Dendritic Cells Maturation of Monocyte-Derived Human IFN

Loss of Type I IFN Receptors and Impaired IFN Responsiveness During Terminal Maturation of Monocyte-Derived Human Dendritic Cells This information is current as of September 25, 2021. Maria Cristina Gauzzi, Irene Canini, Pierre Eid, Filippo Belardelli and Sandra Gessani J Immunol 2002; 169:3038-3045; ; doi: 10.4049/jimmunol.169.6.3038

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Loss of Type I IFN Receptors and Impaired IFN Responsiveness During Terminal Maturation of Monocyte-Derived Human Dendritic Cells1

Maria Cristina Gauzzi,* Irene Canini,* Pierre Eid,† Filippo Belardelli,* and Sandra Gessani2*

Type I IFNs are modulators of myeloid dendritic cell (DC) development, survival, and functional activities. Here we monitored the signal transduction pathway underlying type I IFN biological activities during in vitro maturation of human monocyte-derived DCs. IFN-inducible tyrosine phosphorylation of STAT family members was severely impaired upon LPS-induced DC maturation. This correlated with a marked reduction of both type I IFN receptor chains occurring as early as 4 h after LPS treatment. The reduced receptor expression was a post-transcriptional event only partially mediated by ligand-induced internalization/degrada-

tion. In fact, although an early and transient production of type I IFNs was observed after LPS treatment, its neutralization was Downloaded from not sufficient to completely rescue IFN receptor expression. Notably, neutralization of LPS-induced, endogenous type I IFNs did not interfere with the acquisition of a fully mature surface phenotype, nor did it have a significant effect on the allostimulatory properties of LPS-stimulated DCs. Overall, these data indicate that DCs strictly modulate their responsiveness to type I IFNs as part of their maturation program, underlining the importance of the IFN system in the regulation of DC physiology. The Journal of Immunology, 2002, 169: 3038–3045. http://www.jimmunol.org/ endritic cells (DCs)3 play a pivotal role in linking innate IFNs (in combination with GM/CSF) rapidly induced the differ- and adaptive immunity by their ability to induce appro- entiation of monocytes into partially mature DCs (mDCs) en- D priate immune responses upon recognition of invading dowed with potent functional activities both in vitro and in vivo (7, pathogens. To exert their role, DCs pass through different func- 8). Likewise, IFN-␣ has been described to act as a potent matu- tional states of activation. Resting DCs reside in peripheral tissues ration factor for CD11cϩ DCs (myeloid DCs), directly purified in an immature state, where they are adapted to capture and accu- from the peripheral blood (9) and to enhance the terminal differ- mulate Ags. Upon receipt of a variety of activation signals, these entiation of CD34ϩ-derived DCs (10). Moreover, type I IFNs have cells are induced to mature and migrate to secondary lymphoid been shown to act as adjuvants in the promotion of humoral im- organs. This process is accompanied by a concomitant decrease in mune responses through stimulation of DCs (11). On the other by guest on September 25, 2021 their capacity for Ag uptake and a marked increase in their ability hand, a negative role of type I IFNs in the functional maturation of to present Ags and activate naive T cells. Activated DCs are also DCs induced by pro-inflammatory cytokines has also been de- capable of directing the type of response made by T cells, a process scribed (12, 13). that can be strongly influenced by the cytokine milieu where DC Type I IFNs exert their biological activity through a broadly maturation takes place (1, 2). expressed heterodimeric receptor composed of the IFNAR1 and Type I IFNs are cytokines spontaneously expressed at low levels IFNAR2 subunits, whose mutual interaction activates a series of under physiologic conditions (3) whose expression is highly en- intracellular signaling molecules (14) The major signal transduc- hanced soon after cell exposure to viruses or other stimuli (4). tion pathway used by type I IFNs is triggered upon receptor bind- Although first characterized as potent antiviral molecules, type I ing by the receptor-associated tyrosine kinases Janus kinase type 1 IFNs are also endowed with immunoregulatory activities, and their (JAK1) and tyrosine kinase type 2 (Tyk2). These kinases are ac- important role in linking innate and adaptive immune responses tivated by tyrosine auto/cross-phosphorylation and phosphorylate has been unraveled (5, 6). A number of reports have recently dem- a number of substrates. Among them, a pivotal role is played by onstrated a profound effect of these cytokines on DC maturation, the transcriptional factors of the STAT family. Although STAT-1 survival, and function. In this regard it has been shown that type I and -2 are considered the major factors mediating type I IFN signaling, it has been reported that STAT-3 and, in humans, STAT-4 can be activated in response to these cytokines (15, 16). *Laboratory of Virology, Istituto Superiore di Sanita`, Rome, Italy; and †Viral On- Despite the growing evidence showing the importance of type I cology, Centre National de la Recherche Scientifique, Villejuif, France IFNs in the development, survival, and functional activities of Received for publication March 20, 2002. Accepted for publication July 17, 2002. DCs, no studies are currently available on the expression and sig- The costs of publication of this article were defrayed in part by the payment of page naling activity of type I IFN receptors during the differentiation/ charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. maturation of these cells. Moreover, although many of the stimuli 1 This work was supported in part by the European Community (Contract QLK2- promoting DC terminal maturation are known to induce the pro- CT-2001-02103) and the Italian Project on AIDS (Istituto Superiore di Sanita`-Min- duction of type I IFNs, the role of these endogenously produced istry of Health Contract 40D.7). IFNs in the modulation of DC functions is still poorly studied. In 2 Address correspondence and reprint requests to Dr. Sandra Gessani, Laboratory of this study we report that upon LPS-induced maturation, DCs show Virology, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy. ␤ E-mail address: [email protected] a greatly impaired responsiveness to IFN- , which correlated with 3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; JAK1, a marked reduction of the expression of both IFN receptor subunits. Janus kinase type 1; Tyk2, tyrosine kinase type 2; mDC, mature DC. Down-modulation of receptor chains was only partially mediated by

