TNF-a Downregulates Filaggrin and Loricrin through c-jun N-terminal Kinase: Role for TNF-a Antagonists to Improve Skin Barrier

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1 ORIGINAL ARTICLE TNF- Downregultes Filggrin nd Loririn through -Jun N-terminl Kinse: Role for TNF- Antgonists to Improve Skin Brrier Byung Eui Kim, Mihel D. Howell,, Emm Guttmn,, Ptrii M. Gilleudeu, Irm R. Crdinle, Mrk Boguniewiz,, Jmes G. Krueger nd Donld Y.M. Leung, Filggrin (FLG), loririn (LOR), nd involurin re importnt epiderml rrier proteins. As psorisis is hrterized y overexpression of tumor nerosis ftor- (TNF-) nd impired skin rrier, we investigted the expression of skin rrier proteins in psorisis ptients nd whether their expression ws modulted y TNF-. The expression of FLG nd LOR ws found to e deresed in nd non- skin of psorisis ptients. A orreltion ws found etween the expression of TNF- nd epiderml rrier proteins in psorisis. TNF- ws found to modulte the expression of FLG nd LOR vi -Jun N-terminl kinse-dependent pthwy. Importntly, we report tht linil tretment of psorisis ptients with TNF- ntgonist results in signifint enhnement of epiderml rrier protein expression. Our urrent study suggests tht TNF inhiits rrier protein expression, nd TNF- ntgonists my ontriute to linil improvement in ptients with psorisis y improving rrier protein expression. Journl of Investigtive Dermtology (), 7 79; doi:.8/jid..; pulished online Ferury INTRODUCTION The epidermis provides n importnt physil rrier ginst the environment. Filggrin (FLG), loririn (LOR), nd involurin (IVL) re mjor proteins tht hve n importnt role in formtion of the epiderml skin rrier (Klinin et l., ; Cndi et l., ). FLG ggregtes kertin filments nd provides ytoskeleton for the ornified envelope (Cndi et l., ). LOR is initilly expressed in the grnulr lyer nd omprises 7% of the totl protein mss of the ornified lyer (Klinin et l., ; Cndi et l., ). IVL is n erly omponent in the ssemly of ornified envelope nd provides sffold for ornified envelope (Cndi et l., ). is ommon hroni inflmmtory skin disese nd is hrterized y n impired skin rrier (Huffmeier et l., 7; Proksh et l., 8). The mehnism for redued skin rrier funtion in psorisis is not known. Tumor nerosis ftor- (TNF-) is overexpressed in the Deprtment of Peditris, Ntionl Jewish Helth, Denver, Colordo, USA; Deprtment of Peditris, University of Colordo, Auror, Colordo, USA; Lortory for Investigtive Dermtology, The Rokefeller University, New York, New York, USA nd Deprtment of Dermtology, Weill Cornell Medil College, New York, New York, USA Correspondene: Donld Y.M. Leung, Deprtment of Peditris, Ntionl Jewish Helth, Jkson Street, Room K96i, Denver, Colordo 86, USA. E-mil: leungd@njhelth.org Arevitions: A, ntiody; AD, topi dermtitis; FLG, filggrin; GAPDH, glyerldehyde--phosphte dehydrogense; HBD, humn- defensin; IVL, involurin; KC, kertinoyte; LOR, loririn; TNF, tumor nerosis ftor Reeived 6 Otoer ; revised 7 Deemer ; epted 8 Jnury ; pulished online Ferury epidermis of psorisis skin (Kristensen et l., 99; Ettehdi et l., 99) nd hs pivotl role in the pthogenesis of psorisis, s mny TNF--regulted genes re overexpressed in psorisis nd TNF- ntgonists re highly effetive therpeuti gents in most ptients (Nikoloff et l., 99; Rihrdson nd Gelfnd, 8). TNF- ws funtionlly linked to the IL-/Th7 pthwy in psorisis y its ility to tivte myeloid dendriti ells (Z et l., 7, 9). This study ws onduted to determine whether overexpression of TNF- in psorisis my ontriute to defiieny of epiderml rrier proteins. RESULTS Defiieny of FLG nd LOR in psorisis skin We initilly determined gene expression of FLG nd LOR in skin iopsies from norml sujets nd in ptients with psorisis. Using rel-time RT-PCR, we found tht the gene expression of FLG is signifintly deresed in (7.99±. ng FLG per ng of glyerldehyde--phosphte dehydrogense (GAPDH); Po.) nd non- (8.