl expression at themRNA and protein level in murine bone marrow-derived DC(BMDC) treated with different TLR agonists. Because of this discrep-ancy with our data on human DC, we next studied whether the de-creased amount of cell surface Axl seen in our system resulted fromlower production or increased shedding. To elucidate this issue, wetested Axl mRNA expression in IFN/DC cultured in the presence orin the absence of LPS (100 ng/ml) or poly(I:C) (15 �g/ml) for 6 h. Asshown in Fig. 4A, Axl mRNA was markedly elevated upon treatmentwith both TLR agonists, in line with Rothlin’s results.

Cell surface Axl expression was measured by FACS analysisin IFN/DC after treatment with LPS, poly(I:C), and TLR7/8ligand for different times (1, 3, and 6 h). A decrease in thepercentage of Axl-positive cells was already evident after 3 h oftreatment, and a further decrease was observed after 6 h (Fig.4B). No significant differences in Axl expression were observedat 1 h (data not shown).

The discrepancy between mRNA and cell surface Axl expres-sion can be explained by our additional ELISA findings, whichreveal that sAxl was significantly increased in the supernatants ofIFN/DC treated with LPS, poly(I:C), and TLR7/8 ligand (Fig. 4B).Notably, the augmented amount of sAxl in the culture medium wasconcurrent with a reduced expression of the transmembrane pro-tein at the various times.

Members of the metalloproteinase superfamily have beenshown to be responsible for the cleavage of the majority of shedproteins (43, 44). To test whether the release of sAxl is medi-ated by metalloproteinases, cells were first preincubated for 30min with GM6001, a general inhibitor of matrix metallopro-teinase (MMP) and a disintegrin and metalloproteinase(ADAM) family proteases (7), followed by direct addition ofmaturation stimuli. These experiments demonstrated the abilityof GM6001 to restore the presence of Axl on the cell surfaceand to diminish sAxl release in maturation stimuli-treated cells(Fig. 4B). Furthermore, we observed a capacity of GM6001 tosuppress constitutive Axl shedding in IFN-DC, as demonstratedby a significant reduction in the release of sAxl ( p 0.0002)and by a higher amount of Axl on the cell membrane ( p 0.0462) at 6 h (Fig. 4B). These data indicate that the TLR-induced reduction of surface Axl corresponds to an increasedproteolytic cleavage.

Taking into account that ADAM10 and ADAM17/TACE havebeen implicated in the constitutive and inducible ectodomain shed-ding of a number of cell surface-expressed molecules and murineAxl (7, 43), we investigated the involvement of ADAM proteasesin sAxl generation in human DC. First, we examined the expres-sion of ADAM10 and ADAM17 in IFN/DC unstimulated ortreated with LPS for 6 and 24 h by real-time RT-PCR. ADAM10and ADAM17 mRNA is expressed by unstimulated IFN/DC. Thelevel of ADAM10 expression was not altered by treatment with

FIGURE 3. Maturation/Activation stimuli down-regulate Axl cell surface expression in IFN/DC. A, IFN/DC were cultured for 24 h with the indicatedstimuli, and Axl, Mer, and CD83 expression was determined by flow cytometry. B, IL-4/DC stimulated with LPS, IFN-�, and LPS together with IFN-�for 24 h were stained with anti-Axl, Mer, and CD83 Ab. Indicated markers’ staining (filled histograms) is presented in comparison with isotype-matchedcontrols (empty histograms). Positive cell percentage is reported in each panel. Data shown are representative of three independent experiments.

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LPS, whereas a modest increase in the ADAM17 mRNA level wasobserved 24 h after stimulation (data not shown).

Next, cells were pretreated with the ADAM inhibitor TAPI-1(45) for 30 min, followed by addition of LPS to the culture me-dium for 6 h. As shown in Fig. 4C, TAPI-1 significantly inhibitedLPS-induced Axl shedding, as demonstrated by a dramatic drop

in the amount of sAxl and an increase in the percentage ofAxl-positive cells.

Our data indicate that the activation of several TLR path-ways, despite Axl induction at mRNA level, results in a dra-matic decrease in the expression of cell surface Axl most likelymediated by ADAM family protease activity in human IFN/DC.

FIGURE 4. LPS mediates the decrease of cell surface Axl expression by promoting receptor shedding. A, IFN/DC were cultured for 6 h with theindicated stimuli, and real-time RT-PCR analysis of Axl and Gas6 was performed. B, IFN/DC were stimulated for 3 and 6 h with the indicated stimuli afterpretreatment with GM6001 (�; 25 �M) or vehicle (�; DMSO) for 30 min. Cell surface expression of Axl was analyzed by flow cytometry (n 3), andthe fraction of stained cells is given (upper panels). sAxl concentration in the supernatants was determined by ELISA (lower panels). C, IFN/DC wereincubated with vehicle (�; DMSO) or TAPI (�; 25 �M) for 30 min, followed by LPS (100 ng/ml) stimulation for 6 h. Cell surface expression of Axl wasanalyzed by flow cytometry (left panel), and sAxl production was determined by ELISA (right panel). The results shown are the mean of three independentexperiments. �, p � 0.05; ��, p 0.01; ���, p 0.001.

