- Email: [email protected]
Cutaneous immune responses in CD18-deficient mice
ESDR I JSID I SID Abstracts
0028
0025 PD-1 REGULATES SELF-TOLERANCE TO PRE\‘ENT TISSUE DESTRUCTION. Hirovuki Nshimura. IHiroshi Hiai. *Naeahiro M&to
ofMedical chemisq, Medicine,
Kyot”
‘Pethology
and&uku
and Biology of l%easz.
H”n$.
zInmwxl”gy.
Department Fncil!ty of
University, Kyoro. Japan.
FD-1 is a member of immunoglohulin supxfamily end has an immun”recep:“r tyxxine-based inhibitory motif (ITIM). lt is expressed on not only activated T sod !3 cells but also activated Mac!+ ce!ls. It suggests that I’D-1 mfiy negatively regula;e immune responses. Although PD-1 deficient mice showed slightly ragmented immune nsponses, when crossed v&h au;“-reactix 2C T celi receptor (TCR) transgenic mire r” exarninz i role of PD-I for self-t”kr.mce. they exhibi!ed autoimmune-like symptanr. 2C x?D-I-/- mice developed !“ss of weights by 30% skin lesions especially on tails and eye iesions by the age of IO weeks. 12% oi the mice died. Histological examination showed that B various degrees of lymphoid and myeloid cells inli!trated various tissues systemically. Most outstanding were the my”-en&xardit;s and perivasculx coffings in the lung. Tnere were interstitional in~lnmmntians in ss:ivety g!ands, kidney, 2nd liver. In the skin, mononuclearcell~ infiltrated aroud appendages and into the eplderrnis, resulting in the damages/loss of sebaceous gI?nds and follicul~ shafts, liquefaction degeneration, parakeratosis and acanthosis. They revealed enlarged spleens. In conaast, 2CxPD-l+/+ mice never exhibited evidence of these autoimnune-like symptoms. These results suggestedthat PD-I re.&ualtc~ peripheral se:f tolerance 1” prevent tissu: destruction although mos: Jf ~utoreactive 2C T &Is wcrc also eliminated iTr the thymuses. In addition,, because 2C TCR rccognwd MHC andgel (alloantigen). we proposed that 2CxPD-I-/mice are a new model “fchmnic Graft versus Host Disease
THE T-CELL COS’UMULATORY MOLECULES B7-1 (CD80) ANJI B7-2 (CD861 WHEN EXPRESSED ON KERATINOCYTES DELIVER DIFFERENT SIGNALS DURIN? CONT.$Tx HYPERSENSITIVFyz RESPONSES. &Bums. A.Na.w. YD. Depts. of ‘Dermatology and zCancer Center. Univ. of Rochester School of Medicine and Dentistry. Rochester, NY. and ‘Dept. of Dermatology, Univ. North Carolina, Chapel Hill, NC: The B7 family of costimulatory molecules are responsible for potent activation of helper T cells upon encounter with antigen. Recent findings suggest that the members ?f ths family, B7-1 and B7-2, may transduce qualitatively different signals resulttng in differences in T cell effector Iitnction (Cell 80:707-718, 1995; lmmonity 2:523-532. 1995: JEM 181:1145-1155, 1995). To determine whether B7-1 and B7-2 provide differing signals in the skin, lines of transgenic mice that express similar cell surface levrk of either B7-I or B7-2 on basal and suorabasal KC (Kl4 “remoter) were generated. When sensitized mice were challmged wiih t& hapten 2.4 dinitrofluorobenzene (DNFB). the 87-l Tg mice developed greatly exaggerated and persistent delayed-ti hypersensitivity (DTH). The B7-2 Tg mice developed DTH that was persistent. but intermediate in severity between non-Tg and 87-l Tg mice. RN&se protection analysis of cyto4dne gene transcription showed significant upregulation of the inilammatory cytoldnes TNF-a, lymphotoxin and IL-6 in the affected tissue of the B7-1 Tg mice at 24 brs when compared to expression levels of B7-2 Tg m$e, (p
__.___. _._._. _
~~~
_..-._‘.o.,. _.._.
