Impaired IFN-γ-Dependent Inflammatory Responses in Human Keratinocytes Overexpressing the Suppressor of Signaling 1 This information is current as of September 27, 2021. Monica Federici, Maria Laura Giustizieri, Claudia Scarponi, Giampiero Girolomoni and Cristina Albanesi J Immunol 2002; 169:434-442; ; doi: 10.4049/jimmunol.169.1.434

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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

Impaired IFN-␥-Dependent Inflammatory Responses in Human Keratinocytes Overexpressing the Suppressor of Cytokine Signaling 11

Monica Federici,2 Maria Laura Giustizieri,2 Claudia Scarponi,2 Giampiero Girolomoni, and Cristina Albanesi3

Keratinocytes contribute relevantly to the pathogenesis of inflammatory skin diseases by expressing a variety of proinflammatory molecules, with T cell-derived IFN-␥ being the most potent keratinocyte activator. Suppressor of cytokine signaling (SOCS)1 and SOCS3 are negative regulators of IFN-␥ signaling and are induced in many cell types by IFN-␥ itself or by other . We show in this work that SOCS1, SOCS2, SOCS3, and cytokine-inducible SH2-containing mRNA were up-regulated by

IFN-␥ in normal human keratinocytes, whereas only SOCS1 or SOCS1 and cytokine-inducible SH2-containing protein were Downloaded from induced by TNF-␣ or IL-4, respectively. SOCS1, SOCS2, and SOCS3 were undetectable in healthy skin and highly expressed in the epidermis of psoriasis and allergic contact dermatitis, but were only weakly expressed in atopic dermatitis skin. In keratinocytes transiently transfected with SOCS1 or SOCS3 the IFN-␥-induced transactivation of an IFN-␥-responsive reporter was markedly inhibited. SOCS1 and SOCS3 overexpression in keratinocyte stable clones inhibited IFN-␥-induced phos- phorylation of IFN-␥R␣ and activation of STAT1 and STAT3. Furthermore, SOCS1 and, to a lesser extent, SOCS3 reduced membrane expression of ICAM-1 and HLA-DR, and release of IFN-␥-inducible protein-10, monokine induced by IFN-␥, and http://www.jimmunol.org/ monocyte chemoattractant protein-1 by keratinocyte clones promoted by IFN-␥. SOCS1-expressing keratinocytes showed con- stitutively higher, but not IFN-␥-inducible, IL-8 levels compared with SOCS2 and SOCS3 clones, and were resistant to IFN-␥- mediated growth inhibition. Targeting keratinocyte SOCS1 may represent a novel therapeutic approach to IFN-␥-dependent skin diseases. The Journal of Immunology, 2002, 168: 434Ð442.

hronic inflammatory skin disorders such as psoriasis, al- In the last five years, a large number of studies has focused on lergic contact dermatitis (ACD),4 and atopic dermatitis the mechanisms by which cytokine actions are negatively regu- (AD) are characterized by an intense infiltrate of acti- C lated. Suppressors of cytokine signaling (SOCS) are a family of by guest on September 27, 2021 vated T lymphocytes, which release lymphokines influencing the intracellular molecules comprising at least eight members, SOCS1 immune functions of resident skin cells, including keratinocytes to SOCS7 and cytokine-inducible SH2-containing protein (CIS), (1Ð3). Among T cell-derived cytokines, IFN-␥ is the most potent which share structural similarities and are characterized by a cen- activator of the proinflammatory functions of keratinocytes. In tral SH2 domain and a unique motif, the SOCS box, in their C- fact, IFN-␥-activated keratinocytes express a broad array of che- terminal region. SOCS molecules have been detected in various mokines, cytokines, and membrane molecules that direct the re- tissues and are produced in response to different cytokines. SOCS cruitment, activation, and retention of specific leukocyte subpopu- regulate the magnitude and duration of responses triggered by var- ␥ lations in the skin (4Ð6). Although less potently than IFN- , ious cytokines by inhibiting their signal transduction pathway in a ␣ TNF- and IL-4 can also activate keratinocytes. The reciprocal classic negative feedback loop (7Ð9). At the molecular level, activation of T lymphocytes and keratinocytes has a primary role SOCS/CIS bind directly to cytokine receptors or to the catalytic in the amplification of skin inflammation during immune-mediated domain of (Jak) proteins and impede the recruitment skin diseases. and phosphorylation of STAT (10). SOCS1 induction by IFN-␥ and negative regulation of the IFN-␥ signaling by SOCS1 have been well documented in a wide variety of cell types, including Laboratory of Immunology, Istituto Dermopatico dell’ Immacolata, Istituto di Ricov- M1, HeLa, bone marrow cells, and monocytes (11Ð13). In partic- ero e Cura a Carattere Scientifico, Rome, Italy ular, SOCS1 inhibits IFN-␥ signaling by binding as a pseudosub- Received for publication February 8, 2002. Accepted for publication April 19, 2002. strate to Jak1 and Jak2, which are associated, respectively, with the The costs of publication of this article were defrayed in part by the payment of page IFN-␥R ␣ and ␤ subunits. Disabled Jak1 and Jak2 cannot mediate charges. This article must therefore be hereby marked advertisement in accordance STAT1 phosphorylation, which is necessary for activation of ␥-ac- with 18 U.S.C. Section 1734 solely to indicate this fact. tivated sequences (GAS) in the promoters of target (8, 10, 1 This work was supported by grants from the Italian Ministry of Health. 14). Moreover, constitutive activation of STAT1 and hyperrespon- 2 M.F., M.L.G., and C.S. contributed equally to this paper. siveness to IFN-␥ were found in SOCS1-deficient mice (15). Also, 3 Address correspondence and reprint requests to Dr. Cristina Albanesi, Laboratory of SOCS3 represses signaling induced by IFN-␥, although its inhib- Immunology, Istituto Dermopatico dell’Immacolata, Via Monti di Creta, 104, 00167 Rome, Italy. E-mail address: [email protected] itory activity toward STAT1 activation is weaker than that exhib- 4 Abbreviations used in this paper: ACD, allergic contact dermatitis; AD, atopic der- ited by SOCS1 (12). matitis; PS, phosphoserine; MCP, monocyte chemoattractant protein; Mig, monokine Although the molecular bases of SOCS molecule activity have induced by IFN-␥; SOCS, suppressor of cytokine signaling; CIS, cytokine-inducible SH2 protein; Jak, Janus kinase; GAS, ␥-activated sequence; IP-10, IFN-␥-inducible been extensively investigated, limited information exists on in- protein-10; PY, phosphotyrosine. flammatory mediators affected by SOCS (16, 17) and on SOCS

