IL-13-Stimulated Human Keratinocytes Preferentially Attract CD4 þ CCR4 þ T cells: Possible Role in Atopic Dermatitis

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1 ORIGINAL ARTICLE -Stimulted Humn Kertinocytes Preferentilly Attrct CD4 þ CCR4 þ T cells: Possible Role in Atopic Dermtitis Rhul Purwr 1, Thoms Werfel 1 nd Mirim Wittmnn 1 Skin inflmmtion in topic dermtitis (AD) is chrcterized by the predominnt infiltrtion of T-helper (Th)2- cells in lesionl skin. However, the mechnism of recruitment of these cells in lesionl skin of AD is not yet fully elucidted. In this study, we investigted the role of -stimulted humn primry kertinocytes (HPKs) in the recruitment of lymphocytes nd further delineted the mechnism of enrichment of these cells. In the migrtion ssys, we observed preferentil enrichment of CD4 þ CCR4 þ T cells towrds -stimulted HPKs. Interestingly, CD4 þ CCR4 þ T cells from AD showed higher chemotctic response thn those from helthy individuls. We observed significnt increse in the expression of CCL22 in -stimulted HPKs s compred to unstimulted cells. Blocking of CCL22 in -stimulted HPKs by neutrlizing ntibody resulted in 7 9% inhibition in migrtion of CD4 þ CCR4 þ T cells. Moreover, upregulted IFN-g-induced chemokines, CCL2 nd CCL5, in HPKs. Tken together, our dt suggest tht -stimulted HPKs prticipte in positive feedbck loop by preferentilly enriching Th2-cells in lesionl skin of cute AD ptients. However, in chronic phse, my ct in synergy with IFN-g resulting in lymphocytes recruitment of mixed phenotype t the site of inflmmtion, thus contributing to the chronifiction of eczem. Journl of Investigtive Dermtology (26) 126, doi:1.138/sj.jid.5785; published online 16 Februry 26 INTRODUCTION Atopic dermtitis (AD) is chronic inflmmtory skin disese nd chrcterized prticulrly by infiltrtion of lymphocytes into the skin comprtment. Chemokines re potent chemottrctnts nd ctivting fctors, which ply criticl role in defining the nture of the inflmmtory infiltrte in AD (Ono et l., 23). In cute skin lesions, ccumultion of T-helper (Th)2-lymphocytes producing IL-4 nd/or nd expressing CCR4 hs been observed (Hmid et l., 1996; Nktni et l., 21; Wkugw et l., 21). In contrst to more Th2-like micromilieu in cute lesions, IFN-g hs been reported to be expressed in chronic skin lesions in AD (Werfel et l., 1996). is known s pivotl meditor of Th2 immune responses (Wynn, 23). CD4 þ T cells nd mst cells re mjor sources of in cute lesions nd in peripherl blood of AD (Obr et l., 22). Recently, higher number of -expressing CD4 þ T cells in peripherl blood 1 Deprtment of Dermtology nd Allergology, Hnnover icl School, Hnnover, Germny Correspondence: Dr Rhul Purwr, Deprtment of Dermtology nd Allergology, Hnnover icl School, Ricklinger Str. 5, Hnnover D-3449, Germny. E-mil: purwrrhul@yhoo.com Abbrevitions: AD, topic dermtitis; GAPDH, glycerldehyde-3-phosphte dehydrogense; HPK, humn primry kertinocyte; TNF-, tumor necrosis fctor- Received 6 My 25; revised 14 September 25; ccepted 27 September 25; published online 16 Februry 26 mononucler cells hve been observed in AD ptients thn in helthy individuls (L Grutt et l., 25). Additionlly, it hs lso been described tht, in AD skin lesions, there is higher number of -positive cells in the cute phse thn in the chronic phse (Hmid et l., 1996). does not exert direct effects on T cells, which lck functionl receptor (Zurwski nd de Vries, 1994). Therefore, unlike IL-4, it does not support the prolifertion nd differentition