0029
0026 PHENVR'PIC AND FVJNCTIDNAL
In summary, memory Th-( t&&zdD4+ T cellsinitially activated by superantigens in-vitro became locally re-activated in-y&o and represent the pathqenic T cell subset in psoriasis. The capacity to produce skin lesions using long term T cell lines. will help clarify the nature ofthe activating factor in-wvo, and the effector m&wles responsible for causing psonasis.
ROLE OF MlTF IN MELANOCYTE DEVELOPMENT: A TRANSGENIC ANALYSIS. Theas The Ronald 0. Perelm~ Department of Dermatology. Department of Biochcmishy~, and Howard Hugh= Medical Institute*. New York University Medical Center, New York, NY. Although dvz phenotypes of mice mutant at the &&&&~i8 allele and additional experimental data demonstrate dwt microphthalmia-associated transaiption factor (MlTF) promotes melanocyte development and differentiation, we further define the role of MITF in melanocyte development jn yiva using a transgenic tnouse model. A transgenic mouse line was generated expnzssing bacterial p-galactosidase from a 3.5 kh region of upstream genomic DNA from the dopachrome tautomer&RP-2 gene.. Expression of the transgene in wild-type embryos permits visualization of melancqte oxcursors in the “a-“tic newal crest as ewlv as emhrvonic dav IEl9.5 of development. Thk wtnsgene is also expressed in other neural cr& derivatives such as certain cells of the dorsal mot ganglia and in the wall of the dorsal aorta. serving as a marker of a subset of pred”mi&mtly do& cell types of neural crest origin. Transgenic mice were successively crossed with mice heterozygous for the mutant allele m to generate littas containing transgenic embryos homozygaus for the mutant allele. Mutant embryos at El3 show an absence of melanocyte precwsors, but full expression of the transgene in the dorsal root ganglia. AtB11.5. an absence of melanocyte precursors is also observed, but only scant expression is observed in the dorsal root ganglion. At EIO. mutant emhlyos show markedly reduced numbers of transgene-expressing cells within the dorsal neural tube, delaminating from the dorsal newal tube. and migrating to the region of the “tic vesicle. These results suggest that Ml’TF acts a early as the level of the neural tube to enhance either the specification of or the early survivill of melanccyte precutsors in melanocyte development. and that MlTF promotes migration of a subset of cells to the dasal root aannli&
0027
0030
CHARACTERISTICS OPBLDDD- DERNED CD4+ 7’ CELL LlNES THAT PRODUCE PSORIATIC PLAQUES (PP SKIN). B.J. Nickoloftt Wrone-Smith. DeparQnmt of Pathology, Loyola University, Maywood, IL. To funher understand the rote ofthe immune wItem in wniasis. rymptmnleu skin (PN skin) was engrafted onto SCID mice, and then injected withhighly aa&ws CD4+ &CDS+ T cell lines derivedfromperipheralblood. While PN skindoesnot spontaneously canverl 10 PP skin, inwadcrmal Injection of actwated lymphocytes induces full-fledged plaques with all ofthe relevant clinical and histological hsllmarks of psoriasis. In order to characteri~c tic T cells that EBUES psoriasis, WC investig&tedthe phenotype and functional cytokinc production pmtile of long term Tcell lines from 5 different patients. Initially, F~coll-Hypaque interface cells were activated by adding bacterial superantigens SEW SEC2 and IL-2 nnRPMI+IO% autologwl serum. ARer 7 days, negative ~elect~onWE performed using magnetic beads and appropriate mAbs to separateCW+ and CDS+ (798% pure) T celts. Lines were produced by continuwsly exposing the CD4+ and CDS+ T eellsto IL-Z. A&r 3-6 weeksin culture,7 cell tines(5 x 10’eelIs)were qccted intiermally intOengrafted PN skin. 5 of 5 CD4+ T cell tines produced a PN to PP U)nve&n. butnoneoftke CD*+ T cell lanes induced thus
phitied
(I p&l)
the following’phenot&: CD,+, CDS:, &5<0+, HLA-DR+, CiA+ and CDZS;. 3-4 weeks at?er injection, DeeasionalT cells expressedCM9 and CDZS, particularly at the demnl-epidmnal interface e&n though the cell liner had been thoroughly washed ofall IL-2 aid suspendedin PBS. The appearanceof these T cell acute activation markers in-Go, even though the washed T cells in-wro down modulate these surface molecules, suggesta mondary reactivation event IS occurring during the PN to PP cwwenion m the user catmn of the skin.