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 435 expression in human pathologic conditions (18). In this study, we oxidase system and 3-amino-9-ethylcarbazole chromogen (Vector Labora- sought to determine whether SOCS family members are expressed tories). Sections were counterstained with Mayer’s hematoxylin. As by normal human keratinocytes in vitro and in vivo during im- negative controls, primary Abs were omitted or replaced with control serum. mune-mediated diseases. Furthermore, through establishment of SOCS1-, SOCS2-, and SOCS3-expressing keratinocyte clones, we SOCS plasmids investigated the potential role of SOCS molecules in inhibiting Myc-tagged full-length murine JAK-binding protein/SOCS1 and human IFN-␥ signal transduction as well as IFN-␥-induced production of SOCS2 or SOCS3 in pcDNA3 plasmid (pcDNA-myc/SOCS1-2-3) were a chemokines and adhesion molecules. generous gift of Dr. A. Yoshimura (Kyushu University, Fukuoka, Japan). FLAG epitope-tagged murine SOCS1, SOCS2, and SOCS3 in pEF-BOS expression vector (pEF-FLAG/SOCS1-2-3) were kindly provided by Dr. Materials and Methods D. J. Hilton (Walter and Eliza Hall Institute for Medical Research, Cell cultures and treatments Parkville, Victoria, Australia). Normal human keratinocytes were prepared from plastic surgery skin ob- Transient and stable transfectants tained from healthy individuals (n ϭ 3), as described previously (4). Sec- ond- or third-passage keratinocytes were used in all experiments, with cells Normal human keratinocytes and HaCaT cells were transiently transfected cultured in six-well plates in the serum-free medium, keratinocyte growth in duplicate using Lipofectin and LipofectAMINE PLUS reagents (Invitro- medium (Clonetics, San Diego, CA), for at least 3Ð5 days (at 60Ð80% gen), respectively. Typically, 1.5Ð2 ϫ 105 cells were seeded in six-well confluence) before performing cytokine treatment. Stimulation with 200 plates 24Ð48 h before transfection (60Ð80% confluence). For each well, U/ml human rIFN-␥, 50 ng/ml rTNF-␣, or 50 ng/ml rIL-4 (R&D Systems, 0.5 ␮g pCMV ⅐ SPORT-␤-galactosidase plasmid (Invitrogen) and 0.5 ␮g Abingdon, Oxon, U.K.) was performed in keratinocyte growth medium pGAS-Luc or pNF-␬B-Luc vectors (Stratagene) were cotransfected with devoid of hydrocortisone and bovine pituitary extract, and supplemented increasing concentrations (0.1Ð2 ␮g) of pcDNA3-myc/SOCS1-2-3 or pEF- Downloaded from with 0.1% BSA (Sigma-Aldrich, Milan, Italy). The HaCaT human kera- FLAG/SOCS1-2-3 plasmid sets. Increasing concentrations of pcDNA3 and tinocyte cell line was a gift from N. E. Fusenig (Deutsches Krebsforschungs- pEF-BOS empty vectors were also used as controls. After overnight cul- zentrum, Heidelberg, Germany) and was grown in DMEM (Biochrom, ture, the cells were incubated for 24 h with 200 U/ml IFN-␥ in serum-free Berlin, Germany) supplemented with 10% Fetalclone II serum (HyClone medium and then lysed. ␤-galactosidase and luciferase activities were mea- Laboratories, Logan, UT). When 60Ð80% confluence was achieved, Ha- sured using the ␤-Gal ELISA (Boehringer Mannheim, Mannheim, Ger- CaT cells were stimulated with 200 U/ml IFN-␥. many) and luciferase assay system (Promega, Madison, WI), respectively. ␤ Luciferase activity of each sample was normalized to the -galactosidase http://www.jimmunol.org/ RNase protection and Northern blot analysis activity, and its basal level, in the absence of IFN-␥ and the different SOCS constructs, was given the value of 1. To confirm that increasing the con- Total RNA was extracted from cultured keratinocytes using the TRIzol centration of transfected SOCS plasmids resulted in an increased expres- reagent (Invitrogen, Carlsbad, CA). The human SOCS multiprobe template sion of SOCS proteins, lysates were analyzed by Western blot using anti- set and the complete kit for RNase protection assay were purchased from c-myc 9E10 mAb (Santa Cruz Biotechnology) or anti-FLAG M2 mAb BD PharMingen (San Diego, CA). ␣-32P-Labeled antisense riboprobes (Sigma-Aldrich). SOCS HaCaT cells were permanently transfected with were generated from DNA corresponding to CIS, SOCS7, SOCS6, SOCS5, pcDNA-myc/SOCS1-2-3 or empty pcDNA3 plasmids linearized by ScaI SOCS3, SOCS2, and SOCS1, as well as the housekeeping genes L32 and restriction endonuclease (Boehringer Mannheim). Genetycin-resistant GAPDH, and were in vitro transcribed in the presence of a GACU nucle- clones were selected after ϳ20 days by adding 0.4 mg/ml G418 (Invitro- otide pool precursors using a T7 RNA polymerase. Hybridization of ribo- ␮ gen) to the culture medium. HaCaT clones expressing SOCS1-2 or -3 pro-