±.9 ng; Po.) skin from psorisis ptients, s ompred with skin from norml sujets (.6±7.78 ng; Figure ). In ddition, FLG gene expression ws signifintly deresed in psorisis skin ompred with non psorisis skin (Po.). LOR gene expression ws lso signifintly deresed in (.6±.7 ng LOR per ng of GAPDH; Po.) nd non- (8.±. ng; Po.) skin from psorisis ptients ompred with skin from norml sujets (7.7±6.8 ng; Figure ). However, the gene expression of IVL is not 7 Journl of Investigtive Dermtology (), Volume & The Soiety for Investigtive Dermtology

2 8 6 Norml non- Loririn expression (ng) 8 6 Norml non- Norml non- d Norml non- Filggrin Loririn Isotype Isotype e f Stining intensity of filggrin Norml non- Stining intensity of loririn Norml non- Figure. Expression of filggrin (FLG) nd loririn (LOR) in the skin from norml sujets nd ptients with psorisis. RNA ws isolted from the skin of norml sujets nd ptients with psorisis. The gene expression FLG () nd LOR () ws evluted using rel-time RT-PCR. (, d) Representtive prffinemedded skin iopsies from norml sujets (n ¼ ) nd ptients with psorisis (n ¼ 9) stined for FLG () nd LOR (d) re shown. Imges were olleted t mgnifition. (e, f) The intensities of the stining for FLG (e) nd LOR (f) were grded visully on sle from (no stining) to (the most intense stining). The sle r represents mm. Po.; Po.; Po.. GAPDH, glyerldehyde--phosphte dehydrogense. deresed in skin from psorisis ptients (dt not shown). This ws onfirmed t the protein level using immunostining (Figure nd d). The omposite dt for FLG nd LOR stining in ll smples re shown in Figure e nd f. The stining intensity of FLG nd LOR is signifintly deresed in (FLG: Po.; LOR: Po.) nd non- (FLG: Po.) psorisis skin, s ompred with skin from norml sujets. In ddition, the stining intensity of LOR in psorisis skin ws signifintly deresed ompred with non- psorisis skin (Po.). TNF- inhiits expression of FLG nd LOR is hrterized y overexpression of TNF- (Kristensen et l., 99; Ettehdi et l., 99) nd impired skin rrier (Huffmeier et l., 7; Proksh et l., 8). Therefore, we exmined whether TNF- modultes the expression of FLG, LOR, nd IVL. We differentited primry humn kertinoytes (KCs) with. mmol l CCl for dys, nd then the KCs were inuted with vrious onentrtions of TNF- for hours. The gene expression of FLG ws signifintly inhiited y TNF- with onentrtion s low s ng ml (.6±.8 ng FLG per ng of GAPDH; Po.) ompred with medi lone (.6±.7 ng; Figure ). Similrly, LOR gene expression ws signifintly inhiited y TNF- with onentrtions s low s ng ml ompred with medi lone (dt not shown). However, IVL gene expression ws not modulted y TNF- (dt not shown), suggesting tht TNF- does not hve glol effet on epiderml proteins. To further understnd the modultion of TNF- on FLG nd LOR, humn primry KCs were pre-inuted with TNF--neutrlizing ntiody (A) efore TNF- stimultion. We demonstrted tht FLG gene expression ws not signifintly deresed in the KCs treted with TNF-neutrlizing A (.9±. ng FLG per ng of GAPDH; Po.), s ompred with the untreted KCs (.9±.6 ng; Figure ). Similrly, LOR gene expression ws not signifintly deresed in the KCs pre-treted with TNF--neutrlizing A, s ompred with the untreted KCs (dt not shown). 7