of nive T cells into Th2 phenotype. hs been described to upregulte the expression of vsculr cell dhesion molecule-1 on endothelil cells (Sironi et l., 1994) to enhnce the expression of vrious dhesion molecules on monocytes, such s CD11b, CD11c, CD18, CD29, nd CD49e (VLA-4) (reviewed in de Vries, 1998), nd to upregulte IL-6 (Derocq et l., 1994) s well s IFN-ginduced CCL5 secretion in kertinocytes (Wongpiybovorn et l., 23). Originlly, Th1 nd Th2 subsets hve been functionlly seprted bsed on their profiles of cytokine production. However, it hs now become obvious tht certin types of chemokine receptors re selectively expressed on either Th1- (e.g. CXCR3, CCR5) or Th2-cells (e.g. CCR4, CCR8, CRTH2) (Bonecchi et l., 1998; Ngt et l., 1999; Gombert et l., 25). In vitro studies hve reveled tht high-ffinity lignds for CCR4, thymus, ctivtion-regulted chemokine (CCL17), nd mcrophge-derived chemokine (CCL22) induce selective migrtion of Th2-cells (Imi et l., 1998, 1999). However, expression of CCR4 hs lso been documented & 26 The Society for Investigtive Dermtology 143

2 -Stimulted Kertinocytes Attrct Th2-cells on cutneous lymphocyte ntigen-positive memory T cells nd Th-cells (Cmpbell et l., 1999). Recently, the chemottrctnt receptor-homologous molecule tht expressed Th2- cells (CRTH2) hs been shown to be selectively expressed on Th2-cells (Cosmi et l., 2). Expression of CCR4 hs been speculted to be importnt for T-cell infiltrtion into skin lesions of AD (Wkugw et l., 21). The frequency of CD4 þ CCR4 þ T cells in peripherl blood of AD ptients hs been reported higher thn tht of helthy individuls nd hs been correlted with severity of the disese (Nktni et l., 21). Additionlly, in the epiderml lyer of AD skin, kertinocytes hve been shown to be positive for CCL17 nd CCL22 (Vestergrd et l., 2; Horikw et l., 22). Activted humn primry kertinocytes (HPKs) re n importnt source of chemokines, hence cpble of regulting the progression of the inflmmtory response. Recently, interction of T-lymphocytes nd kertinocytes hs been shown to promote Th1 cell ccumultion in chronic inflmmtory skin diseses (Albnesi et l., 21; Klunker et l., 23). These observtions led us to hypothesize tht plys n importnt role in the recruitment of inflmmtory cells to the sites of AD skin lesions. Co-locliztion of - producing CD4 þ T cells nd kertinocytes expressing receptor in the skin of AD ptients (Wongpiybovorn et l., 23) prompted us to investigte if -stimulted HPKs cn ttrct effector subsets of CD4 þ T cells. In this study, we report tht -stimulted chemokine(s) relesed by HPKs preferentilly enrich CD4 þ CCR4 þ T cells. Higher chemotxis indices of CD4 þ CCR4 þ T cells from AD ptients thn from norml subjects could be observed. In ddition, we hve shown tht lone induces CCL22 nd little but significnt mount of CCL2 in HPKs. Furthermore, enhnces IFN-g-induced CCL2 nd CCL5 in HPKs. Tken together, these findings suggest tht -stimulted chemokine(s) relesed by HPKs my ply role in the initition nd persistence of skin inflmmtion in AD. RESULTS -stimulted HPKs preferentilly ttrct CD4 þ CCR4 þ T cells To determine if -stimulted HPKs could contribute to the recruitment of CD4 þ T cells, we investigted the migrtory cpcity of CD4 þ T cells towrds -stimulted HPKs in trnswell chemotxis ssy. In this direction, we dded purified CD4 þ T cells, from helthy individuls, suspended in kertinocyte growth medium in the upper chmber of trnswell culture insert contining -treted or -untreted HPKs in the lower chmber. After 