probes with 10 g of each RNA sample was performed overnight, followed by guest on September 27, 2021 teins were selected by Western blot analysis with the anti-c-myc Ab. by digestion with RNase A and T1. The samples were treated with pro- teinase K, extracted with Tris-saturated phenol plus chloroform:isoamyl Western blot and immunoprecipitation alcohol (50:1), and finally precipitated in the presence of ammonium ac- etate. Protected fragments were resolved by electrophoresis on 4.5% poly- Protein extracts were prepared by solubilizing cells in RIPA buffer (1% acrylamide-urea gel. For Northern analysis, 5 ␮g of total RNA was frac- Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) containing a tionated on 1% formaldehyde-agarose denaturing gels, blotted on nylon mixture of protease and phosphatase inhibitors. Proteins were subjected to membrane (Amersham Pharmacia Biotech, Milan, Italy), and fixed by UV SDS-PAGE and transferred to polyvinylidene difluoride membrane. Mem- irradiation. SOCS2 probe was obtained by RT-PCR performed on RNA branes were then blocked and probed with various primary Abs diluted in isolated from IFN-␥-stimulated keratinocyte cultures, and using the fol- PBS containing 5% nonfat dried milk or 3% BSA. The latter were as lowing primer pairs: TATCAGGATGGTACTGGGGAAGTA (5Ј) and follows: anti-STAT1 (E-23; Santa Cruz Biotechnology), anti-STAT3 (C20; CTTGTTGGTAAAGGCAGTCCCCAG (3Ј) (GenBank accession no. Santa Cruz Biotechnology), anti-phosphotyrosine (PY) STAT1 (Tyr701) AF037989). SOCS2 amplificate was gel purified, cloned into pCR-TOPO and STAT3 (Tyr705) (both from New England Biolabs, Beverly, MA), vector (Invitrogen), and then subjected to an automated sequence analysis anti-phosphoserine (PS) STAT1 (Ser727; Upstate Biotechnology, Lake using a PerkinElmer Sequencer (model ABI Prism 377 XL; PerkinElmer, Placid, NY), anti-SOCS1 and anti-SOCS3 (both from Immuno-Biological Roche Molecular Systems, Branchburg, NJ). SOCS2 probe was labeled Laboratories), anti-SOCS2 (M-19; Santa Cruz Biotechnology), and HRP- with [32P]dCTP and used for hybridization conducted for1hat68¡Cin conjugated anti-c-myc (9E10; Santa Cruz Biotechnology). Filters were QuickHyb solution (Stratagene, La Jolla, CA), as per the manufacturer’s properly developed with anti-mouse, anti-goat, or anti-rabbit Ig Abs con- protocol. The filters were washed two times at room temperature and once jugated to HRP using the ECL-plus detection system (Amersham Pharma- at 60¡C under high-stringency conditions (0.1ϫ SSC, 0.1% SDS) and cia Biotech), followed by autoradiography. For immunoprecipitation, pro- finally exposed at Ϫ80¡C to Kodak Biomax MS-1 films (Kodak, Roches- tein extracts were incubated with protein G-Sepharose beads (Amersham ter, NY). Pharmacia Biotech) and anti-Jak1, anti-Jak2 (Upstate Biotechnology), and anti-IFN-␥R␣ subunit (C-20; Santa Cruz Biotechnology) polyclonal Abs. Immunohistochemistry Immunoprecipitates were run on 8% SDS-PAGE and probed on polyvi- nylidene difluoride filters with the 4G10 anti-PY (Upstate Biotechnology) Four-millimeter punch biopsies were taken from skin of adult patients with or with anti-c-myc Abs. chronic plaque psoriasis (n ϭ 5) and chronic AD (n ϭ 5) and from healthy control subjects (n ϭ 3). Skin biopsies were also taken from 48-h positive ELISA patch test reactions to NiSO4 from four patients with ACD to nickel. Pa- tients were not receiving any systemic or topical therapy before sampling. IFN-␥-inducible protein-10 (IP-10)/CXCL10 was assayed using the puri- Five-micrometer cryostatic sections were fixed with 5% paraformaldehyde fied 4D5/A7/C5 and the biotinylated 6D4/D6/G2 Abs (BD PharMingen). for 10 min, treated with 0.3% hydrogen peroxide to quench endogenous Monokine induced by IFN-␥ (Mig)/CXCL9 was determined using the Ab peroxidase activity, incubated with normal horse serum (Vectastain ABC pair, 2310D mAb for coating and biotinylated B8-6 (BD PharMingen) for kit; Vector Laboratories, Burlingame, CA) for 20 min, and finally perme- detection. Human recombinant chemokines (BD PharMingen) were used as abilized with 0.05% Triton X-100. Staining was performed with goat poly- standards. IL-8 (CXCL8) and monocyte chemoattractant protein (MCP)- clonal anti-SOCS1 (15 ␮g/ml; C-20; Santa Cruz Biotechnology, Santa 1/CCL2 were measured with OptEIA kits (BD PharMingen), as per the Cruz, CA) and anti-SOCS2 (10 ␮g/ml; M-19; Santa Cruz Biotechnology), manufacturer’s protocol. The plates were analyzed in an ELISA reader (model and rabbit anti-SOCS3 (5 ␮g/ml; Immuno-Biological Laboratories, Ham- 3550 UV; Bio-Rad, Valencia, CA). Cultures were conducted in triplicate for burg, Germany). Immunoreactivity was revealed using avidin-biotin-per- each condition. Results are given as nanograms/106 cells Ϯ SD. 436 SOCS1 INHIBITS IFN-␥ SIGNALING IN HUMAN KERATINOCYTES