3 6 Medi TNF-α Anti-TNF-α TNF-α TNF-α (ng ml ) Anti-TNF-α Figure. Tumor nerosis ftor- (TNF-) inhiits expression of filggrin (FLG). Primry humn kertinoytes (KCs) were stimulted in the presene of vrious onentrtions of TNF- for hours. The gene expression of FLG ws exmined y rel-time RT-PCR (). In ddition, primry humn KCs were pre-inuted with. mgml of TNF--neutrlizing ntiody efore TNF- ( ng ml ) stimultion. The gene expression of FLG ws then exmined y rel-time RT-PCR (). Po.; Po.; Po.. GAPDH, glyerldehyde--phosphte dehydrogense. The effets of TNF, s ompred with Th ytokines, on rrier proteins differ Th ytokines hve previously een reported to downregulte expression of FLG, LOR, nd IVL (Howell et l., 7; Kim et l., 8). To determine whether the mehnism for TNF- differs from effets of Th ytokines on rrier proteins, we exmined the effets of Th ytokines nd TNF- on gene expression of FLG, LOR, nd IVL. Primry KCs were treted with Th ytokines or TNF- for hours nd then mrna ws extrted nd evluted for gene expressions of FLG, LOR, nd IVL. The gene expression of FLG, LOR, nd IVL ws signifintly inhiited in the KCs treted with Th ytokines (FLG:.7±. ng; LOR:.7±.9 ng; IVL: 6.9±. ng) ompred with the untreted KCs (FLG:.6±.9 ng; LOR:.±. ng; IVL: 6.9±. ng; Po. for ll omprisons; Figure ). In ontrst, the gene expression of FLG nd LOR ws signifintly inhiited in the KCs treted with TNF- (FLG:.±.6 ng, Po.; LOR:.9±.8 ng, Po.) ompred with the untreted KCs (FLG:.6±.9 ng; LOR:.±. ng; Figure nd ). However, TNF- did not ffet IVL expression (Figure ), suggesting tht TNF- modultes rrier proteins in mnner different thn Th ytokines. In ddition, we determined the effets of TNF- nd Th ytokines on humn -defensin (HBD-), potent ntimiroil peptide in the epidermis. We found tht HBD- expression ws signifintly inresed in the KCs treted with TNF- (.89±. ng HBD- per ng of GAPDH; Po.), ompred with the untreted KCs (.6±.7 ng), wheres Th ytokines did not indue HBD- (Figure d). This suggests tht TNF- potentilly indues ntimiroil peptides s well s inhiits rrier proteins in KCs. -Jun N-terminl kinse inhiitor loks TNF--medited inhiition of FLG nd LOR As NF-kB is downstrem trget of TNF- (Hsu et l., 99; Clrke et l., ), we treted primry KCs with NF-kB inhiitor ( nm) efore TNF- stimultion to exmine whether NF-kB inhiitor loks the effet of TNF- on rrier proteins. NF-kB inhiitor did not lok the TNF- effets on gene expression of FLG (Figure ) nd LOR (dt not shown), suggesting tht TNF- does not downregulte FLG nd LOR y tivting NF-kB. It is known tht TNF- lso tivtes the mitogen-tivted protein kinse pthwy (Chng nd Krin, ; Lin, ; Zruin nd Hn, ). Therefore, we pre-treted primry KCs with n extrellulr signl-regulted kinses / inhiitor ( mm), p8 inhiitor ( mm), nd sp6 (-Jun N-terminl kinse inhiitor, mm) efore TNF- ( ng ml ) stimultion. Extrellulr signl-regulted kinses / inhiitor nd p8 inhiitor did not lok the TNF- effets on gene expression of FLG (Figure nd ) nd LOR (dt not shown). However, FLG gene expression ws not signifintly deresed in the KCs pre-treted with sp6 (.7±. ng FLG per ng of GAPDH; Po.), s ompred with untreted KCs (.±.6 ng; Figure d). Similrly, LOR gene expression ws not signifintly deresed in the KCs pre-treted with sp6, s ompred with the untreted KCs (dt not shown), wheres IVL gene expression ws not ffeted y TNF- or sp6 (dt not shown). TNF- ntgonist upregultes FLG nd LOR On the sis of our oservtions tht TNF- inhiits the in vitro expression of FLG nd LOR, we further investigted the linil signifine of this oservtion y determining whether TNF- monolonl A (etnerept) ould lok the TNF--medited inhiition of these rrier proteins in vivo in humns with psorisis. For this study, six independent psorisis ptients followed up t the Rokefeller University were given mg etnerept, i-weekly for weeks. We studied skin iopsies, efore nd fter they reeived etnerept. The gene expression of FLG ws signifintly inresed in skin from fter tretment (.89±.78 ng FLG per ng of GAPDH; Po.) ompred with skin from efore tretment (.8±.77 ng; Figure ). Similrly, LOR gene expression ws signifintly inresed in skin from fter tretment (7.9±6.9 ng LOR per ng GAPDH; Po.) ompred with skin from efore tretment (.±. ng; Figure ). Interestingly, the inrese in gene expression of FLG nd LOR is positively orrelted with hnge in psorisis 7 Journl of Investigtive Dermtology (), Volume