3 hours of trnsmigrtion, we observed significnt increse in the bsolute number of migrted CD4 þ T cells in response to -stimulted HPKs s compred to unstimulted controls (P ¼.1; Figure 1). We further chrcterized whether chemokines produced by -stimulted HPKs could in turn promote the preferentil migrtion of Th1-lymphocytes (CD4 þ CXCR3 þ ) versus Th2- lymphocytes (CD4 þ CCR4 þ ). We used the CXCR3 nd CCR4 chemokine receptor to chrcterize Th1- nd Th2-cells. As depicted in Figure 1 nd b, significntly higher number Absolute counts b 1,3, 1,2, 1,1, 1,, 9, 8, 7, 6, 5, 4, 3, 2, 1, Chemotxis index CD4 CXCR3 CCR4 CXCR3 CCR4 IFN-γ Figure 1. Chemotxis of CD4 þ T cells nd preferentil recruitment of CD4 þ CCR4 þ T cells in response to stimultion of HPKs. HPKs were treted with (5 ng/ml) or IFN-g (1 ng/ml) for 24 hours. Purified CD4 þ T cells from helthy donors (n ¼ 6) were dded in the upper chmber nd -treted or -untreted HPKs in the lower chmber of the 24-well trnswell plte. Migrtion ws performed for 3 hours t 371C in humidified environment. (, b) For phenotype nlysis, trnsmigrted CD4 þ T cells were stined for CXCR3 (Th1) nd CCR4 (Th2) by using respective mabs nd nlyzed by flow cytometry. The numbers of cells trnsmigrted into the lower chmber were counted, nd bsolute numbers of ech subtype nd chemotxis indices were clculted s described in Mterils nd Methods. The gry lines depict men vlues. NS ¼ nonsignificnt. P-vlue o.2, nd *Po.1. of CD4 þ CCR4 þ T cells migrted towrds -stimulted HPKs thn towrds unstimulted cells (P ¼.2). There ws bsl migrtion of CD4 þ CXCR3 þ T cells towrds unstimulted nd -stimulted HPKs; however, there ws no significnt difference in migrtion of these cells towrds -stimulted HPKs thn to unstimulted cells (Figure 1). In contrst to this, s depicted in Figure 1b, significntly higher migrtion of CD4 þ CXCR3 þ T cells nd CD4 þ CCR4 þ T cells ws observed in response to IFN-g-stimulted HPKs s compred to unstimulted cells (P ¼.1). Chemotxis index of CD4 þ CXCR3 þ T cells ws higher s thn tht of CD4 þ CCR4 þ T cells in IFN-gstimulted HPKs (Figure 1b). -stimulted HPKs ttrct more CD4 þ CCR4 þ T cells from AD ptients thn from helthy individuls In the next series of experiments, we compred migrtion of CD4 þ CCR4 þ T cells nd CD4 þ CXCR3 þ T cells from AD ptients with those from helthy individuls (Figure 2). We observed significntly higher chemotxis index of CD4 þ CCR4 þ T cells in response to -stimulted HPKs thn to unstimulted cells with CD4 þ T cells derived from AD ptients (chemotxis index , P ¼.1) s well s with CD4 þ T cells freshly isolted from helthy NS 144 Journl of Investigtive Dermtology (26), Volume 126

3 -Stimulted Kertinocytes Attrct Th2-cells b CXCR3 FL1-H Chemotxis index 3. * CXCR3 CCR4 CXCR3 CCR4 Helthy AD Helthy AD FL1-H CCR4 individuls (chemotxis index , P ¼.2). Interestingly, s shown in Figure 2, chemotxis indices of CD4 þ CCR4 þ T cells from AD were significntly higher thn those from helthy individuls (P ¼.44). However, there ws no observble effect in migrtion of CD4 þ CXCR3 þ T cells derived from AD nd helthy subjects towrds - stimulted HPKs s compred to unstimulted cells (Figure 2). Moreover, Figure 2b depicts the representtive experiment of -induced trnsmigrtion of CD4 þ CXCR3 þ nd CD4 þ CCR4 þ T cells derived from AD (n ¼ 6 for CXCR3, n ¼ 8 for CCR4) nd helthy subjects (n ¼ 6 for CXCR3, n ¼ 8 for CCR4). Recently, it hs been shown tht CCR4 could lso be expressed on Th- or Th1-cells (Cmpbell et l., 1999). To confirm tht