Flow cytometry analysis SOCS2-specific probe. As shown in Fig. 1B, keratinocytes tran- ␥ HaCaT clones were stained with FITC-conjugated anti-CD54 (84H10; Im- siently up-regulated SOCS2 only after IFN- stimulation, with munotech, Marseille, France) and anti-HLA-DR (L243; BD PharMingen) mRNA peaking at 2 h after treatment and returning to basal levels mAbs. In control samples, staining was performed using isotype-matched within 10 h. Similarly, IFN-␥-induced expression of SOCS3 and control Abs. Cells were analyzed with a FACScan equipped with CellQuest CIS was detectable as early as 30 min after stimulation, peaked at software (BD Biosciences, Mountain View, CA). 2 h, and decreased thereafter. Finally, IL-4 strongly increased CIS Cell proliferation analysis mRNA expression, which persisted for at least 48 h upon treatment. A total of 104 cells were seeded in 24-well plates in triplicate for each condition, and after 3 days medium was changed with fresh medium with SOCS1, SOCS2, and SOCS3 are overexpressed in the epidermis or without 200 U/ml IFN-␥. Clones were cultured for 1Ð7 days and the number of viable cells was determined by a trypan blue exclusion test. of psoriasis and ACD lesion Experiments were performed on three SOCS1, SOCS2, SOCS3, or control To see whether keratinocytes express SOCS molecules in vivo, we clones. examined the presence of SOCS1, SOCS2, and SOCS3 proteins in Statistical analysis normal skin from healthy subjects and in the skin affected with psoriasis, ACD to nickel and AD. As shown in Fig. 2, AÐC, ker- Wilcoxon’s signed rank test (SigmaStat; Jandel, San Rafael, CA) was used to compare differences in luciferase activity of transiently transfected ker- atinocytes of healthy skin did not stain for SOCS1, SOCS2, or atinocytes and chemokine release from SOCS clones. Values of p Յ 0.05 SOCS3, although rare and scattered immunoreactive cells were were considered significant. present in the epidermis. In contrast, psoriatic epidermis showed a diffuse and intense SOCS1, SOCS2, and SOCS3 reactivity, with Downloaded from Results keratinocytes displaying a cytoplasmic staining more evident in Expression pattern of SOCS genes in cultured human the basal and suprabasal epidermal layers (Fig. 2, DÐF). Keratin- keratinocytes ocytes in ACD reactions also stained for SOCS1, SOCS2, and The presence of SOCS mRNA was analyzed in normal human SOCS3 molecules, with the staining more intense in discrete areas keratinocytes in resting conditions and upon IFN-␥, TNF-␣,or of the basal and suprabasal layers (Fig. 2, GÐI). Finally, epidermis IL-4 treatment (Fig. 1A). Low levels of SOCS1, SOCS2, SOCS5, of chronic AD skin showed only some restricted areas of faint http://www.jimmunol.org/ SOCS6, SOCS7, and CIS mRNA were found to be constitutively positivity for SOCS molecules (Fig. 2, JÐL). A portion of leuko- expressed by keratinocytes. Expression of SOCS1, SOCS2, cytes infiltrating the dermis in all three diseases was also immu- SOCS3, and CIS was markedly increased following IFN-␥ stim- noreactive for SOCS1, SOCS2, and SOCS3. Nonlesional skin ulation, whereas only SOCS1 or SOCS1 and CIS were signifi- from patients with psoriasis or AD did not stain for SOCS mole- cantly up-regulated in keratinocytes exposed to TNF-␣ or IL-4, cules (data not shown). respectively. In contrast, SOCS5Ð7 were not affected by cytokine Forced expression of SOCS1 and SOCS3, but not SOCS2, ␥ ␣ treatments. Induction of SOCS1 mRNA by IFN- , TNF- , or IL-4 inhibits the IFN-␥-induced transactivation of a STAT1-binding was increased soon after a 1-h activation and was maintained at a promoter in keratinocytes by guest on September 27, 2021 high level for at least 48 h. Because riboprobe for RNase protec- ␣ tion did not permit clear evaluation of SOCS2 expression in ker- Although SOCS molecules may also be involved in TNF- or atinocytes, mRNA was also analyzed by Northern blot using a IL-4-mediated keratinocyte activation, we concentrated on the role of SOCS molecules in the regulation of IFN-␥ signaling, because IFN-␥ is the most powerful proinflammatory cytokine on keratin- ocytes. To this end, keratinocytes were cotransfected with the IFN- ␥-inducible reporter plasmid, pGAS-Luc, in the absence or pres- ence of increasing concentrations (0.1Ð2 ␮g/well) of pcDNA3- myc/SOCS1-2-3 or empty pcDNA3 vectors. The pGAS-Luc reporter gene contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of the GAS, which are a prototypical STAT1-binding site (14). Ker- atinocytes transfected with the pGAS-Luc and stimulated with IFN-␥ showed a 20-fold increase in luciferase activity (Fig. 3). Transfection of SOCS1 decreased the luciferase activity of pGAS- Luc plasmid very efficiently and in a dose-dependent manner (Fig. 3B; p Ͻ 0.002). Also, SOCS3 overexpression reduced IFN-␥ sig- naling in keratinocytes, although less potently than SOCS1 (Fig. 3C; p Ͻ 0.03). In contrast, SOCS2 or pcDNA3 empty vector did not affect the activation of luciferase gene transcription by IFN-␥ (Fig. 3, A and D). An identical inhibitory pattern by SOCS on IFN-␥ signaling was obtained when the pEF-FLAG/SOCS1-2-3 plasmid set was used to transfect keratinocytes (data not shown). To test whether the SOCS1- or SOCS3-dependent inhibition of FIGURE 1. SOCS mRNA induction in human keratinocytes activated IFN-␥ signaling was exerted specifically on STAT1-activated with IFN-␥, TNF-␣, or IL-4. Keratinocyte cultures were treated for the ␬ ␥ ␣ pathway, we also used the pNF- B-Luc reporter plasmid that is indicated time periods with human rIFN- (100 U/ml), rTNF- , or rIL-4 ␬ (both at 50 ng/ml). Total RNA was used in RNase protection assay con- activated specifically by NF- B transcription factor. Luciferase ␥ ducted with a human SOCS multiprobe template set as well as the house- levels resulting from transfection of IFN- -treated keratinocytes keeping gene L32 (A). Total RNA were also analyzed by Northern blot with pNF-␬B-Luc were lower (6-fold induction) compared with using a SOCS2-specific probe. Eight-hour exposures of films for both that obtained with pGAS-Luc, and, more importantly, did not RNase protection and Northern blot were conducted. change in keratinocytes overexpressing SOCS1, SOCS2, or The Journal of Immunology 437