ligand-induced receptor internalization consequent to LPS-stimulated Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing a type I IFN secretion. cocktail of protease and phosphatase inhibitors (10 ␮g/ml aprotinin, 10 ␮ ␮ Overall, these data indicate that DCs strictly modulate their re- g/ml leupeptin, 2 g/ml pepstatin, 2 mM PMSF, 2 mM NaF, and 0.4 mM Na3VO4). The protein concentration was determined using the Bio-Rad sponsiveness to type I IFNs as part of their maturation program, protein assay (Hercules, CA) according to the manufacturer’s instructions. strongly supporting the importance of the IFN system for the reg- Forty micrograms of lysate was boiled for 4 min in Laemmli sample buffer, ulation of DC physiology. fractionated on an 8% SDS-PAGE, and electroblotted to nitrocellulose filter (Protran BA 85, Schleicher & Schuell, Keene, NH). The following Abs were used for the immunoblots: rabbit anti-phospho-STAT-1 (Y701) and Materials and Methods rabbit anti-phospho-STAT-2 (Y689) (Upstate Biotechnology, Lake Placid, Cell separation and culture NY), rabbit anti-phospho-STAT-3 (Y705; Cell Signaling Technology, Beverly, MA), and anti-STAT-1, -2, and -3 mAbs (Transduction Labora- Monocytes were isolated from the peripheral blood of healthy donors by tories, Lexington, KY). An ECL Western blot detection system (Amer- counterflow centrifugal elutriation and were further purified by depleting sham, Piscataway, NJ) was used according to the manufacturer’s the nonmonocytic population by immunomagnetic beads selection (MACS instructions. monocyte isolation kit from Miltenyi Biotec, Auburn, CA) using the manufacturer’s procedure. To obtain immature DCs (iDCs), monocytes were Immunoprecipitation cultured at 1 ϫ 106 cells/ml in RPMI 1640 (Life Technologies, Gaithers- burg, MD) medium containing 10% FBS supplemented with 50 ng/ml re- For IFNAR1 and IFNAR2 immunoprecipitation, cells were lysed with combinant human GM-CSF and 500 U/ml recombinant human IL-4. Both Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris-Cl (pH 8), 0.5% cytokines were provided by Schering-Plough (Dardilly, France). GM-CSF Nonidet P-40, 10% glycerol, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 2 and IL-4 were added to the cultures every 2Ð3 days. On days 4Ð6, iDCs ␮g/ml pepstatin, and 2 mM PMSF). IFNAR1 and IFNAR2 were immuno-