migrted CD4 þ T cells re of Th2 origin, we studied nother Th2 mrker, CRTH2. Initilly, we investigted the expression of CRTH2 in freshly isolted purified CD4 þ T cells nd observed tht AD ptients hve higher number of CD4 þ CRTH2 þ T cells thn tht of helthy individuls (Figure 3), s previously observed by others (Cosmi et l., 2). Furthermore, we investigted the - induced trnsmigrtion of CD4 þ CRTH2 þ T cells of AD nd helthy individuls. Consistently, there ws significnt increse in the migrtion of bsolute numbers of CD4 þ CRTH2 þ T cells in response to -stimulted HPKs s compred with negtive controls (P ¼.1, chemotxis index ) in helthy individuls s well s in AD FL1-H FL1-H Figure 2. Higher migrtion of CD4 þ CCR4 þ T cells derived from AD ptients thn from helthy individuls towrds -stimulted HPKs. Purified CD4 þ T cells either from helthy donors or AD ptients were dded in the upper chmber nd superntnts derived from -treted (5 ng/ml, 24 hours) or -untreted HPKs in the lower chmber of the 24-well trnswell plte. Migrtions were performed for 3 hours t 371C in humidified environment. For phenotype nlysis, trnsmigrted CD4 þ T cells were stined for CXCR3 (n ¼ 6) nd CCR4 (n ¼ 8) by using respective mabs nd nlyzed by flow cytometry. The numbers of cells trnsmigrted into the lower chmber were counted, nd bsolute numbers of ech subtype nd chemotxis indices were clculted s described in Mterils nd Methods (). Horizontl line shows men vlues. Representtive dotplots of n AD ptient nd helthy individul re shown (n ¼ 6 for CXCR3, n ¼ 8 for CCR4) (b). The numbers in ech qudrnt re indicted in percentges of respective subpopultions. *P-vlue o.5. CRTH2 c CRTH2 Helthy AD Helthy AD Forwrd sctter Forwrd sctter , 8, 7, 6, 5, 4, 3, 2, 1, Figure 3. Migrtion of CD4 þ CRTH2 þ T cells in response to - stimulted HPKs. () Freshly isolted CD4 þ T cells either from peripherl blood of helthy individuls or from AD ptients were stined for CRTH2. Representtive dotplots of helthy individul (n ¼ 6) nd n AD ptient (n ¼ 6) re shown. (b) Purified CD4 þ T cells either from helthy donors (n ¼ 5) or from AD ptients (n ¼ 3) were dded in the upper chmber nd - treted or -untreted HPKs (5 ng/ml) in the lower chmber of the 24-well trnswell plte. Migrtion ssys were performed for 3 hours t 371C in humidified environment. Trnsmigrted CD4 þ T cells were stined for CRTH2, the numbers of cells trnsmigrted into the lower chmber were counted, nd the bsolute numbers of migrted CD4 þ CRTH2 þ T cells nd chemotxis indices were determined. (c) Representtive dotplots of n AD (n ¼ 3) nd norml donor (n ¼ 5) re shown. The numbers in ech qudrnt re indicted in percentges of respective subpopultions. The gry lines depict men vlues. P-vlue o.2. ptients (Figure 3b nd c). In ddition, Figure 3c depicts the representtive experiment of -induced trnsmigrtion of CRTH2 cells derived from AD (n ¼ 4) nd helthy subjects (n ¼ 3). Induction of CCL22 by in HPKs ttrct CD4 þ CCR4 þ T cells To confirm tht the observed effect on migrtion of Th2-cells ws due to chemokine production by HPKs, we studied the bility of HPKs to synthesize Th2-type C C chemokines such s CCL22 nd CCL17 in vitro. HPKs were subjected to, tumor necrosis fctor- (TNF-), IL-1b, or combintion of these cytokines for 4 hours. The mrna levels of CCL22 nd CCL17 were then determined by rel-time quntittive RT- PCR. We used TNF- nd IL-1b stimultion, becuse HPKs respond well to these cytokines, which re known to be upregulted during inflmmtion. We showed tht CCL22 mrna ws upregulted 2- to 8-fold by stimultion (Figure 4). However, TNF-, IL-1b or its combintion with did not show synergistic effects within sme time spn (dt not shown). In contrst to CCL22, CCL17 ws not detected in -stimulted HPKs (Figure 4). b Absolute count 145