FIGURE 2. SOCS expression in in- flammatory skin lesions. Immunohisto- chemistry was performed on frozen sec- tions from normal skin (AÐC), psoriasis vulgaris (DÐF), 48-h patch test reactions to nickel sulfate (GÐI), and AD lesions (JÐL). SOCS reactivity (red staining) was revealed by using avidin-biotin-peroxi- dase complexes and 3-amino-9-ethylcar- bazole as substrate. Normal skin did not stain for SOCS1 (A), SOCS2 (B), or SOCS3 (C) molecules. SOCS1 positivity Downloaded from was found in keratinocytes of psoriasis (D), ACD (G), and, to a lesser extent, AD lesions (J). A similar pattern of expres- sion was revealed for SOCS2 (E, H, and K) and SOCS3 (F, I, and L). Some infil- trating dermal leukocytes were also reac-

tive for SOCS in all the diseases exam- http://www.jimmunol.org/ ined. Sections were counterstained with Mayer’s hematoxylin. Similar results were observed in skin biopsies from five patients. by guest on September 27, 2021

SOCS3 (Fig. 3E). Thus, SOCS1 and SOCS3, but not SOCS2, im- imal steps of IFN-␥ signaling appear to be identical in normal pair the ability of IFN-␥ to transactivate luciferase keratinocytes and HaCaT cells. from STAT1- but not NF-␬B-binding promoters in keratinocytes. Inhibition of the IFN-␥-dependent phosphorylation of IFN-␥R␣ Early molecular responses to IFN-␥ in HaCaT cells, and subunit, STAT1, and STAT3 in keratinocytes overexpressing establishment of SOCS-expressing HaCaT clones SOCS1 and SOCS3 To study the functional role of SOCS proteins in IFN-␥ signaling SOCS1 and SOCS3 have been demonstrated to inhibit signaling in keratinocytes, the keratinocyte-like cell line HaCaT was stably induced by IFN-␥ by inactivating Jak-mediated phosphorylation transfected with SOCS1, SOCS2, or SOCS3 cDNAs. HaCaT and homodimerization of STAT proteins (19, 20). After a 5-min clones permanently expressing SOCS were generated by transfect- stimulation with IFN-␥, Jak1 and Jak2 became phosphorylated in ing cells with linearized pcDNA3-myc/SOCS1-2-3 and, as control, control keratinocyte clones, as assessed by immunoprecipitation empty pcDNA3 vector. Genetycin-resistant clones were screened experiments (Fig. 5). Unexpectedly, only a slight reduction of Jak1 for SOCS expression by Western blot analysis with the anti-c-myc phosphorylation was observed in SOCS1 and SOCS3 clones com- Ab, and 20 SOCS1 and SOCS2 clones and nine SOCS3 clones pared with controls or SOCS2 clones (Fig. 5A). Consistently, Jak2 were obtained and included in this study. Before establishing phosphorylation was unaffected in SOCS1 and SOCS3 clones (Fig. clones, HaCaT cells were analyzed and compared with normal 5B). Although we did not observe a significant down-regulation of keratinocytes in SOCS expression and IFN-␥-induced Jak/STAT Jak activation, SOCS molecules bound tightly to Jak1 and Jak2 signaling pathways. IFN-␥ promoted the same pattern of SOCS because they were coimmunoprecipitated by anti-Jak1 or Jak2 Abs gene expression in HaCaT cells and normal keratinocytes, and (Fig. 5). The molecular mechanisms by which SOCS inhibit Jak IFN-␥ signaling was inhibited in transiently transfected HaCaT by activity are not fully understood. It has been hypothesized that SOCS1 and SOCS3, but not by SOCS2 (data not shown). Like SOCS molecules can function as pseudosubstrates for Jaks, which normal keratinocytes, 5 min after IFN-␥ treatment HaCaT cells in turn cannot phosphorylate their natural substrates (20). A strong showed Jak1, Jak2, and IFN-␥R␣ subunit phosphorylated in ty- inhibition of IFN-␥R␣ phosphorylation was observed in SOCS1 rosine residues (Fig. 4). In both keratinocytes and HaCaT cells, and, to a lower extent, in SOCS3 clones. In contrast, we could not STAT1 and, to a lesser degree, STAT3 proteins became phosphor- detect any changes in the phosphorylation of IFN-␥R␣ of SOCS2 ylated 15 min after IFN-␥ stimulation (Fig. 4). Therefore, the prox- clones (Fig. 6). These data were confirmed in four SOCS1 and 438 SOCS1 INHIBITS IFN-␥ SIGNALING IN HUMAN KERATINOCYTES

FIGURE 4. The proximal steps of IFN-␥ signaling are identical in nor- mal keratinocytes and HaCaT cells. Cultured keratinocytes and HaCaT cells were left untreated or stimulated with 200 U/ml IFN-␥ for 5Ð15 min.

Lysates were immunoprecipitated with anti-Jak1, -Jak2, and -IFN-␥R␣ Downloaded from Abs and Western blotted with anti-PY Ab. Lysates were also subjected to Western blot analysis performed with anti-PY-STAT1, -PS-STAT1, and -PY-STAT3 Abs. The positions of activated Jak1 (130 kDa), Jak2 (130 kDa), IFN-␥R␣ (90 kDa), STAT1 (91 and 84 kDa), and STAT3 (92 kDa) are indicated. http://www.jimmunol.org/

