Oct. 28/Concurrent Session 1 - Immunology: Cytokines

001

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004 THE RELATIVE ROLES OF TUMOR NECROSIS FACTOR ALPHA (TNF-A) AND INTERLEUKIN-1 (ILl) IN THE INDUCrION OF INTERCELLULAR ADHESION MOLECULE-I (ICAM-I) IN HUMAN EFIDBRMIS BY ULTRAVIOLET RADIATION Davldpmtmeandts of Dermatology and Intemal Medicine. Umverstty of Colorado School of Medune, Denver , Colorado. USA

Low corwmtbve levels of expressme of the adhesmn molecule ICAM-I (CD54) on the,surface of keratmocy?es wtthm the epidermis is felt to be protecttve amnst cytotoxtc damage by acttvated leukocw lnducbon of keratmocyxe ICAM-I is an early event m mflammat”~ skm diseases. eomlatod with keratinocyte activation and cyratoxicrty.

Ultravmlet radratioo (UVR) IS an tmportant inducer of ICAM- expresston in the eptdetis In an extenswe study of 21 different stmtns of hunta kerattttc+es dewed from different donors. we found that the level of mdwtion of lteratinocyte ICAM- expresston m individual cell strams was sam&ar with UVR and TNF-a (R=591 p= 0347) Donors could be characterized as htgh responders or low responders to both TNF-a and UVR. Donors who were high responders to TNF-a m vitro also showed stgmfarit inductton of !%ratinocyte [CAM-I expression m the epdetmxs after UVR radiation an viv” law responders Ld not upregulate [CAM-I m v~Y”.

Convemely. 10 cultured human neonati and adult kerarinccytes. IL-1 drd not induce slgmficant ICAM-I express&on These cells cadam large amounts of inuacellular IL-l= (icIL-lra), the receptor antagoorst for IL-1 In three dtfferettt transfomKd human keratmcc>?e cell hnes. the level of ICAM-I mduetxon by IL-1 vaned ID move= r&bon to the level of IcIL-lra measured by ELISA. Iotraczllular IL-lm m keranoocytes may rnfluence IL-I receptor expression (or funcuon) and thus IL1 responaveness

We conclude that UVR-mduced ICAM-I expresston m human kera!moc)tes can be medtated by both TNF-a and IL-l. but that the IL-I effect 1s irthibtted by endogenous ,clL-lra TNF-a response IS a resproducible mdivtdual charactenstic of cells from dtfferent donors and correlates wltb the level of UVR-induced ICAM-I

002

PENTOXIFYLLINE @‘TX) SUPPRESSES UVB-INDUCED CYTOKINE RELEASE BY KERATINOCYTES Aeatha Schwarz. Yoshinori Araeane. Manuel Simon. Thomas A. Lueer and Thomas Schwarz Department of Dermatology and LB1 for Cellbiology and Immunobiology of the Skin, University Miinster. Miinster. Germany

Currently we could show that the phosphodiesterase inhibitor PTX is able to suppress the effector phase of contact hypersensitivity and irritant reactions. This activit

r may be explained by the recently described ability of PTX to downregulate

the re ease of tumor necrosis factor a (TNFa) by leukocytes because TNFu is a critical mediator in these events. As keratinocytes are known to be a source of a variety of cytokines like interleukin (IL) I, IL-6 and TNFa the effect of PTX on the release of these medtators by keratinocytes was studied. Thus several keratinocyte cell lmes (A431, KB. HaCaT) and normal human keratinocytes were exposed to IJVB-light (FS40) and supernalants tested for IL-I, IL-6 and TNFu levels. UVB-irradiation caused a signtficant upre u&ion, while addition of PTX

J immediately after UVB-exposure resulted in a r uctton of IL-l, IL-6 and TNFu release. In contrast. addttion of PTX to mm-radiated cells slightly increased consututive cytokme secretion. The inhibttory effect of UVB-mediated cytokine production by PTX was maximally pronounced between 50 and 100 nglml. suppression was even observed when PTX was added I hr after irradiation. However, treatment of cells with PTX at later time points had no effect. To study whether downregulation by PTX is a general phenomenon keratinocytes were treated with PMA, another well known cytokine inducer. Addition of PTX to PMA treated keratinocybs. however, showed no suppression or an even sbghtly increased release of IL-l. IL-6 and TNFa. In summary, these data demonstrate PTX as a substaoce which is able to downregulate UVB-induced cytokine production, a phenomenon which has been so far only described for corticosteroids. The fact that PTX blocks UVB-induced but stimulates PMA-mediated cytokine release supports the concept that PMA and UVB-light work via dtfferent pathways.

003

REGULATION OF CYTOKINE GENES INVOLVED IN CYTOKINE NETWORK IN THE ECCRINE SWEAT GLAND. F. Sat”. G Soos. and K. Sat” Marshall Dermatology Research laboratories, University’of Iowa College “f’Medicine. Iowa City. Inwl t1.u