were induced to final maturation by adding LPS (200 ng/ml; Sigma-Al- precipitated from 1 mg whole cell lysate with 10 ␮g anti-IFNAR1 64G12 Downloaded from drich, St. Louis, MO) and were harvested 24 h later. In some experiments mAb or 5 ␮g anti-IFNAR2 mAb (Calbiochem), respectively, followed by DCs were generated by culturing monocytes in the presence of GM-CSF a mix of protein A and protein G (1/1)/Sepharose. The immunoprecipitates (50 ng/ml) and IFN-␣2b (PeproTech, Rocky Hill, NJ). To neutralize type were resolved on SDS-PAGE, electroblotted, and subjected to Western blot I IFNs during the course of LPS-induced maturation, the following Abs analysis as described above. The following mAbs were used for detection: were used: anti-IFNAR1 mAb 64G12 (20 ␮g/ml) (17, 18) or a mixture of 64G12 and anti-IFNAR2 (Calbiochem). sheep anti-human IFN-␣ and sheep anti-IFN-␤ (PBL; Biomedical Labo- ratories, New Brunswick, NJ) Abs, each at the concentration of 200 neu- MLR assay tralizing units/ml. One neutralizing unit is defined as the amount of Ab that http://www.jimmunol.org/ reduces 10 IU/ml IFN to 1 IU/ml. MLR was performed in RPMI culture medium supplemented with 5% heat-inactivated normal human AB serum. Allogeneic T cells (1Ð2 ϫ 105/ Type I IFN determination well) were cultured for 5 days in 96-well culture microplates as responder cells with 1Ð20 ϫ 103 stimulatory cells (DCs). [3H]Thymidine incorpora- Type I IFNs secreted in culture supernatants were measured using a cyto- tion (sp. act., 5 Ci/mM; Amersham) was measured after a 16-h pulse with pathic effect reduction assay with HeLa cells (1 ϫ 104 cells/well in 96-well 0.5 ␮Ci/well. Results are shown as mean counts per minute of triplicate microplates) and vesicular stomatitis virus at a multiplicity of infection of determinations. [3H]Thymidine incorporation in negative control wells, 0.1 PFU/cell as challenge virus. Human IFN-␣ reference standard (Ga23- with responder T cells, or with stimulators DCs alone was always 902-530; National Institutes of Health, Bethesda, MD) was used at a di- Ͻ800 cpm. lution of 500 IU/ml. The sensitivity of the assay ranged from 5 to 15 IU/ml. by guest on September 25, 2021 FACS analysis of DC surface phenotype Results Type I IFN signaling is severely impaired in LPS-matured DCs Cell staining was performed using FITC-conjugated mAbs (BD PharMin- gen, San Diego, CA). The following mAbs were used: CD14 (M5E2), Human peripheral blood monocytes were cultured with GM-CSF/ CD1a (HI149), CD80 (L307.4), CD86 (2331), CD83 (HB15e), HLA-DR IL-4 to generate iDCs. Incubation of these cells with LPS for an 5 (G46-6), and CD40 (5C3). Briefly, 1Ð2 ϫ 10 cells were preincubated for additional 24 h led to a significant up-modulation of cell surface 30 min on ice with PBS/10% human serum to block nonspecific Ig binding markers, such as CD80, CD86, HLA-DR, CD83, and CD40, in- and then incubated for an additional 30 min with the appropriate mAb. Cells were washed in PBS/10% human serum, fixed in 1% formaldehyde, dicating the acquisition of the well-defined phenotype of mDCs and analyzed with a FACS flow cytometer (BD Biosciences, Mountain (Table I). View, CA). In a first series of experiments the signaling activity of type I FACS analysis of type I IFN receptor expression IFN receptor during the course of DC maturation was evaluated by monitoring the IFN-␤-induced tyrosine phosphorylation of STAT Surface receptor expression was monitored by staining cells with anti- family members, which are among the most proximal targets of the IFNAR1 64G12 mAb (17, 18) or anti-IFNAR2 mAb (Calbiochem, La Jolla, CA), followed by biotinylated polyclonal anti-mouse IgG Ab (BD PharMingen) and FITC-streptavidin (BD PharMingen). Before staining, cells were blocked with PBS, 50% human serum, and 10% FBS. At the end Table I. Phenotypic characterization of immature and mature DC of each incubation, cells were washed with PBS/10% human serum. After populationsa FITC-streptavidin incubation, cells were fixed and analyzed as described above. iDCs LPS-mDCs RT-PCR % Positive Fluorescence % Positive Fluorescence Cell Surface Marker cells intensity cells intensity The total RNA used for the RT reaction was extracted using the RNeasy ϭ Ϯ Ϯ Ϯ Ϯ Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instruc- CD14 (n 5) 6 2150.5 3 1172 ϭ Ϯ Ϯ Ϯ Ϯ tions. RT-PCR analysis of IFNAR1, IFNAR2, and GAPDH was performed CD80 (n 7) 15 3161922463 ϭ Ϯ Ϯ Ϯ Ϯ as previously described (19). IFNAR1 cDNA was amplified using primers CD86 (n 7) 9 2202932 130 24 ϭ Ϯ Ϯ Ϯ Ϯ 2 and 5 as described by Abramovich et al. (20) For IFNAR2 cDNA am- CD83 (n 7) 3 0.4 15 0.9 86 3366 ϭ Ϯ Ϯ Ϯ Ϯ plification, primers DN11 and DN12 were used as previously described CD40 (n 3) 73 13 22 0.6 91 0.9 55 3 ϭ Ϯ Ϯ Ϯ Ϯ (21). The sequence of GAPDH primers has been previously described (22). HLA-DR (n 4) 90 4678933 148 47 a Peripheral blood monocytes were cultured with GM-CSF (50 ng/ml) and IL-4 Immunoblotting (500 U/ml) for 4Ð6 days. Cells were then stimulated with LPS (200 ng/ml) for an additional 24 h. At the end of incubation, cells were directly stained with specific Abs For the immunoblotting analysis of STAT tyrosine phosphorylation, cells ␤ and analyzed by flow cytometry. The percentage of cells positive for the expression were left untreated or were treated for 45 min with different doses of IFN- of each surface Ag is calculated by differences between the level of staining with a (provided by Serono, Ardea, Italy), washed three times with ice-cold PBS, specific Ab and the baseline of the control isotype. The values represent the mean Ϯ and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl (pH 7.5), 1% SE of n separate experiments. 3040 DC MATURATION AND IFN RESPONSIVENESS