␥ FIGURE 3. SOCS1 and SOCS3 forced expression strongly reduces the IFN- -induced ICAM-1 and HLA-DR membrane molecules and IFN-␥-induced activation of a STAT1-responsive reporter plasmid in tran- chemokine production are inhibited in SOCS1 keratinocyte siently transfected keratinocytes. Cultured human keratinocytes were co- clones ␮ transfected with increasing amounts (0Ð2 g/well) of pcDNA3 (A), IFN-␥-activated keratinocytes express membrane Ags, such as pcDNA3-myc-SOCS1 (B), -SOCS3 (C), or -SOCS2 (D) vectors and 0.5 ␮g ICAM-1 and HLA-DR (5, 21), and a broad array of chemokines, pGAS-Luc reporter plasmid. Keratinocytes were also cotransfected with a ␮ including MCP-1, IP-10, Mig, and IL-8 (4Ð6). Because many of

fixed amount (1 g/well) of pcDNA3, pcDNA3-myc-SOCS1, -SOCS2, and by guest on September 27, 2021 ␥ -SOCS3, and 0.5 ␮g pNF-␬B-Luc reporter plasmid (E). After overnight the IFN- -induced genes in keratinocytes are transcriptionally reg- culture, cells were incubated for 24 h with 200 U/ml IFN-␥ and then lysed ulated by STAT1 (22, 23) and STAT1 is disabled in SOCS1 and to determine ␤-galactosidase and luciferase activities. Luciferase activity SOCS3 keratinocyte clones, we tested whether SOCS clones could of each sample was normalized to the ␤-galactosidase activity, and its basal still express inflammatory mediators in response to IFN-␥. Flow level, in the absence of IFN-␥ and the different SOCS constructs, was given cytometry analysis revealed that all 20 SOCS1 clones examined the value of 1. showed a significant reduction (50Ð65%) of ICAM-1 and an al- most complete abrogation of HLA-DR expression (Fig. 8). Al- though less efficiently, ICAM-1 and HLA-DR were reduced also in some (four of nine) SOCS3 clones, whereas SOCS2 expressed three SOCS2 or SOCS3 clones. As a consequence of the inhibition ICAM-1 and HLA-DR levels comparable to that of control (Fig. of IFN-␥R␣ phosphorylation, STAT1 and STAT3 proteins cannot 8). Interestingly, in some SOCS2-expressing clones (4 of 20) a be recruited and activated in IFN-␥-stimulated SOCS1 and SOCS3 superinduction of ICAM-1 and HLA-DR was revealed (data not transfectants (Fig. 7). Following IFN-␥ stimulation, control and shown). Similar results were obtained when we tested chemokine SOCS2 keratinocyte clones phosphorylated latent STAT1 at both content in supernatants from IFN-␥-activated keratinocyte clones. tyrosine 701 and serine 727 residues. While tyrosine phosphory- IP-10, Mig, and MCP-1 release was greatly impaired in all SOCS1 lation of STAT1 occurred in keratinocytes only upon treatment clones, with 86Ð90% reduction for IP-10 and 75Ð80% reduction with IFN-␥ (Fig. 7A), serine phosphorylation of STAT1 was con- for Mig and MCP-1 (Fig. 9; p Ͻ 0.002). A 50Ð80% and 44Ð80% stitutive and up-regulated by IFN-␥ (Fig. 7B). Low amounts of reduction of IP-10 and Mig, respectively, was observed in five of tyrosine phosphorylated STAT3 were also detected in keratino- nine IFN-␥-stimulated SOCS3 clones ( p Ͻ 0.05). In contrast, cytes following IFN-␥ stimulation (Fig. 7C). The IFN-␥-induced MCP-1 release by SOCS3 clones was similar to that of controls. STAT1 and STAT3 activation was greatly inhibited in all keratin- Chemokine release was not affected in the majority of IFN-␥-stim- ocyte clones expressing SOCS1 (Fig. 7, AÐC). Six of nine SOCS3 ulated SOCS2 clones (Fig. 9). Similar to what we observed for stable clones showed a significant reduction of STAT1 and STAT3 ICAM-1 and HLA-DR expression, four SOCS2-expressing clones phosphorylation in response to IFN-␥, whereas no differences with showed an enhanced release of IP-10, Mig, and MCP-1 compared controls were observed for cells permanently transfected with with controls (data not shown). SOCS clones not exposed to IFN-␥ SOCS2 (Fig. 7, AÐC). Keratinocyte clones expressed comparable secreted very limited amounts of IP-10, Mig, and MCP-1 (data not levels of SOCS1, SOCS2, and SOCS3 proteins (Fig. 7D). Taken shown). Unexpectedly, IL-8 release was higher in IFN-␥-stimu- together, these results indicate that SOCS1 and, less efficiently, lated SOCS1 clones compared with control and SOCS2 or SOCS3 SOCS3 block the IFN-␥-dependent phosphorylation of IFN-␥R␣ clones (Fig. 10A). However, a more detailed study of IL-8 revealed by binding to Jak1 and Jak2, and thus prevent STAT1 and STAT3 that unstimulated SOCS1 clones secreted higher levels of IL-8 activation in keratinocytes. compared with control, SOCS2, or SOCS3 clones (3.2Ð6vs The Journal of Immunology 439 Downloaded from

FIGURE 6. Keratinocytes permanently transfected with SOCS1 and SOCS3, but not with SOCS2, do not phosphorylate the IFN-␥R␣ subunit in response to IFN-␥. Clones were left untreated or stimulated with 200 U/ml IFN-␥ for 5 min before lysis. Immunoprecipitation of samples with ␥ ␣ the IFN- R chain Ab was followed by Western blot analysis with anti-PY http://www.jimmunol.org/ Ab, and the membranes were stripped and reprobed with the anti-IFN-␥R␣ subunit Ab.

trast, a dramatic and progressive reduction of cell number was observed in control, SOCS2, and SOCS3 clones following IFN-␥ stimulation (Fig. 11).

FIGURE 5. Jak1 and Jak2 phosphorylation status does not significantly by guest on September 27, 2021 change in IFN-␥-stimulated keratinocytes permanently transfected with SOCS1, SOCS2, or SOCS3. pcDNA3, SOCS1, SOCS2, and SOCS3 clones were left untreated or stimulated with 200 U/ml IFN-␥ for 5 min. Lysates were immunoprecipitated with the anti-Jak1 (A) or anti-Jak2 (B) Abs and subjected to Western blot analysis with anti-PY Ab. To confirm the pres- ence of similar amounts of Jak proteins in all samples, anti-PY-stained filters were stripped and reprobed with anti-Jak1 or anti-Jak2 Abs. SOCS1, SOCS2, and SOCS3 binding to Jak proteins were assessed by probing filters of blotted Jak1 and Jak2 immunoprecipitates also with the anti-myc Ab. Myc-tagged SOCS1, SOCS2, and SOCS3 showed molecular masses of 35, 34, and 40 kDa, respectively. Similar results were obtained with four SOCS1 and three SOCS2 or SOCS3 clones.