4 Involurin expression (ng) Medi TNF-α IL-/ Loririn expression (ng) d HBD- expression (ng) Medi TNF-α IL-/ Medi TNF-α IL-/ Medi TNF-α IL-/ Figure. The effets of tumor nerosis ftor- (TNF-) nd Th ytokines on rrier proteins. Primry humn kertinoytes were stimulted with TNF- ( ng ml ) or omintion of IL- ( ng ml ) nd IL- ( ng ml ) for hours. The gene expression of filggrin (FLG) (), loririn (LOR) (), involurin (IVL) (), nd humn- defensin (HBD-) (d) were exmined y rel-time RT-PCR. Po.; Po.. GAPDH, glyerldehyde--phosphte dehydrogense. Medi TNF-α Medi TNF-α NF-κB inhiitor p-8 inhiitor TNF-α NF-κB inhiitor TNF-α p8 inhiitor d Medi Medi TNF-α ERK / TNF-α ERK / TNF-α sp6 TNF-α sp6 Figure. -Jun N-terminl kinse (JNK) inhiitor loks the tumor nerosis ftor- (TNF-)-medited inhiition of filggrin (FLG). Primry humn kertinoytes were pre-inuted with NF-kB inhiitor ( nm, ), extrellulr signl-regulted kinses / (ERK/) inhiitor ( mm, ), p8 inhiitor ( mm, ), nd JNK inhiitor sp6 ( mm, d) efore stimultion with TNF- ( ng ml ). The gene expression of FLG ws exmined y rel-time RT-PCR. Po.; Po.. GAPDH, glyerldehyde--phosphte dehydrogense. re nd severity index (dt not shown). However, IVL gene expression ws not ffeted (Figure ). In ddition, we lso exmined the gene expression of FLG nd LOR in nd non- psorisis skin from efore tretment. The gene expression of FLG nd LOR in psorisis skin ws signifintly deresed ompred with non- psorisis skin (FLG: Po.; LOR: Po.; Figure nd ). Interestingly, the gene expression of TNF- in psorisis skin ws signifintly inresed ompred with non- psorisis skin (Po.; dt not shown). Immunostining onfirmed inresed expression of oth FLG nd LOR in skin fter tretment, s ompred with skin from efore tretment (Figure 6 nd ). The omposite dt in ll smples re shown in Figure 6 nd d. The men fluoresent intensity of FLG nd LOR ws signifintly inresed in skin from fter tretment ompred with skin from efore tretment (FLG: Po.; LOR: Po.). However, the men fluoresent intensity of IVL ws not ffeted (dt not shown). In ddition, the men fluoresent intensity of LOR in psorisis skin ws 7

5 No tretment non- Before tretment Involurin expression (ng) 8 6 No tretment non- Loririn expression (ng) 6 Before tretment No tretment non- Before tretment Figure. Tumor nerosis ftor- (TNF-) ntgonist inresed the gene expression of filggrin (FLG), loririn (LOR), nd involurin (IVL). Ptients with psorisis (n ¼ 6) were treted with TNF- monolonl ntiody for weeks. RNA ws isolted from the nd non- skin of the ptients with psorisis. Gene expression of FLG (), LOR (), nd IVL () ws evluted using rel-time RT-PCR. Po., Po.. GAPDH, glyerldehyde--phosphte dehydrogense. No tretment non- Before tretment Filggrin ntiody Isotype Filggrin men fluoresent intensity 6 No tretment non- Before tretment No tretment non- Before tretment d Loririn ntiody Isotype Loririn men fluoresent intensity 8 6 No tretment Before tretment non- Figure 6. Tumor nerosis ftor- (TNF-) ntgonist inresed the protein expression of filggrin (FLG) nd loririn (LOR). (, ) Representtive skin iopsies from ptients with psorisis stined for FLG () nd LOR () re shown. (, d) The men fluoresent intensity is shown for FLG () nd LOR (d) in the