4 -Stimulted Kertinocytes Attrct Th2-cells CCL22/GAPDH Fluorescence (F1) CCL CCL22 (pg/ml) 2 Fluorescence (F1) Fluorescence (F1) (ng/ml) b 18 * c d CCL22 (pg/ml) 2 CCL17 GAPDH Chemotxis index 7 5 +mab Figure 4. Induction of CCL22 by in HPKs ttrct CD4 þ CCR4 þ T cells. HPKs were stimulted with (5 ng/ml) for 4 hours for RNA nd with different doses of for 24 hours for protein mesurement. Totl RNA ws isolted nd subjected to rel-time quntittive LightCycler RT-PCR. () Dt re shown s normlized rtio (CCL22/GAPDH), nd representtive grph of the intensity of the fluorescence signl of the originl dt of CCL22 (n ¼ 6), CCL17 (n ¼ 3), nd housekeeping gene GAPDH with their corresponding melting curves re shown (n ¼ 6) fter (5 ng/ml) stimultion s compred to un-stimulted cells (n ¼ 6). (b nd c) In cell-free superntnts, CCL22 protein ws mesured by specific ELISA (n ¼ 9). The gry lines depict men vlues. (d) Superntnt from - treted HPKs ws preincubted for 2 hours with nti-ccl22 ntibody before dding to the bottom chmber. Migrtion ssys were performed with purified CD4 þ T cells derived from helthy donors for 3 hours t 371C. Trnsmigrted CD4 þ T cells were stined for CCR4. Originl dt (un-processed) were normlized to bsl migrtion (un-stimulted HPKs, dotted line) for ech experiment nd shown s chemotxis index. P-vlue o.2 nd *Po.1. The next question we ddressed ws whether CCL22 mrna expression in unstimulted versus -stimulted HPKs could be confirmed t the protein level. We mesured CCL22 in culture superntnts of HPKs collected 24 hours fter (5 ng/ml) stimultion by specific ELISA (Figure 4b). HPKs produced pg/ml quntities of CCL22 (men , rnge pg/ml, P ¼.4) upon stimultion with s compred to unstimulted HPKs. We further studied dose-dependent effects of on CCL22 genertion in HPKs. As depicted in Figure 4c, 1 ng/ml of IL- 13 ws insufficient to induce CCL22 production in HPKs, wheres 5 ng/ml of could enhnce the production of CCL22 in HPKs significntly. Induction of CCL22 by in HPKs my be responsible for the ccumultion of CD4 þ CCR4 þ T cells; therefore, to prove this hypothesis, we performed blocking experiments with neutrlizing nti- CCL22 ntibody. As depicted in Figure 4d, CD4 þ CCR4 þ T cells migrted in response to -treted HPKs, wheres pre-incubtion with nti-ccl22 mab significntly inhibited the migrtion of CD4 þ CCR4 þ T cells (8 9%). With this set of experiments, we conclude tht migrtion of these cells in response to -stimulted HPKs ws due to CCL22. As ll migrted CD4 þ cells were not CD4 þ CCR4 þ T cells, we studied other T-cell chemottrctnts, CCL2, CCL5, nd CCL2, fter stimultion with (5 ng/ml). At the mrna level, CCL2 ws upregulted bout 2- to 4-fold by IL- 13 stimultion (Figure 5), but induction of CCL5 by could not be detected in the sme smples (Figure 5c). Furthermore, we observed little upregultion of CCL2 production upon stimultion with s compred with negtive control (Figure 5b). In blocking experiments using neutrlizing nti-ccl2 mab, we could not detect consistent 146 Journl of Investigtive Dermtology (26), Volume 126