0.6Ð1.6 ng/106 cells; p Ͻ 0.01). After IFN-␥ stimulation, IL-8 secretion was not up-regulated in SOCS1 clones, whereas it was significantly enhanced in control, SOCS2, or SOCS3 clones (Fig. 10B). Therefore, SOCS1 overexpression in keratinocytes can en- hance IL-8 production through a STAT1-independent mechanism, and, at the same time, can block the IFN-␥-induced IL-8 up-reg- ulation in a STAT1-dependent manner.

SOCS1 prevents the IFN-␥-induced inhibition of keratinocyte growth Other than inducing proinflammatory genes, IFN-␥ exerts an an- FIGURE 7. Inhibition of STAT1 and STAT3 activation induced by IFN-␥ in keratinocytes permanently transfected with SOCS1 and SOCS3, tiproliferative activity on a number of cell types including kera- but not with SOCS2. Control and SOCS clones were untreated or stimu- tinocytes, and STAT1 activation is required for mediating this ef- lated with 200 U/ml IFN-␥ for 15 min. Western blot analyses were per- ␥ fect (24). The antiproliferative activity of IFN- was completely formed on lysates using anti-PY-STAT1 (A), anti-PS-STAT1 (B), and anti- abolished in SOCS1 clones. In fact, the number of unstimulated PY-STAT3 (C). Each filter was stripped and reprobed with Abs against the and IFN-␥-stimulated SOCS1-expressing keratinocytes was com- nonphosphorylated forms of STAT1 and STAT3. SOCS levels in SOCS1, parable over all the time points of experiments (Fig. 11). In con- SOCS2, and SOCS3 clones were detected using the anti-c-myc 9E10 Ab (D). 440 SOCS1 INHIBITS IFN-␥ SIGNALING IN HUMAN KERATINOCYTES Downloaded from

FIGURE 8. Keratinocytes permanently transfected with SOCS1 and SOCS3, but not with SOCS2, express reduced ICAM-1 and HLA-DR fol- lowing IFN-␥ stimulation. Control and SOCS1, SOCS2, or SOCS3 clones were analyzed for ICAM-1 and HLA-DR expression by flow cytometry http://www.jimmunol.org/ 48 h after treatment with medium alone or 200 U/ml IFN-␥. A, Histogram plots relative to ICAM-1 and HLA-DR expression by representative con- trol and SOCS clones. Thin lines represent staining with matched isotype Ig, whereas gray and bold lines represent staining of unstimulated and IFN-␥-treated cells, respectively. The x-axis and the y-axis indicate the relative cell number and fluorescence intensity, respectively. The numbers indicate the net mean fluorescence intensity of IFN-␥-treated samples. B, Net mean fluorescence intensities (⌬MFI) for ICAM-1 and HLA-DR ex- pression of two control, four SOCS1, three SOCS2, and three SOCS3 by guest on September 27, 2021 clones after IFN-␥ treatment.

Discussion In this study we focused on the role of the cytokine negative reg- ulators SOCS in IFN-␥-driven activation of keratinocytes. Under- standing the mechanisms by which IFN-␥ signaling is switched off in keratinocytes is fundamental because this cytokine potently transforms keratinocytes in strong producers of inflammatory me- FIGURE 9. Keratinocyte clones overexpressing SOCS1 show an im- diators during immune-mediated skin diseases (4Ð6). That IFN-␥ paired IFN-␥-induced release of IP-10, Mig, and MCP-1. Supernatants activates keratinocytes in a dominant fashion compared with from IFN-␥-treated keratinocyte cultures were tested by ELISA for IP-10 ␣ TNF- and IL-4 becomes evident also from SOCS mRNA analysis (A), Mig (B), and MCP-1 (C) content. Data are expressed as mean nano- upon cytokine treatment. IFN-␥ induced SOCS1, SOCS2, SOCS3, grams per 106 cells Ϯ SD of triplicate cultures. and CIS in keratinocytes, whereas TNF-␣ promoted only SOCS1 and IL-4 promoted only SOCS1 and CIS. A high expression of SOCS1, SOCS2, and SOCS3 molecules was also found in epider- lation and triggering of STAT1 and small amounts of STAT3. mal keratinocytes of psoriasis and ACD lesions, whereas chronic Once phosphorylated in tyrosine 701 and serine 727 residues, AD skin showed only a faint epidermal staining. These differences STAT1 is fully active and can thus induce a variety of inflamma- are likely due to the prominent infiltration of IFN-␥-producing tory genes. For instance, ICAM-1, class II transactivator, IP-10, type 1 lymphocytes in ACD and psoriasis compared with AD (1, Mig, and MCP-1 genes are all strongly expressed by IFN-␥-treated 3). Indeed, studies performed on IFN-␥-stimulated keratinocytes keratinocytes and tightly regulated at the transcriptional level by cultured from normal-appearing skin of healthy subjects and pa- STAT1 (22, 23, 25Ð27). Regulation by STAT1 was also observed tients with psoriasis or AD demonstrated no significant differences for several cell cycle and proapoptotic genes, including cyclin ki- in SOCS1, SOCS2, and SOCS3 mRNA induction (data not nase inhibitor p21 (WAF1), c-myc, and caspase 1 (28Ð30), mod- shown). Therefore, the reduced keratinocyte expression of SOCS ulating the IFN-␥-induced antiproliferative effect. In transiently molecules in the epidermis of AD compared with psoriasis or ACD transfected keratinocytes, IFN-␥-induced STAT1-dependent gene cannot be attributed to altered properties of keratinocytes to re- activation was impaired in the presence of SOCS1 and SOCS3, but spond to IFN-␥ but rather to the lower amount of IFN-␥ produced not of SOCS2. SOCS1- and SOCS3-dependent inhibition of IFN-␥ locally by activated T lymphocytes. signaling was exerted specifically on the STAT1 and not the Key events in IFN-␥ signal transduction in keratinocytes in- NF-␬B pathway. A discrete portion of SOCS1 and SOCS3, called volve Jak1 and Jak2 activation followed by IFN-␥R␣ phosphory- kinase inhibitory region, contributes to high-affinity binding to the The Journal of Immunology 441