CUTANEOUS
IMMUNE
It~4’&NSES
IN
CDIS-DEFl$IENT
MICE.
Scharffetter-Kochanek Dqwtmmts
S. Grabbe. S. B&sea M. SteinaL arr. K. of I LBILUfMUns dCI German CD18 ?!y& &mzL ‘chain “YE ~2”%&% a &II of baemdimeric adhesion mokculcs including LFA-1 (CDlla/C!JJl8). Mac.1 ( & llb/CD18) and p150.95 (CDllc/CDl8). CD18deliciat mice [CD18(-/-)I were recently generated and display greatly iocreased nom&s of perip+il, blood neuuo#~’ 11s lugbly ekvikd smm immunoglobulin levels and exhibt greatly Lmtmsbzd neutmp II vansmt tton through blood veals. To fort& cheracterize cutawous immune responses. itvitant P rmattus ” (ID) awl allergic contact dmnatitis (ACD) were aswssed in tbesc mu. CDI8G/-) mice were almost completely unable to mount tqten-speeific ACD. but displayed unaltered ear swelling after irritant application, whmas the inflammat~ infiltrate was greatly nducal in both ACD and ID. Bone marmwderived dendritic antigen ting cells and epidermal Langerhans eelIs were largely oonnal with rcganl to tnorp %“” ology and presentation of protein antigen or alloantigen. T cells from CDls(_/-) mice failed to cluster afta mitogen stimulation and showed signiticantly weaker pIolif+iv~ rey aftu T cell receptor stimulation with immobilized anti-CD3 or m an al ogenw nnxed ukocytc reacdon, but exhibited enhanced proliferative activity after mite UI stimulation. Maover. blood. spleen and peripheral lymph nodca in most of the CD&+) mice contained much lower numbers of T cells end almost cmnpktely lacked CD& T cells To distinguish lx&van defects in priming or clicitaticm BS the reason for absent ACD in tbzse mice. regional lymph node cells from sensitized CD18(-/-) and control mice, rcspcctivcly. were injecti S.C. into em-s of syngeneic mke. which wwe subsequently challenged with hapten. Afta S.E. injection of smsitizd syngeneic I difference in ACD was found betwrm CDl8(-/-) and mmroi mice. suggesting ?h??es* at D18(-/-) no mice were adcquatetyprimed but t&at immtmoeompetent cells WQF tumble to emigrate into the 6” of ~apten T lication to elicit ACD. Mormvcx, regional LN cells obtained from senstuzed race pmh sated normally in nsponse to hapten challenge. Taken together. these data suggestthat CD18 controls leukocyte trammigration into inflammatory skin sites and thus is mdispensibls for elicitation of ACD. Moreover. CD18 is of central importance for other leukocyte functions including T cell clustering, activation and differentiation into CD8+ T cells.
,
- _
CYTOKINE-RESISTANT BINDING OF STAT3 KINASE +CTIVITY.