5 -Stimulted Kertinocytes Attrct Th2-cells * b 1,4 1,2 1, CCL2/GAPDH CCL2 (pg/ml) Fluorescence (F1) Fluorescence (F1) Fluorescence (F1) c CCL2 CCL5 GAPDH Figure 5. Induction of CCL2 mrna nd protein but not CCL5 by in HPKs. Similr to CCL22, CCL2 nd CCL5 were quntified in HPKs stimulted with or without (5 ng/ml). () Dt re shown s normlized rtio (CCL2/GAPDH), nd representtive grph of the intensity of the fluorescence signl of the originl dt with the corresponding melting curves is shown (n ¼ 5). (b) In cell-free superntnts, CCL2 protein ws mesured by specific ELISA (n ¼ 8). The gry lines depict men vlues. (c) Representtive grph of the intensity of the fluorescence signl of the originl dt with the corresponding melting curves of CCL5 (n ¼ 3) nd GAPDH (n ¼ 5) re shown. *P-vlue o.5 nd Po.2. inhibition of CD4 þ T-cell migrtion. Furthermore, there ws no induction of CCL2 in - or IL-4-stimulted HPKs lone or in combintion with other pro-inflmmtory cytokines (dt not shown). -positive cells hve been shown to be present in the chronic phse of AD ptients where IFN-g, Th-1 signture cytokine, domintes (Hmid et l., 1996). Therefore, in the next series of experiments, we investigted the effect of on IFN-g-induced chemokines synthesis in HPKs. As shown in Figure 6, nd IL-4 mrkedly enhnced the IFN-ginduced CCL5 nd CCL2. However, neither nor IL-4 could modulte the IFN-g-induced CCL22 in HPKs. DISCUSSION HPKs ply pivotl role in skin inflmmtion through the regulted expression of chemokines ttrcting vrious leukocyte subsets, including monocytes, dendritic cells, nd T-lymphocytes. Severl evidences confirm T-cell-medited pthogenesis for inflmmtory skin diseses such s AD, psorisis, nd llergic-contct dermtitis. Accumultion of T cells in the skin comprtment from peripherl blood is highly dependent on chemokines nd expression of chemokine receptors. Our study demonstrtes tht -stimulted HPKs express CCL22, which preferentilly ttrct CD4 þ CCR4 þ T cells. Furthermore, CD4 þ CCR4 þ T cells from AD ptients showed higher chemotctic indices towrds -stimulted HPKs thn those from helthy individuls. We initilly used trnsmigrtion ssy to confirm preferentil migrtion of Th2-cells towrd -stimulted HPKs. Previously, hs been shown to be relevnt in the recruitment of T cells nd eosinophils to the inflmmtory site by upregulting vrious dhesion molecules, nd 147

6 -Stimulted Kertinocytes Attrct Th2-cells CCL2 (pg/ml) 6, 5, 4, 3, 2, 1, IFN-γ IFN-γ IFN-γ + + IL-4 CCL5 (pg/ml) 6, 5, 4, 3, 2, 1, chemokines (Li et l., 2; Lukcs, 2). In AD ptients, higher numbers of -positive cells pper to be present in cute lesions thn in chronic lesions s shown on the mrna level previously (Hmid et l., 1996). Recently, it hs been shown tht is mjor inducer of Th2 milieu in the cutneous microenvironment, being required independent of IL-4 (Herrick et l., 23). This fct, in combintion with the reltive bundnce of over IL-4 in subcute nd chronic AD, emphsizes the potentilly importnt role of in the pthogenesis of AD (Tzw et l., 24). To investigte the phenotype of migrted CD4 þ T cells (Th1/Th2), we used CXCR3 nd CCR4 surfce mrkers s the inflmmtory cells express different chemokine receptors depending on their differentition nd ctivtion stte. In prticulr, Th1-cells express high levels of CCR5 nd CXCR3, wheres Th2-cells express high CCR4 nd CCR8 levels (Ymmoto et l., 2; Gombert et l., 25). We observed higher chemotctic indices of CD4 þ CCR4 þ T-cell migrtion in AD s compred to helthy individuls. It hs been shown tht CCR4 plys n importnt role in the migrtion of Th2- cells from peripherl blood into the inflmed skin (Reiss et l., 21). Our results point to the fct tht the higher rte of migrtion of CD4 þ CCR4 þ T cells in AD ptients my not be only due to higher frequency of CCR4 þ cells in AD ptients (Nktni et l., 21) but lso becuse of higher migrtory cpcity of CCR4 þ cells from AD thn helthy individuls. To further confirm tht migrted CD4 þ CCR4 þ T cells re of Th2 phenotype, we used nother Th2 mrker, CRTH2, tht hs been shown to be expressed selectively on subpopultion of Th2-cells but not on Th- nd Th1-cells (Cosmi et l., 2). Consistently, we observed higher frequency of CRTH2-positive cells in AD ptients thn in helthy individuls. Moreover, we observed higher migrtion of CRTH2 cells towrds -stimulted HPKs in AD s well s in helthy individuls. For CD4 þ CXCR3 þ T cells, we observed lmost the sme or mrginl increse of migrtion towrds -stimulted HPKs s compred to unstimulted cells. Moreover, no significnt difference could be observed in AD versus helthy individuls. Recently, it hs been shown tht IL-4 upregultes the IFN-g- or TNF--induced CXCR3 gonist expression IFN-γ IFN-γ IFN-γ + + IL-4 * CCL22 (pg/ml) IFN-γ IFN-γ IFN-γ + + IL-4 Figure 6. enhnces IFN-c-induced chemokines CCL2 nd CCL5 in HPKs. HPKs were stimulted with IFN-g (1 ng/ml) lone nd in combintion with (5 ng/ml) or IL-4 (5 ng/ml) for 24 hours. In cell-free superntnts, CCL2, CCL5, nd CCL22 t protein levels were mesured by specific ELISA (n ¼ 4). Dt re expressed s men pg/ml7stndrd error of men. *P-vlue o.5, nd Po.2 when compred with IFN-g-stimulted cells. (Albnesi et l., 2). As the cytokine micromilieu nd timing of signl re importnt for the induction of severl genes (Wittmnn et l., 1999), this my explin why we hve observed preferentil enrichment of CD4 þ CCR4 þ T cells towrds -stimulted HPKs (no pre-incubtion with TNF- or IFN-g); however, no significnt effect on migrtion of CD4 þ CXCR3 þ T cells. To understnd the mechnism of migrtion of Th2-cells, we hve evluted the bility of HPKs to newly synthesize nd relese CCL22 nd CCL17. Results of in vitro experiments demonstrted tht HPKs were cpble of generting significnt quntities of CCL22 protein, but induction of CCL17 could not be observed in the sme experimentl settings. Recently, in vivo presence of CCL22-positive cells in skin lesions of AD in both the epidermis (kertinocytes) (Horikw et l., 22) nd the dermis (mture DCs) (Vulcno et l., 21) hs been demonstrted. The in vitro secretion of CCL22 by -stimulted HPKs in this study strongly suggests tht CCL22 expressed by HPKs my function to recruit CCR4 þ lymphocytes into inflmed epidermis. In monocytes, IL-4 nd hve been reported to ugment CCL22 expression (Bonecchi et l., 1998b), wheres in HCT (spontneously immortlized humn kertinocytes) cells, hs been shown to downregulte the CCL22 relese (Fujii-Med et l., 24). HCTs re humn kertinocytes cell line, which we hve not used in this study. Here, we used HPKs, hence differences in the result could be becuse of differences between cell lines nd primry cells. Recently, differences in the NF-kb pthwy hve lso been shown between HCTs nd HPKs (Muller et l., 23). In our study, itself exerted significnt effects on HPKs, but TNF- or IL-1b did not show synergy with in the induction of CCL22, unlike in bronchil epithelil cells (Sekiy et l., 2). Severl recent studies demonstrted tht infiltrtion of IFN-g-positive cells into the epidermis leds to poptosis of kertinocytes, nd thus contributing to the skin inflmmtion in AD (Klunker et l., 23). Our study demonstrtes tht CD4 þ CXCR3 þ nd CD4 þ CCR4 þ T cells migrte in response to IFN-g-treted HPKs. IFN-g induced CCL22 production