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The TLR-mediated regulation of cell surface Axl expression inhuman DC seems to be distinct from that in murine DC. To betterunderstand these differences, mouse BMDC were stimulated withLPS for 24 h and analyzed for Axl cell surface expression (sup-plemental Fig. A).5 Axl was expressed on mouse DC in both un-stimulated and stimulated conditions; only a decrease in MFI wasobserved in LPS-treated compared with untreated cells (MFI ofunstimulated BMDC was 347 � 23 vs 208 � 4 (n 4) in LPS-stimulated BMDC). Similarly to the results obtained in humanIFN/DC, mouse DC constitutively released considerable amountsof sAxl, and treatment with LPS further elevated sAxl production.The inhibitor GM6001 significantly reduced both the constitutiveand LPS-inducible shedding of Axl (supplemental Fig. B).

Gas6-mediated Axl stimulation makes IFN/DC unresponsive tosubsequent LPS-induced activation

Because LPS activation induced the cleavage of Axl ectodomainon IFN/DC (Figs. 3A and 4, B and C), we investigated whether,vice versa, the Axl ligand, Gas6, interfered with TLR-mediatedsignaling. This was assessed by measuring TNF-� and IL-6 levelsin the supernatants of IFN/DC that had been concomitantly stim-ulated with LPS and Gas6, or that had been pretreated with Gas6for 8 h and then stimulated with LPS (Fig. 5). Coincubation ofGas6 with LPS did not affect TNF-� and IL-6 release, as comparedwith LPS alone. However, Gas6 pretreatment significantly inhib-ited LPS-induced TNF-� and IL-6 production. Thus, the engage-ment of Axl by Gas6 renders IFN/DC unresponsive to subsequentLPS-mediated activation.

Gas6-stimulated Axl inhibits apoptosis induced by growth factordeprivation in IFN/DC

We investigated whether Gas6 stimulation affected apoptosis in-duced by growth factor deprivation in IFN/DC. Apoptosis wasinduced in IFN/DC by lowering the serum concentration from 10to 1%, and the proportion of apoptotic cells was assessed by flowcytometry by annexin V-FITC and propidium iodide (PI) staining.

After 15 h of culture in low-serum conditions, 11% of IFN/DCwas annexin V� and PI�, reflecting the percentage of cells under-going early stages of apoptosis, and 23% was already double pos-itive (annexin V�-PI�), indicating a late stage of apoptosis. Ahigher percentage of early apoptotic cells was observed after 30 hof culture (18%). However, when IFN/DC were incubated in the

presence of 50 nM Gas6, we observed a reduction in the percent-age of early apoptotic cells: thus, only 6 and 8% of IFN/DC wereannexin V�/PI� at 15 and 30 h, respectively (Fig. 6). These resultswere consistently observed with three different donors.

Therefore, we can conclude that Gas6/Axl-mediated signalingpromotes survival of human DC.

Gas6 stimulates Axl� DC chemotaxis

Because migration is pivotal for DC function as immune sentinels,we analyzed whether Gas6 exerted chemotactic effects on thesecells, comparing the capacity of Axl� (IFN-�-activated IL-4/DCand IFN/DC) and Axl� (IL-4/DC) human DC to migrate in re-sponse to exogenous Gas6 in a Transwell system. As shown in Fig.7, Gas6 induced the chemotaxis of IFN-�-activated IL-4/DC in adose-dependent manner, generating a marked chemotactic re-sponse to increasing concentrations (5, 25, and 50 nM) of Gas6(Fig. 7A). Similar migratory response to Gas6 was obtained withIFN/DC (data not shown). In contrast, IL-4/DC did not show anychemotactic response to Gas6, suggesting that Axl expression isessential for Gas6 chemotactic effects. Both DC populations mi-grated in response to CCL4 used as positive control. As previouslyshown for IFN/DC, IFN-�-activated IL-4/DC displayed a higherspontaneous migration and chemotactic response to CCL4 com-pared with IL-4/DC (30). The role of Axl is supported by the ob-servation that cell pretreatment with an excess of rGas6 abrogated thechemotactic response to Gas6 (Fig. 7B). Final confirmation thatthese effects are Axl dependent was obtained by the demonstrationthat neutralizing anti-Axl Ab blocked Axl� DC Gas6-driven mi-gration (Fig. 7B). Migration toward Gas6 was not affected by pre-treatment with isotype-matched control Ab. This is the first evi-dence that Axl stimulation can mediate DC chemotaxis.