MELANOMA CELLS EXHIBIT REDUCED DNA AND LACK ?F INHIBITION ?F CYCLIN E-ASSOCIATED M. Jens Funkz. h&&t& t skt.I&B&mwn.P.CH or??%mmas h an. m: t&atol”gy and Ludwi; :l& Iostitote?“rwCell Biolopv and Immunobioloev of the Skin. Universitv of Miinster. %e.“t. of Bio&&istry, RWTH. A&e”, Germany id ‘Fred I&chinson Can& R&atzh Center, Seattle, WA, USA. It is well established that melanoma cells during progression from early primary to metastatic lesions undergo a phenomenon called ,,cytokine resistance”, i. e. loss of growth inhibition by cytokines such as transforming growth factor-b (TGF-P) or interleukind (n-6). In order to elucidate the mechanism responsible for this phenontenor~ we examined ctitical players in the signal transduction and ceU cycle progression m response to IL-6 in two well characterized melanoma cell lines, WM35 (derived, form the radial growth phase of melanoina) and wM9 (established fioom a m&str& lesion and unresponsive to IL-6). We show that the signal transducer and activator of tralsai ption (STAT3) whose function is pivotal in II&mediated Gl arrest is significantly less tyrosyl-phosphorylated in WM35 cells compared to WM9 cells. Accordingly, biding of STAT3 to its DNA response element is reduced. No major differences in the nuclear uanslocation of STAT3 after stbnulation with IL-6 were found in both cell lines. We next analyzed potential targets in the IL-6 mediated cell cycle arrest. WM35 cells displayed marked suppression of cyclic E-associated kinax activity within the fust 24 hours after IL-6 treatwnt compared to only mwginal suppression in WM9 cells. In addition, phosphotylation of the tumor suppressor gene product pRblO5 was retained in Wh49 cells. Expression of ~16’~‘~. ~21”’ and ~27’“’ as well as cd&A, on the other ban$ was not regulated by IL-6. Taken together, our data provide an insight into the mechamsm of IIxCnsistance of advanced m=elanoma cells and show for the first time distinct molecular players of the cell cycle to be regulated by IL-6.
0028
0025 PD-1 REGULATES SELF-TOLERANCE TO PRE\‘ENT TISSUE DESTRUCTION. Hirovuki Nshimura. IHiroshi Hiai. *Naeahiro M&to
ofMedical chemisq, Medicine,
Kyot”
‘Pethology
and&uku
and Biology of l%easz.
H”n$.
zInmwxl”gy.
Department Fncil!ty of
University, Kyoro. Japan.
FD-1 is a member of immunoglohulin supxfamily end has an immun”recep:“r tyxxine-based inhibitory motif (ITIM). lt is expressed on not only activated T sod !3 cells but also activated Mac!+ ce!ls. It suggests that I’D-1 mfiy negatively regula;e immune responses. Although PD-1 deficient mice showed slightly ragmented immune nsponses, when crossed v&h au;“-reactix 2C T celi receptor (TCR) transgenic mire r” exarninz i role of PD-I for self-t”kr.mce. they exhibi!ed autoimmune-like symptanr. 2C x?D-I-/- mice developed !“ss of weights by 30% skin lesions especially on tails and eye iesions by the age of IO weeks. 12% oi the mice died. Histological examination showed that B various degrees of lymphoid and myeloid cells inli!trated various tissues systemically. Most outstanding were the my”-en&xardit;s and perivasculx coffings in the lung. Tnere were interstitional in~lnmmntians in ss:ivety g!ands, kidney, 2nd liver. In the skin, mononuclearcell~ infiltrated aroud appendages and into the eplderrnis, resulting in the damages/loss of sebaceous gI?nds and follicul~ shafts, liquefaction degeneration, parakeratosis and acanthosis. They revealed enlarged spleens. In conaast, 2CxPD-l+/+ mice never exhibited evidence of these autoimnune-like symptoms. These results suggestedthat PD-I re.