epidermis of eh iopsy. The sle r represents mm. Po., Po.. Arrows in pnel nd indite protein expression of filggrin nd loririn, respetively. signifintly deresed ompred with non- psorisis skin (Po.). DISCUSSION In our urrent study, we demonstrted tht there is defiieny of FLG nd LOR in nd non- psorisis skin. IVL expression, however, ws not deresed in psorisis skin. We demonstrted these findings using oth rel-time RT-PCR nd immunostining. This finding is similr to our previous dt showing reltive derese of FLG nd LOR in psorisis skin in omprison with norml skin (Guttmn-Yssky et l., 9). Therefore, our dt suggest 76 Journl of Investigtive Dermtology (), Volume

6 tht the well-estlished skin rrier defet (Huffmeier et l., 7; Proksh et l., 8), known to our in psorisis, is the result of defiieny in epiderml rrier proteins. In ontrst, it is well known tht psorisis is rrely ssoited with miroil infetion nd overexpress ntimiroil peptides. Therefore, this skin disese hs different profile of epiderml rrier proteins thn found in topi dermtitis (AD; Ong et l., ). KCs re the primry ells of the epidermis nd express skin rrier proteins inluding FLG, LOR, nd IVL (Wtt, 989; Cndi et l., ). For this reson, we used primry humn KCs to exmine why FLG nd LOR re deresed in the skin of psorisis ptients. skin is hrterized y overexpression of TNF- (Kristensen et l., 99; Ettehdi et l., 99). Therefore, we investigted whether skin rrier proteins re modulted y TNF-. We found tht the gene expression of FLG nd LOR ws signifintly inhiited in the KCs treted with TNF- following dose-dependent pttern. The importne of TNF- in downregulting these rrier proteins ws supported y the oservtion tht TNF-neutrlizing A loked the in vitro TNF--medited inhiition of FLG nd LOR. It hs een reported tht o% of ptients with psorisis hve FLG muttions nd 8% of psorisis ptients hve FLG defiieny in their skin (Huffmeier et l., 7). Our urrent dt suggest tht the defiieny of FLG nd LOR in most ptients with psorisis is quired s the TNF- modultion. In ddition, our lortory hs previously demonstrted tht defiienies of rrier proteins in AD skin re quired rther thn onstitutive (Howell et l., 7; Kim et l., 8). On the sis of our urrent dt nd previous dt (Howell et l., 7; Kim et l., 8), oth TNF- nd Th ytokines modulte rrier proteins. To determine whether the mehnisms for ytokine modultion of epiderml proteins re different, we stimulted KCs with TNF- or Th ytokines nd exmined the effets of these ytokines on rrier proteins. Interestingly, TNF- only redued FLG nd LOR. In ontrst, Th ytokines inhiited FLG, LOR, nd IVL. Therefore, we ould suggest tht TNF- modultes rrier proteins in mnner different thn Th ytokines. Indeed previous reports hve demonstrted tht Th ytokines inhiit rrier proteins vi the signl trnsduer nd tivtor of trnsription 6 in vitro in KCs nd in vivo in mie tht overexpress signl trnsduer nd tivtor of trnsription 6 (Kim et l., 8; Sehr et l., ). Both AD nd psorisis re ommon hroni inflmmtory skin diseses nd re hrterized y impired rrier proteins. Importntly, % of AD ptients suffer from reurrent skin infetions, wheres only 6.7% of psorisis ptients suffer from skin infetion (Christophers nd Henseler, 987). To explin this disrepny, we exmined the expression of HBD-, potent ntimiroil peptide, in the KCs treted with TNF- or Th ytokines, whih re overexpressed in AD skin. Importntly, HBD- ws inresed y TNF-; however, Th ytokines did not inrese HBD-. Therefore, we postulte tht the reson psorisis ptients hve rrier defet, ut re less suseptile to miroil infetions, thn AD ptients is tht TNF- only inhiits rrier proteins while induing ntimiroil peptides. Indeed our