FIGURE 1. In vivo tyrosine phosphorylation state of STAT-1, -2, and -3 in iDCs and LPS-mDCs. Peripheral blood monocytes were cultured in the presence of GM-CSF (50 ng/ml) and IL-4 (500 U/ml) for 5 days (iDCs) and stimulated with LPS (200 ng/ml) for an additional 24 h (LPS-mDCs). Immature DCs and LPS-mDCs were left untreated or treated with the indicated doses of IFN-␤ for 45 min. A, Whole cell extracts (40 ␮g) were fractionated on 8% SDS-PAGE and transferred to nitrocellulose ﬁlter. The tyrosine phosphorylation of STAT-1 and -3 was detected using phosphorylation-speciﬁc Abs. The total level of expression of each protein is shown below its phosphorylated form. B, Whole cell extracts (40 ␮g) were processed as described in A and analyzed for STAT-2 expression and tyrosine phosphorylation.

receptor-associated tyrosine kinases, rapidly activated upon IFN IFNAR1 and IFNAR2 mRNA accumulation were observed inde- Downloaded from binding. pendently of the DC maturation stage. Fig. 1 (A and B) shows the results of a representative experiment in which we analyzed the in vivo tyrosine phosphorylation of LPS induces an early and transient production of type I IFNs, STAT-1, -2, and -3 in response to different doses of IFN-␤ in iDCs whose neutralization only partially restores type I IFN receptor and LPS-mDCs 24 h after stimulation. In iDCs, a marked tyrosine expression phosphorylation of all STATs analyzed was observed at low LPS is a well-known type I IFN inducer in a variety of murine and http://www.jimmunol.org/ IFN-␤ doses (ranging from 10 to 50 IU/ml), which only slightly human cell models, including monocytes/macrophages and DCs increased at higher doses of this cytokine. On the contrary, LPS- (22, 23). Since it has been described that type I IFN binding to mDCs displayed a constitutive level of tyrosine phosphorylation of both STAT-1, -2, and -3, which was not further induced even upon stimulation with high IFN-␤ concentrations (up to Ͼ1000 IU/ml). Notably, a higher expression of STAT-1 was consistently detected in LPS-mDCs with respect to iDCs.

Expression of type I IFN receptor chains is rapidly down- by guest on September 25, 2021 modulated during LPS-induced DC maturation To gain insight into the mechanism(s) involved in the impaired STAT activation by LPS-mDCs in response to IFN-␤, time-course experiments were conducted to investigate the surface expression of type I IFN receptor chains, IFNAR1 and IFNAR2, during LPS- induced DC maturation. As shown in Fig. 2, a remarkable percentage of iDCs (CD86low and CD83Ϫ) expressed both receptor components. However, as soon as 4 h after LPS stimulation, a marked reduction in the surface expression of both receptor chains was observed, concomitantly to a signiﬁcant increase in CD86 and CD83 expression. At later time points (8 and 24 h), the surface expression of IFNAR1 and IFNAR2 remained barely detectable, whereas a progressive increase in the expression of costimulatory and maturation markers was observed.

Down-modulation of type I IFN receptor chains is mediated by post-transcriptional mechanisms To characterize the mechanism(s) responsible for the reduced expression of type I IFN receptor chains at the plasma membrane, the steady-state level of expression of the receptor subunits was evaluated by immunoprecipitation assays. Both IFNAR1 and IFNAR2 are glycosylated proteins that migrate with an apparent molecular mass of 110Ð150 kDa (IFNAR1) and 90Ð100 kDa (IFNAR2), depending on the cell type. As shown in Fig. 3A, the total content of FIGURE 2. Time-course analysis of IFNAR1 and IFNAR2 subunit sur- both IFN receptor subunits was severely reduced upon LPS-in- face expression during the course of LPS-induced maturation. Immature duced maturation. To establish whether the marked reduction in DCs, generated as described in Fig. 1, were left untreated or were treated the total content of the IFN receptor chains was associated with a for the indicated times with 200 ng/ml LPS. At the end of treatment, cells decreased expression of the corresponding mRNA, we performed were labeled with the indicated Abs, ﬁxed, and analyzed by ﬂow cytom- semiquantitative RT-PCR analysis using RNA isolated from iDCs etry. Filled histograms represent background staining of isotype-matched and LPS-mDCs. As shown in Fig. 3B, comparable levels of control Ab. The Journal of Immunology 3041

of LPS and cultured until 24 or 48 h from the first stimulation, when supernatants were collected to measure the residual type I IFN production. As shown in Fig. 4, type I IFN production was barely detectable between 8 and 24 h poststimulation and was below detection limits between 24 and 48 h. These results suggest that type I IFN production is an early and transient phenomenon, starting between 2 and 4 h after LPS stimulation, peaking between 4 and 8 h, and rapidly extinguishing between 8 and 24 h. We then conducted experiments aimed at evaluating the role of LPS-induced type I IFNs in the observed IFN receptor down-modulation. To this purpose, iDCs were stimulated to undergo maturation by LPS in the presence or the absence of a mixture of IFN-␣- and IFN-␤-neutralizing Abs. As shown in Fig. 5, the marked reduction in the percentage of cells expressing IFNAR1 or IFNAR2 observed in LPS-mDCs was only partially reverted when LPS-induced type I IFNs were neutralized by specific Abs. Nota- bly, IFNAR2 expression appeared to be almost completely independent of the presence of type I IFNs. No changes were observed for IFNAR1 and IFNAR2 expression in cells cultured with a con- Downloaded from trol Ab (Fig. 5). FIGURE 3. Expression of type I IFN receptor mRNAs and proteins in iDCs and LPS-mDCs. A, Whole cell extracts (1 mg) were immunoprecipi- Endogenous type I IFNs, secreted during LPS-induced tated with anti-IFNAR1 or anti-IFNAR2 mAbs. Immunoprecipitates were maturation of iDCs, are not required for acquisition of the fully fractionated on 8% SDS-PAGE, transferred to nitrocellulose filter, and im- munoblotted with the indicated Abs. B, Total RNA was extracted from mature DC phenotype and allostimulatory capacity iDCs or LPS-mDCs and analyzed for the expression of IFNAR1 and We finally evaluated whether the LPS-induced endogenous type I http://www.jimmunol.org/ IFNAR2 mRNA by semiquantitative RT-PCR. The level of expression IFNs played a role in phenotypic and functional DC maturation. of the housekeeping gene GAPDH is shown as internal control. Immature DCs were stimulated to undergo terminal maturation by LPS in the presence of Abs directed to the IFNAR1 subunit or in the presence of a control Ab. As shown in Fig. 6A, neutralization their receptor induces IFNAR1 internalization and degradation of the biological activity of endogenous type I IFNs did not result (24), we asked whether the LPS-induced type I IFNs could be responsible for the down-modulation of the IFN receptor chains observed during DC maturation. To this aim, we initially analyzed the kinetics of type I IFN production by DCs stimulated with LPS. by guest on September 25, 2021 In our experimental cell model iDCs did not secrete any detectable level of biologically active type I IFNs (data not shown). On the contrary, biologically active type I IFNs were detected in the culture supernatant 4 h after LPS stimulation, reached a plateau at 8 h, and remained stable until 48 h poststimulation (Fig. 4). To dis- criminate between de novo production and accumulation of these cytokines in the culture medium, after supernatant collection 1, 2, 4, 8, and 24 h poststimulation, cells were reseeded in the presence