DiscussionIn this study, we demonstrate that Axl is up-regulated on humanDC during differentiation and activation in an IFN-�-dependentmanner. Furthermore, we show that Gas6 inhibits apoptosis andstimulates migration of human DC via Axl activation, providingthe first evidence that Gas6/Axl-mediated signaling regulates hu-man DC activities and can be considered a novel chemotactic path-way for DC. Previous detection of Axl on murine macrophages(20, 21, 38) and on BMDC (20) fostered the idea that Axl was ofimportance in murine APC biology (20). Moreover, it has recentlybeen found that Gas6 regulates TLR-induced cytokine release bymurine DC in vitro (22). Our new findings relative to human DCfurther corroborate the importance of Gas6/Axl-mediated signal-ing in DC biology.

IFN/DC and IL-4/DC share typical DC characteristics, but,probably due to a distinct transcriptional signature of IFN-� incomparison with other cytokines, IFN/DC express higher levels ofseveral maturation markers involved in T cell activation (CD80and CD86) (26–34), in the migratory capacity to the lymph node(CCR7) (30), in cell survival (32), and show a plasmacytoid-likephenotype associated with NK cell characteristics (31). We ob-served that Axl was absent on IL-4/DC, but within the IFN/DC wewere able to identify an Axl-expressing subpopulation that dis-played high migratory activity toward Gas6.

IL-15 up-regulates Axl expression in mouse fibroblasts (46), andIL-15-mediated NK cell development from hematopoietic progen-itor cells is impaired upon blockade of the Axl/Gas6 pathway (47).In this study, we report that IFN/DC express high levels of IL-15,with no differences between Axl� and Axl� cells. Thus, it seemsunlikely that IL-15 is involved in Axl up-regulation in IFN/DC.Interestingly, Axl� and Axl� differ in IFNAR expression. Current5 The online version of this article contains supplemental material.

FIGURE 5. Gas6 pretreatment inhibits LPS-induced TNF-� and IL-6production. Relative production of TNF-� and IL-6 after 15-h stimulationof IFN/DC with LPS (100 ng/ml), either pretreated for 8 h (preGas6) orconcomitantly treated (coGas6) with Gas6 (50 nM). Results were normal-ized to the production of the corresponding cytokines in the presence ofLPS alone and are represented as mean � SEM of three experiments(�, p 0.002).

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investigations are underway to elucidate whether this differencereflects some peculiar response.

Rothlin et al. (22) reported that Axl mRNA and protein expres-sion was increased in murine BMDC treated with different TLRagonists. Our study demonstrates that, even though maturationstimuli such as LPS induce Axl gene transcription, they lower theexpression of cell surface Axl by inducing its shedding. Humanmonocytes and DC express several metalloproteinases, some ofwhich are increased following LPS-induced maturation (48).ADAM10-mediated proteolysis has been reported to constitute amajor mechanism in sAxl generation by Axl shedding in mice (7).The inhibition of both basal and TLR agonist-inducible sheddingby the general MMP and ADAM family inhibitor, GM6001, dem-onstrated that Axl shedding is metalloproteinase dependent. More-over, also a broad ADAM inhibitor TAPI-1, which is extensivelyused to block ADAM17/TACE-mediated TNF-� shedding (45),inhibited the release of sAxl, suggesting a role of ADAM proteasesin the Axl cleavage in human DC. However, because of the notcomplete selectivity of TAPI-1, additional experiments with a tar-geted deletion of ADAMs or the use of compound that preferen-tially blocks their activity are required to understand the individualcontribution(s) of ADAM members to Axl release in IFN/DC.

The decreased amount of Axl on the cell membrane could resultin a reduced sensitivity to Gas6. Thus, the sequence in which hu-man DC encounter TAM ligands and LPS might influence DCactivation. Our findings that Gas6 pretreatment inhibits LPS-in-duced production of TNF and IL-6 support this hypothesis. Theinhibitory effect of Gas6 could be mediated by the induction ofTwist transcriptional repressor that has been implicated in the sup-pression of TNF by type I IFN and Axl (38). Coincubation of Gas6and TLR ligands does not inhibit IL-6 and TNF production inhuman DC, but does inhibit the production of these cytokines inmurine DC, as reported by Rothlin et al. (22). We show that dif-

ferently from the human DC in murine BMDC, Axl is expressedon the surface of all CD11c� cells. Therefore, a more efficientGas6-mediated Axl stimulation, compared with human DC, couldresult in an inhibitory signal for TNF-� and IL-6 production.