lortory hs previously demonstrted tht TNF- indues HBD- through signl trnsduer nd tivtor of trnsription nd NF-kB (Alnesi et l, 7). Furthermore, our dt reently showed tht TNF- synergizes with IL-7 in indution of ntimiroil peptides in humn KCs (Chiriozzi et l, ). In ontrst, in AD, Th ytokines inhiit oth rrier proteins nd ntimiroil peptides needed to fight infetion. Beuse TNF- tivtes NF-kB (Hsu et l., 99; Clrke et l., ) nd mitogen-tivted protein kinse (Chng nd Krin, ; Lin, ), we further investigted the effet of NF-kB inhiitor or mitogen-tivted protein kinse inhiitors on TNF- modultion of rrier proteins. Of note, only the -Jun N-terminl kinse inhiitor loked the TNF-medited inhiition of FLG nd LOR in the KCs. This oservtion strongly suggests tht TNF- modultes the expression of FLG nd LOR through the -Jun N-terminl kinse-dependent pthwy. Perhps, the most importnt oservtion mde in our urrent study ws to exmine the role of TNF ntgonism in vivo on skin rrier protein expression in the skin of ptients with psorisis. Tretment of ptients with psorisis for weeks with nti-tnf- signifintly inresed the expression of FLG nd LOR in their psorisis lesions. Therefore, our dt strongly suggest tht TNF- ntgonists ontriute to linil improvement in ptients with psorisis y improving rrier protein expression. We lso demonstrte diretly in linilly relevnt setting tht TNF- hs key role in driving rrier dysfuntion in psorisis. We onlude tht rrier protein defiieny in ptients with psorisis n e quired vi TNF--medited downregultion. MATERIALS AND METHODS Sujets Sujets inluded helthy persons with no history of skin disese nd psorisis ptients with moderte-to-severe psorisis. In ddition, we studied skin iopsies from six dult psorisis ptients with moderte-to-severe psorisis (men psorisis re nd severity index:.9±.) who reeived weeks of the nti-tnf- etnerept therpy in vivo under Rokefeller University Institutionl Review Bord-pproved protool. Skin iopsies (6 mm) were otined from nd non- skin of these ptients efore etnerept therpy ws initited nd fter weeks of tretment. The ptients were treted with mg of etnerept, i-weekly, s previously desried (Z et l., 7). Overll, 69% derese in psorisis re nd severity index in ll study prtiipnts ws oserved with etnerept therpy, s previously desried (Z et l., 7, 9). None of the ptients hd previously reeived systemi ortiosteroids or ylosporine, nd none hd reeived topil ortiosteroid or lineurin inhiitors for t lest week efore enrollment. These studies were onduted ording to the Delrtion of the Helsinki Guidelines nd were pproved y the institutionl review ord t the Ntionl Jewish Helth in Denver. All sujets gve written informed onsent efore prtiiption in these studies. 77

7 From ll other norml sujets nd ptients with nd non- psorisis, mm punh skin iopsies were otined. The skin iopsies were immeditely sumerged in ml Tri Regent (Moleulr Reserh Center, Cininnti, OH) nd frozen t 8 C for future RNA isoltion nd immunostining, or in ml of % uffered formlin for immunohistohemistry. RNA preprtion nd rel-time RT-PCR Totl RNA ws isolted from skin iopsies y hloroform/phenol extrtion nd isopropnol preipittion ording to mnufturer s guidelines (Sigm Chemil, St Louis, MO). RNesy Mini Kits (Qigen, Vleni, CA) were used ording to the mnufturer s protool to isolte RNA from ell ultures nd to further purify RNA from skin iopsies. RNA ws reverse trnsried into DNA nd nlyzed y rel-time RT-PCR y using n ABI Prism 7 sequene detetor (Applied Biosystems, Foster City, CA) desried erlier (Nomur et l., ). Primers nd proes for humn GAPDH, FLG, LOR, IVL, nd HBD- were purhsed from Applied Biosystems. To llow for omprisons etween smples