FIGURE 4. Kinetics of type I IFN production during the course of LPS- induced DC maturation. Immature DCs, generated as described in Fig. 1, FIGURE 5. Effect of type I IFN neutralization on the down-modulation were treated with LPS (200 ng/ml), and culture supernatants were collected of IFNAR1 and IFNAR2 observed in the course of LPS-induced DC mat- for IFN titration at 1, 2, 4, 8, 24, and 48 h poststimulation. Cells were then uration. Immature DCs or LPS-mDCs generated in the presence or the reseeded in the presence of LPS and cultured 24 or 48 h. At the end of the absence of Abs directed to IFN-␣ and IFN-␤ were assessed 24 h after incubation, culture supernatants were collected again and assessed for the induction of maturation for the surface expression of IFNAR1 and IFNAR2 presence of biologically active type I IFNs as described in Materials and as described in Fig. 2. Filled histograms represent background staining of Methods. A representative experiment of three performed is shown. isotype-matched control Ab. 3042 DC MATURATION AND IFN RESPONSIVENESS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Effect of Ab to IFNAR1 subunit on the phenotypic maturation of monocyte-derived DCs generated in the presence of GM-CSF/IL-4 or GM-CSF/IFN-␣. A, Peripheral blood monocytes were cultured in the presence of GM-CSF (50 ng/ml) and IL-4 (500 U/ml) for 5 days and stimulated with LPS (200 ng/ml) in the presence or the absence of 20 ␮g/ml of an anti-IFNAR1 neutralizing mAb or control IgG1. Cells were harvested 24 h later, stained with FITC-conjugated mAbs, and analyzed by ﬂow cytometry. Filled histograms represent the background staining of isotype-matched control mAbs. B, Peripheral blood monocytes were cultured in the presence of GM-CSF (50 ng/ml) and IFN-␣ (1000 IU/ml) for 3 days in the presence or the absence of 20 ␮g/ml of the anti-IFNAR1 neutralizing mAb or control IgG1. FACS analysis was performed as described in A.

in any signiﬁcant phenotypic change as the same pattern of surface Ab to GM-CSF/IFN-␣ monocyte cultures completely reversed the marker expression was found independently of the presence of the IFN-induced up-modulation of CD86 and CD83 as well as the anti-IFNAR1 Ab. Similar results were obtained when LPS matu- down-modulation of CD14. The addition of a control Ab had no ration occurred in the presence of a mixture of Abs directed to effect on the phenotypic maturation induced by IFN-␣. IFN-␣ and IFN-␤ (data not shown). To exclude that the lack of We then performed functional experiments aimed at comparing effect on the LPS-induced DC phenotypic maturation could result the ability of DCs undergoing LPS-induced maturation under ex- from incomplete blockage of IFN activity, DCs were derived from perimental conditions in which the biological activity of type I monocytes cultured with GM-CSF and IFN-␣ in the presence or IFNs was abrogated to stimulate proliferation of allogeneic T cells. the absence of the speciﬁc Ab. As previously described, this ex- As shown in Fig. 7, at all stimulator/responder ratios, LPS-mDCs perimental procedure leads to the generation of partially mature exhibited a higher capacity to stimulate the proliferation of allo- DCs expressing CD83 and exhibiting marked allostimulatory geneic lymphocytes than iDCs. LPS-mDCs generated in the pres- properties (7). As shown in Fig. 6B, the addition of anti-IFNAR1 ence of Ab to IFNAR1 showed a similar allostimulatory capacity The Journal of Immunology 3043