in the phosphorylated tyrosine 1007 residue, known as one of the major sites of autophosphorylation of Jak2 (20). The reduced IFN- ␥R␣ phosphorylation in SOCS1 and SOCS3 keratinocyte clones led to a substantial inhibition of STAT1 and STAT3 activation. In particular, SOCS1 overexpression in keratinocytes markedly in- hibited the IFN-␥-induced STAT1 phosphorylation on both ty- rosine 701 and serine 727 residues. Although the mechanisms through which STAT1 is phosphorylated on serine are not yet fully understood, it is known that Jak2 is required for STAT1 serine phosphorylation in response to IFN-␥ (32). Therefore, the inhibi- tion of the IFN-␥-induced serine phosphorylation of STAT1 by SOCS1 and SOCS3 can occur indirectly through the abrogation of Jak2 activity. Inhibition of STAT1 and STAT3 phosphorylation was observed in all the SOCS1-expressing keratinocytes clones, in six of nine SOCS3 clones, and in none of the 20 SOCS2 clones examined, although the expression of SOCS2 protein was compa- rable with those of SOCS1 and SOCS3. Compared with SOCS1, ␥ SOCS3 appears to be a weaker inhibitor of IFN- signaling, pos- Downloaded from sibly because SOCS3 is not very efficient in binding and inhibiting the catalytic domain of Jak proteins (20). As a direct consequence of inhibition of STAT1 activation, we found a significant reduction of ICAM-1 and HLA-DR expression and IP-10 and Mig produc- tion in IFN-␥-activated SOCS1 clones and in four of nine SOCS3

clones. Moreover, SOCS1, but not SOCS3, overexpression inhib- http://www.jimmunol.org/ ited IFN-␥-induced MCP-1 release in keratinocytes. Consistent FIGURE 10. IL-8 constitutive release is enhanced in keratinocyte with these results, mice lacking SOCS1 die of a complex disease clones overexpressing SOCS1 and is not up-regulated by IFN-␥. A, IL-8 characterized by a massive T cell, macrophage, and eosinophil release was examined in supernatants from SOCS clones treated for 48 h infiltration of visceral organs and the skin (15, 33). Membrane ␥ with 200 U/ml IFN- by ELISA. B, IL-8 secretion was also tested in molecule and chemokine production was not affected by SOCS2 supernatants from control (circles), SOCS1 (squares), SOCS2 (triangles), constitutive expression. Interestingly, in a small percentage of and SOCS3 (diamonds) clones treated with medium alone (open symbols) or 200 U/ml IFN-␥ (filled symbols) for the indicated time periods. Data are SOCS2 clones (4 of 20) a superinduction of ICAM-1, HLA-DR, expressed as mean nanograms per 106 cells Ϯ SD of triplicate cultures. IP-10, Mig, and MCP-1 was observed. These findings can be ex- plained by considering SOCS2 as a negative regulator of SOCS1 by guest on September 27, 2021 and SOCS3. Indeed, previous works showed the SOCS2 capacity Jak2 tyrosine kinase domain and is required for the inhibition of of restoring sensitivity of 293 cells to prolactin and growth hor- Jak2 activity by preventing the access of substrates and/or ATP in mone suppressed by SOCS1 (34, 35). Because SOCS2 protein the catalytic pocket (20, 31). As a result of Jak1 and Jak2 inacti- does not contain kinase inhibitory region domain (10) but can vation, the phosphorylation in tyrosine 440 residue of IFN-␥R␣ interact with Jak1 and Jak2 (Fig. 5), it is possible that SOCS2 subunit induced by IFN-␥ was impaired in keratinocyte clones functions as an inefficacious pseudosubstrate for Jak1 and Jak2 overexpressing SOCS1 and reduced in SOCS3, but not in SOCS2 or as a competitor of SOCS1 or SOCS3. Another interesting clones. Therefore, we could not observe a direct reduction of Jak1 feature of SOCS1 clones was their capacity to produce higher and Jak2 phosphorylation, but rather a decrease of Jak1 and Jak2 amounts of IL-8 compared with SOCS2 and SOCS3 clones and ␥ intrinsic activities in IFN- -treated SOCS1 and SOCS3 clones. In to actively proliferate even in the presence of IFN-␥. Despite contrast, SOCS1 binds to the catalytic domain of Jak2 specifically the higher constitutive production of IL-8, SOCS1 clones did not up-regulate this chemokine in response to IFN-␥.Weare currently investigating the mechanisms by which SOCS1 influ- ences IL-8 production, and whether enhanced IL-8 production and abrogated IFN-␥-induced growth inhibition in SOCS1 clones are related. Targeting of SOCS molecules can be an important strategy for the control of cytokine-induced disorders. Indeed, SOCS3 overex- pression with blockade of the IL-6/STAT3 pathway improved ex- perimental intestinal inflammation and arthritis (18, 36). Our study provides the first evidences of SOCS expression in human kera- tinocytes and in immune-mediated skin diseases. Keratinocytes overexpressing SOCS1 are particularly resistant to the proinflam- ␥ FIGURE 11. The antiproliferative effect of IFN- is abrogated in ker- matory effect of IFN-␥. Interestingly, this resistance is achieved at F f atinocyte clones overexpressing SOCS1. Control ( ), SOCS1 ( ), SOCS2 the most proximal step of IFN-␥ pathway, namely at the receptor (Œ), and SOCS3 (᭜) clones were cultured for the indicated time periods in medium with or without 200 U/ml IFN-␥. Viable cells were determined by level, and it is effective in preventing downstream expression of trypan blue exclusion test. Data are expressed as the ratio between the relevant inflammatory genes. These findings identify SOCS1 as a number of IFN-␥-treated and untreated cells (relative cell number) Ϯ SD. potential molecular target for the treatment of IFN-␥-dependent Experiments were conducted in triplicate for each condition. skin diseases. 442 SOCS1 INHIBITS IFN-␥ SIGNALING IN HUMAN KERATINOCYTES

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