&ualtc~ peripheral se:f tolerance 1” prevent tissu: destruction although mos: Jf ~utoreactive 2C T &Is wcrc also eliminated iTr the thymuses. In addition,, because 2C TCR rccognwd MHC andgel (alloantigen). we proposed that 2CxPD-I-/mice are a new model “fchmnic Graft versus Host Disease
THE T-CELL COS’UMULATORY MOLECULES B7-1 (CD80) ANJI B7-2 (CD861 WHEN EXPRESSED ON KERATINOCYTES DELIVER DIFFERENT SIGNALS DURIN? CONT.$Tx HYPERSENSITIVFyz RESPONSES. &Bums. A.Na.w. YD. Depts. of ‘Dermatology and zCancer Center. Univ. of Rochester School of Medicine and Dentistry. Rochester, NY. and ‘Dept. of Dermatology, Univ. North Carolina, Chapel Hill, NC: The B7 family of costimulatory molecules are responsible for potent activation of helper T cells upon encounter with antigen. Recent findings suggest that the members ?f ths family, B7-1 and B7-2, may transduce qualitatively different signals resulttng in differences in T cell effector Iitnction (Cell 80:707-718, 1995; lmmonity 2:523-532. 1995: JEM 181:1145-1155, 1995). To determine whether B7-1 and B7-2 provide differing signals in the skin, lines of transgenic mice that express similar cell surface levrk of either B7-I or B7-2 on basal and suorabasal KC (Kl4 “remoter) were generated. When sensitized mice were challmged wiih t& hapten 2.4 dinitrofluorobenzene (DNFB). the 87-l Tg mice developed greatly exaggerated and persistent delayed-ti hypersensitivity (DTH). The B7-2 Tg mice developed DTH that was persistent. but intermediate in severity between non-Tg and 87-l Tg mice. RN&se protection analysis of cyto4dne gene transcription showed significant upregulation of the inilammatory cytoldnes TNF-a, lymphotoxin and IL-6 in the affected tissue of the B7-1 Tg mice at 24 brs when compared to expression levels of B7-2 Tg m$e, (p
__.___. _._._. _
~~~
_..-._‘.o.,. _.._.
0029
0026 PHENVR'PIC AND FVJNCTIDNAL
In summary, memory Th-( t&&zdD4+ T cellsinitially activated by superantigens in-vitro became locally re-activated in-y&o and represent the pathqenic T cell subset in psoriasis. The capacity to produce skin lesions using long term T cell lines. will help clarify the nature ofthe activating factor in-wvo, and the effector m&wles responsible for causing psonasis.
ROLE OF MlTF IN MELANOCYTE DEVELOPMENT: A TRANSGENIC ANALYSIS. Theas The Ronald 0. Perelm~ Department of Dermatology. Department of Biochcmishy~, and Howard Hugh= Medical Institute*. New York University Medical Center, New York, NY. Although dvz phenotypes of mice mutant at the &&&&~i8 allele and additional experimental data demonstrate dwt microphthalmia-associated transaiption factor (MlTF) promotes melanocyte development and differentiation, we further define the role of MITF in melanocyte development jn yiva using a transgenic tnouse model. A transgenic mouse line was generated expnzssing bacterial p-galactosidase from a 3.5 kh region of upstream genomic DNA from the