receptor chains. The steady-state level of the corresponding tran- scripts was not affected, suggesting that down-modulation is mediated by post-transcriptional mechanisms. Although further studies are needed to precisely define the step(s) at which receptor down-modulation takes place, our data are consistent with the ex- istence of multiple regulatory mechanisms operating during the course of DC maturation. In this regard, a common mechanism involved in the modulation of cytokine and chemokine responsiveness is represented by regulation of their receptor expression through autocrine ligand-mediated loops. With respect to the IFN system, it has been described that IFNAR1 is internalized and de- graded following ligand binding in Daudi cells (24). Moreover, we have previously reported that IFNAR1 expression is regulated in human monocytes/macrophages by differentiation-dependent post-transcriptional mechanism(s) at least in part involving intracellular se- FIGURE 7. Effect of neutralization of LPS-induced type I IFNs on the questration of receptor components (19), and more recently, an allostimulatory properties of DCs. Purified lymphocytes were seeded into ϫ 5 increase in type I IFN receptor binding sites has been observed upon 96-well microplates at a concentration of 1 10 cells/well. Allogeneic ϩ iDCs and LPS-mDCs, generated in the presence or the absence of 20 ␮g/ml in vitro differentiation of CD34 cells (27). Finally, a role for the neutralizing anti-IFNAR1 mAb, were added at graded doses to allogeneic receptor-associated Tyk2 tyrosine kinase in sustaining IFNAR1 ex- Downloaded from T lymphocytes. After 5 days, [3H]thymidine incorporation was measured pression has been demonstrated (28, 29). The finding that LPS-stim- as described in Materials and Methods. ulated DCs rapidly and transiently produce type I IFNs and that their maturation in the presence of a mixture of neutralizing Abs directed to IFN-␣ and IFN-␤ only partially restored IFNAR1 expression sug- with respect to LPS-mDCs derived in the absence of the Ab. No gests that ligand-mediated receptor internalization/degradation is one differences were observed when LPS-mDCs were generated in the of the mechanisms involved in the regulation of receptor expression http://www.jimmunol.org/ presence of a control Ab with respect to control cultures (data not during DC maturation. Although IFNAR1 down-modulation cannot shown). be explained by a reduced Tyk2 expression in LPS-mDCs, as Tyk2 levels did not change upon LPS stimulation (data not shown), a re- Discussion duced intracellular availability of Tyk2 for IFNAR1, due to compe- In this study we report that LPS-induced maturation of monocyte- tition with other cytokine receptor chains interacting with Tyk2, can- derived DCs results in impaired activation of STAT-1, -2, and -3 not be excluded (29). Notably, the expression of IFNAR1 and in response to exogenous type I IFN, which correlated with an IFNAR2 appears to be independently regulated in this cellular model. impressive and rapid down-modulation of both IFN receptor sub- In fact, neutralization of the endogenous LPS-induced type I IFNs did units, IFNAR1 and IFNAR2. The analysis of the signal transduc- not rescue the surface expression of IFNAR2. To the best of our by guest on September 25, 2021 tion pathway typically triggered through type I IFN receptor re- knowledge, this is the first report describing the differential regulation vealed that STAT-1, -2, and -3 were tyrosine phosphorylated in a of type I IFN receptor chains and an IFN-independent regulation of dose-dependent manner upon exposure to IFN-␤ in iDCs, whereas IFNAR2 expression. The observation that a fine regulation of receptor no inducible phosphorylation of any of these STATs was detected expression occurs in DCs during the course of their maturation pro- in LPS-mDCs. Moreover, STAT-1, -2, and -3 were constitutively vides a strong additional evidence on the important role that type I activated in LPS-mDCs. These observations do not necessarily im- IFNs may play in DC biology. In this regard, it is tempting to spec- ply that DC maturation results in the complete loss of type I IFN ulate that this reduced type I IFN sensitivity may contribute to protect sensitivity, but