It has been shown that Gas6-Axl exerts antiapoptotic effects ona variety of different cell types, including endothelial cells andvascular smooth muscle cells (15–18). In this study, for the firsttime, we report that the Gas6/Axl pathway regulates IFN/DC sur-vival by inhibiting apoptosis induced by growth factor deprivation.Significant lifespan differences have been reported for various DCsubsets and mature vs immature DC, but the underlying mecha-nisms remain unclear (49). Although it remains to be investigatedwhether Gas6 can prevent apoptosis induced by different condi-tions, our results strongly suggest that Gas6/Axl can selectivelyinfluence the DC lifespan.

Compared with IL-4/DC, IFN/DC exhibit enhanced chemotaxisto the inflammatory chemokines CCL3 and CCL5 (30, 50). Thissuggests a selective recruitment of IFN/DC with a highly efficientAg-presenting function at the inflammatory site. Because of theirhomology to cell adhesion molecules, the Axl family receptorshave been implicated in cell adhesion and motility (see Refs. 7–9for reviews). Axl promotes fibroblast intercellular adhesion viahomophilic and heterophilic mechanisms (13), whereas Gas6 in-duces vascular smooth muscle cell and neuronal cell migration (14,15). Gas6 inhibition of vascular endothelium growth factor-A-de-pendent endothelial cell chemotaxis has also been reported (51).Interestingly, Gas6�/� mice show impaired leukocyte recruitment(52). TAM�/� mice with homozygous defects in each one of thethree TAM receptors develop autoimmune diseases (21). How-ever, the extent to which functional defects of individual TAMfamily members contribute to the development of human autoim-mune diseases remains to be determined. It has recently been re-ported that high levels of Gas6 are present in the cerebrospinal

FIGURE 6. Gas6 rescues DC fromgrowth factor deprivation-inducedapoptosis. IFN/DC were cultured inmedium containing 1% FBS in theabsence or in the presence of Gas6(50 nM) for 15 and 30 h. Apoptosiswas measured by PI and annexin Vstaining, and percentage of apoptoticcells is indicated. Lower left quad-rant, Shows the viable cells, whichexclude PI and are negative for FITC-annexin V binding. Upper right quad-rant, Represents late apoptotic cells,positive for FITC-annexin V bindingand for PI uptake. Lower right quad-rant, Represents the early apoptoticcells, FITC-annexin V positive, andPI negative. A representative experi-ment of three independent experi-ments is shown.

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fluid of patients with inflammatory autoimmune demyelinatingdiseases (53). This leads to the suggestion that in certain autoim-mune conditions characterized by both high IFN-� (54) and Gas6levels, Gas6 might affect IFN/DC functions. In this study, we re-port that Axl� DC migrate in response to Gas6 and that this effectis Axl mediated. Therefore, DC responsiveness to Gas6 may alsomodulate DC trafficking during autoimmune conditions. In addi-tion, preliminary data from our laboratory indicate that sAxl is2-fold increased in a small cohort of patients with systemic lupuserythematosus (our unpublished observations). This encouragesone to systematically assess the Axl expression patterns and Gas6-induced migratory properties of circulating DC in patients withautoimmune diseases so as to further explore the hypothesis thatexcessive inappropriate Gas6/Axl-mediated signaling may be in-volved in the pathogenesis of some autoimmune diseases.

In summary, our study reveals that functional, type I IFN-de-pendent Gas6/Axl signaling is required for human DC survival and

migration in vitro. This novel pathway in the regulation of humanDC biology deserves to be explored as a potential therapeutictarget in autoimmune diseases in which type I IFN plays animportant role.

DisclosuresThe authors have no financial conflict of interest.

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FIGURE 7. The Gas6/Axl pathway regulates the chemotaxis of DC. A,A total of 5 � 105 IL-4/DC and IFN-�-treated IL-4/DC was seeded in theupper compartments of a 24-well Transwell cell culture chamber whileincreasing concentrations of Gas6 (5, 25, and 50 nM) were added to thelower compartments. CCL4 (250 ng/ml) was used as positive control. Cellsmigrated to the lower compartments after 4-h incubation were counted byflow cytometry. �, p 0.03; ��, p 0.003 vs medium. B, IFN-�-treatedIL-4/DC were preincubated with anti-Axl pAb (10 �g/ml), control pAb(10 �g/ml), or excess of Gas6 (250 nM) for 30 min at 4°C. Thereafter,chemotaxis was measured in response to Gas6 (25 nM). Assays were per-formed in triplicate. Mean value � SEM of three independent experimentsare shown. ��, p 0.003; ���, p 0.001.

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