nd group, quntities of ll trgets in test smples were normlized to the orresponding GAPDH levels in the skin iopsies nd ultured KCs, nd expressed s trget gene. Immunohistohemil stining Prffin-emedded tissues were ut t mm nd pled on frosted mirosope slides. Using toluene nd series of ethnol wshes slides were deprffinized nd rehydrted. Slides were inuted with monolonl mouse nti-humn A direted ginst FLG (: dilution; Am, Cmridge, MA) or polylonl rit nti-humn A diret ginst LOR (: dilution; Am) t C overnight. The ell nd tissue stining kits (R&D systems, Minnepolis, MN) were used ording to the mnufturer s protool. A speifiity ws determined y repling the primry A with n isotype-mthed ontrol (purified non-immune mouse IgG or rit IgG; Southern Biotehnology, Birminghm, AL). All slides were oded efore the smples were evluted so tht the identity of the study sujets ws not reveled. Imges were olleted t mgnifition nd the intensity of the immunostining ws sored on sle from to, with inditing no stining nd the most intense stining. Immunofluoresent stining Frozen tissues from six ptients were ut t mm nd fixed in % prformldehyde for minutes t room temperture. Skin setions were then loked s desried ove. Slides were then stined with monolonl mouse nti-humn A direted ginst FLG (: dilution; Am), polylonl rit nti-humn A diret ginst LOR (: dilution; Am), nd monolonl mouse nti-humn A direted ginst IVL (: dilution; Am) t C overnight. Slides were then wshed with phosphte-uffered sline/tween (.%), followed y inution with Cy-onjugted donkey ntimouse IgG (Jkson Lortories, West Grove, PA) or Cy- onjugted donkey nti-rit IgG (Jkson Lortories). The slides were visulized with onfol mirosopy (Lei, Wetzlr, Germny). Slides were oded to ensure ptient nonymity. Imges were olleted t, nd levels of men fluoresene intensity were mesured with Slideook. (Intelligent Imging Innovtions, Denver, CO). Men fluoresene intensity ws determined for eh exposure group nd ws reported s men men fluoresene intensity±se. KC ell ulture Primry humn KCs were grown in serum-free EpiLife ell ulture medium (Csde Biologis, Portlnd, OR) ontining.6 mmol l lium hloride, % humn KCs growth supplement V (Csde Biologis), nd % ntiiotis (peniillin/streptomyin) under stndrd tissue ulture onditions. To investigte the effets of the TNF- on rrier proteins, primry KCs were differentited with. mmol l CCl for dys nd then inuted in vrious onentrtions of TNF- (R&D systems) for hours. To further exmine the modultion of TNF- on rrier proteins, KCs were stimulted in the presene nd sene of TNF- ( ng ml ), nti- TNF- (. mgml, R&D systems), NF-kB tivtion inhiitor ( nm, EMD Chemils, Drmstdt, Germny), IL- nd IL- ( ng ml, R&D systems), or their omintion for hours. In ddition, the primry KCs were stimulted with TNF- in the presene or sene of mitogen-tivted protein kinse inhiitors (EMD Chemils), inluding extrellulr signl-regulted kinses / inhiitor ( mm), p8 inhiitor ( mm), nd -Jun N-terminl kinse inhiitor ( mm). Totl RNA ws isolted from KCs y using RNesy kits ording to the mnufturers guidelines (Qigen) for rel-time RT-PCR. Sttistil nlysis Sttistil nlysis ws onduted using Grph Pd Prism, version. (Sn Diego, CA). Sttistil differenes in gene expression or protein stining etween multiple groups ws determined y using one-wy nlysis of vrine, nd signifint differenes were determined y Tukey Krmer test. CONFLICT OF INTEREST The uthors stte no onflit of interest. ACKNOWLEDGMENTS This reserh ws supported y NIAMS RO AR6. We thnk Mureen Sndovl for her ssistne in the preprtion of this mnusript. REFERENCES Alnesi C, Firhild HR, Mdonn S et l. 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