clearly indicate that the capacity of mDCs to re- mDCs from IFN-induced apoptosis. The pro-apoptotic activity of type spond to this cytokine by activating the relevant STATs is mark- I IFNs on DCs has been independently described by different groups edly reduced. This could be due to the fact that upon LPS stimu- (7, 8, 13) that observed a reduced cell recovery after DC culture in the lation a number of cytokines, including type I IFNs, are induced, presence of type I IFNs. In addition, it has been recently reported that many of which signal through the JAK/STAT pathway. This mas- type I IFNs in combination with LPS induce apoptosis of monocyte- sive activation of STATs could transiently exhaust the capacity of derived DCs (30). Consistent with these reports we reproducibly ob- mDCs to respond to cytokines through these transduction factors. served a reduced number of viable cells in DC cultures after 24 h of Cytokine-mediated JAK/STAT signaling pathways have been little LPS stimulation with respect to control iDCs. Moreover, when LPS- studied in monocyte-derived human DCs. Interestingly, it has been driven DC maturation took place in the presence of IFN-␣- and IFN- reported that LPS-mDCs fail to activate STAT-1 and -3 in re- ␤-neutralizing Abs, comparable numbers of viable cells were recov- sponse to IL-10 (25) and become unresponsive to IFN-␥ by down- ered in the two maturation stages (data not shown). Protection from regulating membrane expression of IFN-␥R1 (26), suggesting that type I IFN-induced apoptosis could be particularly important in the modulation of receptor expression and/or STAT activation may inflamed lymph node when, upon arrival of plasmacytoid DCs se- represent common mechanisms to regulate cytokine responsive- creting large amounts of type I IFNs, the local concentrations of these ness during the course of DC maturation. Of note, IFN-␥, IL-10, cytokines become very high (31, 32). and type I IFN receptors all belong to the class II cytokine receptor In the last few years the role of type I IFNs in the regulation of family and share many similarities in their structure (14). More- DC development and functional maturation has been the object of over, both IL-10 and type I IFN receptors use the same JAK, Tyk2, an intense investigation that yielded apparently contrasting results and JAK1 for signal transduction. (7Ð10, 12, 13, 23). Although many of the stimuli used for trigger- The absence of IFN-inducible STAT activation correlated with a ing DC maturation are well-known type I IFN inducers, very few clear-cut down-modulation of the membrane expression of IFNAR1 data are available in the literature about the contribution of the and IFNAR2, which occurred as early as 4 h after LPS treatment, endogenous type I IFNs to the maturation process of monocyte- and with a severe reduction of the total intracellular content of both derived DCs. In this regard, type I IFNs were characterized as 3044 DC MATURATION AND IFN RESPONSIVENESS soluble mediators released following measles virus infection of Acknowledgments monocyte-derived iDCs, which could potentially contribute to DC We thank Giorgio Trinchieri for kindly providing the IL-4 and GM-CSF. maturation. However, their neutralization only partially reversed We are indebted to Sabrina Tocchio for excellent editorial assistance, and the induction of CD86 observed in DC stimulated with superna- to Alessandro Spurio and Stefano Billi for preparing the drawings. We also tants from infected cultures, and no effect was observed on DC thank Sandra Pellegrini for critical reading of the manuscript. allostimulatory activity (33). We report here that LPS-induced endogenous type I IFNs are dispensable for accomplishment of the References 1. Lanzavecchia, A., and F. Sallusto. 2001. Regulation of T cell immunity by den- phenotypic and functional changes associated with DC maturation. dritic cells. Cell 106:263. In fact, DCs induced to terminal maturation in the presence of 2. Pulendran, B., K. Paluka, and J. Banchereau. 2001. Sensing pathogens and tuning neutralizing Abs to the IFNAR1 receptor subunit or to IFN-␣ and the immune responses. Science 293:253. 3. Belardelli, F., F. Vignaux, E. 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