Concentrations of imerleukin (IL)-la in homao sweat are much higher than that in plasma and are secreted into sweat on a persistent basis, suggesting that sweat IL-I has biological functions in health and disease. We therefore addressed the questions: is IL la synthesized de nova?; which cytokines are present in sweat?; is IL-la part of a cytokine network?: and what is the pharmacological and molecular basis for the regulation of such a cytokine network? Using Western blot analysis, we demonstrated the presence of IL-6. IL-8. and U-la and B in freshly secreted human sweat and in the tissue homogenates as well as in the sweat gland cultox medium (both the duct and the coil). Furthermore, mRNAs for IL-I, -6 and -8 were also found in cul~red sweat gland cells using Northern blot analysis. This indicates that the sweat cytokines are produced de nov”. IL-la (0.5-l ng/ml,recombinant or of sweat origin) stimulated mRNA levels for IL-lb. IL-6, and IL-X, peaking at 2-6 h after stimulation. suggesting that these cytokines are pan of a cytoldne network where U-la plays a pivotal role as a master trigger. IL6, which is mainly regulated by sweat pmlactin. was a saong mhibitor of IL- 8 mRNA expression while IL-8 had no effect on IL-6 production, indicating that IL-6 is a regulator of IL-g. CAMP strongly inhibited mRNA levels for IL-la and IL-S, but slightly stimulated U-6 mRNA. Phorbol ester (Tf’A) and a Ca-ionaphore (A23187) (both of which are mimicked by high [AChl) weakly elevated [L-la mRNA at 6 h. The role of U-la as a master regulator of the cytokme network in the sweat gland IS further supported because sweat IL-la is the strongest inducer of DNA binding protems such as CRE (CAMP-biding element), CTFMF-I (CCAAT sequence biding element). and NF- rB (r-light chain enhancer) in the gel-retardation assay of nuclear exmcts We conclude that cytokines in sweat are produced de nova and form a network, where IL- la regulates the gene expression of other cytokines at the tmnssrrption level.

005 Parasite Infections Break T Cell Tolerance - a Model for Autoimmunity Induced by Infectious Diseases? Manin~&kenX*. Jose&F. Urban* w M. #Depanrnent of Dermatology. LMU Mlinchen, Mumch Germany: * Laboratory of Immunology. NlAID, NIH Bethesda MD, USA.

The origine of autoirnrnune diseases remains an enigma since non deleted. potentially autoreactive T cells arc nortttally tolerized. Potentially autoreactive, but rolerid T cells could be considered as a source of autoimmunity only if tolerance can be reversed under physiologic conditions. T cell tolerance has previously been reported to affect exclusively the interleuktn-2 (IL-Z) pathway and it has been suggested that tolerant T cells could be interleukin-4 (IL-4) producing or Th2 cells. To characterize more precisely the mechanisms that induce and reverse T cell tolerance we mvestigated rhe state of tolerance established in CD4+ T cells that follows after priming of mice with staphylocx‘occus enterotoxin A or B (SEA or SEB). CD4+ T cells from ammals primed with repetitive injections of SEA or SEB (100 pg) were incapable of prcducmg IL-?, IL 3, IL-4 or inuerferon-r. Thus the state of tolerance Induced by the iniection of superantigens closely r&cts the state of selftolerant CD4+ T fells and does not reoresenr a differentiation of IL-2 oroducine CD4+ T cells toward a Th2 uhenotvve. life&on of SEB/SEA-tolerant an&Is with-parasites could break T cell t”&nc~.‘by induction of CD4+ T cells that produced IL-4 in response to the tolenzing anugen. However, the mducrion of the Th2 phenotype tn tolerant CD4+ T cells required polyclonrd activation and expansion of the tolerant T cell population. Thus the infection did not circumvent T cell tolerance but reversed tolerance by activation of a silenced lymphokine pathway. These findings should funher our understanding of autoimmune phenomena that frequently complicate parasitic diseases such as malaria, leishmania or uypanosom,as,s. after a strong polyclonal T cell activation.

006 KERATINOCYTE-DERIVED TGFB SERVES AS A GROWTH SUPI’RESSIVE FACTOR FOR MOUSE DENDRITIC EPIDERMAL T CELLS. T Kawashima, K

Ar1wml. PR Bergstrrsser. A Takashima, UT Southwestern. Dallas. TX. USA and

Hokknido Umversity. Sappuro, Japan

Dendritic epidermal T cells (DETC) are 76 T cells that reside normally in nmuse epiderrms. We have observed that DETC growth is supported hy IL-2 produced by DETC and by IL-7 produced by keratinocytas (KC). In this srudy. we sought to ldenofy an epidermal cyrokme that might down-regulate their growth. Transforming growth factor p (TGFPI is a family of structurally homologous dimeric pmtems fTGF@l through TFFfl.5).

which &play potent immunosuppressive actwlties. We observed TGFBI InRNA rxprrssiun in Pam 212 KC and m freshly isolated mouse epidermal cells (Northern hioxing) TGFBI bioactivity (0 I nglml range) and mununorractivity were detected in Pam 212 supernarants (CCL64 bloarray and antihody blockingl. We then tested whether rTGF@l affects DETC growth. Proltferauve responses “t the 7-17 DETC line and freshly isolated DETC to nutogens (Con A. Immuhilizrd am-CD3. or PM.4 plus lanophorr) were hhrcked (80%) by 0 I-1 ngiml rTCFBl (‘H-thymldmr incarpnratlon) TGFal also blocked &n Ailriven entry of DETC into S phase (cell cycle andysisl With respect to its mrchamsm of action, TGF@l blocked almosl uvnplrrdly the producrlon of II=2 (ELISA) and the expresston of high aftinily IL-2 receptors (FACS and binding assay,. both of wh,<h are early evem\ ordinar,,y Induced by Gun A Funhrrmore. proliterarlvr responses at prractivdtul DETC to rIL-2 or ro rlL-7 were mh#hued (30.50%) hy TGFBI as well as by rTGFB2. 03 ,>r 85. These resu,t\ mdicare rha TCFD suppress mirogcn-induced DETC growth hy dnwn~regulating IL-2 producnon and IL-2 receptor expresslo” TGFB also mhlh,ts DETC recpnnslvmess to IL-,. suggesting thar KC-drrlved IL-7 and KC-derlvti TGFli rrgularc DETC growth ?II kby reciprocal mechanism\