dopachrome tautomer&RP-2 gene.. Expression of the transgene in wild-type embryos permits visualization of melancqte oxcursors in the “a-“tic newal crest as ewlv as emhrvonic dav IEl9.5 of development. Thk wtnsgene is also expressed in other neural cr& derivatives such as certain cells of the dorsal mot ganglia and in the wall of the dorsal aorta. serving as a marker of a subset of pred”mi&mtly do& cell types of neural crest origin. Transgenic mice were successively crossed with mice heterozygous for the mutant allele m to generate littas containing transgenic embryos homozygaus for the mutant allele. Mutant embryos at El3 show an absence of melanocyte precwsors, but full expression of the transgene in the dorsal root ganglia. AtB11.5. an absence of melanocyte precursors is also observed, but only scant expression is observed in the dorsal root ganglion. At EIO. mutant emhlyos show markedly reduced numbers of transgene-expressing cells within the dorsal neural tube, delaminating from the dorsal newal tube. and migrating to the region of the “tic vesicle. These results suggest that Ml’TF acts a early as the level of the neural tube to enhance either the specification of or the early survivill of melanccyte precutsors in melanocyte development. and that MlTF promotes migration of a subset of cells to the dasal root aannli&
0027
0030
CHARACTERISTICS OPBLDDD- DERNED CD4+ 7’ CELL LlNES THAT PRODUCE PSORIATIC PLAQUES (PP SKIN). B.J. Nickoloftt Wrone-Smith. DeparQnmt of Pathology, Loyola University, Maywood, IL. To funher understand the rote ofthe immune wItem in wniasis. rymptmnleu skin (PN skin) was engrafted onto SCID mice, and then injected withhighly aa&ws CD4+ &CDS+ T cell lines derivedfromperipheralblood. While PN skindoesnot spontaneously canverl 10 PP skin, inwadcrmal Injection of actwated lymphocytes induces full-fledged plaques with all ofthe relevant clinical and histological hsllmarks of psoriasis. In order to characteri~c tic T cells that EBUES psoriasis, WC investig&tedthe phenotype and functional cytokinc production pmtile of long term Tcell lines from 5 different patients. Initially, F~coll-Hypaque interface cells were activated by adding bacterial superantigens SEW SEC2 and IL-2 nnRPMI+IO% autologwl serum. ARer 7 days, negative ~elect~onWE performed using magnetic beads and appropriate mAbs to separateCW+ and CDS+ (798% pure) T celts. Lines were produced by continuwsly exposing the CD4+ and CDS+ T eellsto IL-Z. A&r 3-6 weeksin culture,7 cell tines(5 x 10’eelIs)were qccted intiermally intOengrafted PN skin. 5 of 5 CD4+ T cell tines produced a PN to PP U)nve&n. butnoneoftke CD*+ T cell lanes induced thus
phitied
(I p&l)
the following’phenot&: CD,+, CDS:, &5<0+, HLA-DR+, CiA+ and CDZS;. 3-4 weeks at?er injection, DeeasionalT cells expressedCM9 and CDZS, particularly at the demnl-epidmnal interface e&n though the cell liner had been thoroughly washed ofall IL-2 aid suspendedin PBS. The appearanceof these T cell acute activation markers in-Go, even though the washed T cells in-wro down modulate these surface molecules, suggesta mondary reactivation event IS occurring during the PN to PP cwwenion m the user catmn of the skin.
CUTANEOUS
IMMUNE
It~4’&NSES
IN
CDIS-DEFl$IENT
MICE.
Scharffetter-Kochanek Dqwtmmts
S. Grabbe. S. B&sea M. SteinaL arr. K. of I LBILUfMUns dCI German CD18 ?!y& &mzL ‘chain “YE ~2”%&% a &II of baemdimeric adhesion mokculcs including LFA-1 (CDlla/C!JJl8). Mac.1 ( & llb/CD18) and p150.95 (CDllc/CDl8). CD18deliciat mice [CD18(-/-)I were recently generated and display greatly iocreased nom&s of perip+il, blood neuuo#~’ 11s lugbly ekvikd smm immunoglobulin levels and exhibt greatly Lmtmsbzd neutmp II vansmt tton through blood veals. To fort& cheracterize cutawous immune responses. itvitant P rmattus ” (ID) awl allergic contact dmnatitis (ACD) were aswssed in tbesc mu. CDI8G/-) mice were almost completely unable to mount tqten-speeific ACD. but displayed unaltered ear swelling after irritant application, whmas the inflammat~ infiltrate was greatly nducal in both ACD and ID. Bone marmwderived dendritic antigen ting cells and epidermal Langerhans eelIs were largely oonnal with rcganl to tnorp %“” ology and presentation of protein antigen or alloantigen. T cells from CDls(_/-) mice failed to cluster afta mitogen stimulation and showed signiticantly weaker pIolif+iv~ rey aftu T cell receptor stimulation with immobilized anti-CD3 or m an al ogenw nnxed ukocytc reacdon, but exhibited enhanced proliferative activity after mite UI stimulation. Maover. blood. spleen and peripheral lymph nodca in most of the CD&+) mice contained much lower numbers of T cells end almost cmnpktely lacked CD& T cells To distinguish lx&van defects in priming or clicitaticm BS the reason for absent ACD in tbzse mice. regional lymph node cells from sensitized CD18(-/-) and control mice, rcspcctivcly. were injecti S.C. into em-s of syngeneic mke. which wwe subsequently challenged with hapten. Afta S.E. injection of smsitizd syngeneic I difference in ACD was found betwrm CDl8(-/-) and mmroi mice. suggesting ?h??es* at D18(-/-) no mice were adcquatetyprimed but t&at immtmoeompetent cells WQF tumble to emigrate into the 6” of ~apten T lication to elicit ACD. Mormvcx, regional LN cells obtained from senstuzed race pmh sated normally in nsponse to hapten challenge. Taken together. these data suggestthat CD18 controls leukocyte trammigration into inflammatory skin sites and thus is mdispensibls for elicitation of ACD. Moreover. CD18 is of central importance for other leukocyte functions including T cell clustering, activation and differentiation into CD8+ T cells.
,
- _
CYTOKINE-RESISTANT BINDING OF STAT3 KINASE +CTIVITY.
MELANOMA CELLS EXHIBIT REDUCED DNA AND LACK ?F INHIBITION ?F CYCLIN E-ASSOCIATED M. Jens Funkz. h&&t& t skt.I&B&mwn.P.CH or??%mmas h an. m: t&atol”gy and Ludwi; :l& Iostitote?“rwCell Biolopv and Immunobioloev of the Skin. Universitv of Miinster. %e.“t. of Bio&&istry, RWTH. A&e”, Germany id ‘Fred I&chinson Can& R&atzh Center, Seattle, WA, USA. It is well established that melanoma cells during progression from early primary to metastatic lesions undergo a phenomenon called ,,cytokine resistance”, i. e. loss of growth inhibition by cytokines such as transforming growth factor-b (TGF-P) or interleukind (n-6). In order to elucidate the mechanism responsible for this phenontenor~ we examined ctitical players in the signal transduction and ceU cycle progression m response to IL-6 in two well characterized melanoma cell lines, WM35 (derived, form the radial growth phase of melanoina) and wM9 (established fioom a m&str& lesion and unresponsive to IL-6). We show that the signal transducer and activator of tralsai ption (STAT3) whose function is pivotal in II&mediated Gl arrest is significantly less tyrosyl-phosphorylated in WM35 cells compared to WM9 cells. Accordingly, biding of STAT3 to its DNA response element is reduced. No major differences in the nuclear uanslocation of STAT3 after stbnulation with IL-6 were found in both cell lines. We next analyzed potential targets in the IL-6 mediated cell cycle arrest. WM35 cells displayed marked suppression of cyclic E-associated kinax activity within the fust 24 hours after IL-6 treatwnt compared to only mwginal suppression in WM9 cells. In addition, phosphotylation of the tumor suppressor gene product pRblO5 was retained in Wh49 cells. Expression of ~16’~‘~. ~21”’ and ~27’“’ as well as cd&A, on the other ban$ was not regulated by IL-6. Taken together, our data provide an insight into the mechamsm of IIxCnsistance of advanced m=elanoma cells and show for the first time distinct molecular players of the cell cycle to be regulated by IL-6.