in HPKs (our own observtion) nd in HCT kertinocytes (Fujii-Med et l., 24). These results were rther surprising, becuse CCL22 nd CCL17 re usully considered to ply role in Th2-dominted disorders such s llergic responses, but not in Th1-medited disorders. IFN-g inhibits the development of Th2-cells, ntgonizes Th2- dependent effects such s inhibition of signl trnsducer nd ctivtor of trnscription 6 nd its downstrem genes, nd downregultes CCL22 in monocytes (Bonecchi et l., 1998b; Heller et l., 24). Therefore, it ppers tht IFN-g my exert dichotomous effects on the pthogenesis of AD. This might be n importnt clue in understnding the pthogenesis of the chronic phse of AD where mixed (Th1 nd Th2) cytokine milieu hs been observed. Besides CD4 þ -lymphocytes, CCL2 potently ttrcts monocytes nd dendritic cells. In this study, we could observe little induction of CCL2 in HPKs fter stimultion. The slight induction of CCL2 by lone my 148 Journl of Investigtive Dermtology (26), Volume 126

7 -Stimulted Kertinocytes Attrct Th2-cells not ccount for biologiclly importnt effects; however, in concert with IFN-g, we observed mrked induction of CCL2 s well s CCL5 in HPKs. This suggests tht CCL2 nd CCL5 my contribute to the mixed Th1/Th2 infiltrte tht chrcterizes the chronic phse of AD. Bsl kertinocytes strongly express CCL2 in ptients with AD (Giustizieri et l., 21). IL-4 hs lso been shown to induce mrginl increse in IFN-g-induced CCL2 in orl kertinocytes (Li et l., 2). Therefore, it is nother insight of the potentil role of in mplifying the inflmmtory response t the trget site by recruiting vrious leukocytes, thereby fcilitting the chronifiction of inflmmtory eczemtous response. Although it is well estblished tht multiple C C chemokines re produced by HPKs, there were remrkble vritions within the HPKs isolted nd cultured from different helthy individuls in the induction level of CCL22 nd CCL2. These probbly represent heterogeneity in the popultion with respect to genetic bckground. In greement to our results, previous study lso observed the donordependent vribility in the expression of CCR-3 in HPKs (Petering et l., 21). Tken together, our results indicte the mechnisms of regultion of epithelil chemokine production nd thereby mplifying the inflmmtory response in the skin. We hve demonstrted tht -ctivted HPKs represent n importnt source of the chemokine CCL22 nd preferentilly enrich Th2-cells s observed in the migrtion ssys. Moreover, in concert with IFN-g induces high mounts of CCL2 nd CCL5 in HPKs. With the recent evidences, we conclude tht plys dichotomous role in the pthogenesis of AD by enriching Th2 cells in cute phse nd mixed lymphocytes popultion in chronic phses of llergic skin inflmmtion. MATERIALS AND METHODS Cytokines nd regents All cytokines were used s purified recombinnt humn preprtions. Humn ws purchsed from PromoCell GmbH (Heidelberg, Germny); IFN-g from ImmunoTools (Friesoythe, Germny); nd IL-4 from R&D Systems (Wiesbden, Germny). Cell isoltion nd culture Peripherl blood mononucler cells either from helthy individuls or AD ptients were seprted by Ficoll Hypque density-grdient centrifugtion. AD ptients suffered from the extrinsic form of the disese. SCORing Atopic Dermtitis were in the rnge of At the time point blood smples were tken, none of the ptients received systemic mediction, but most of them used locl steroids (clss II III). The study ws pproved by the ethics committee of the Hnnover icl School nd ws conducted ccording to the Declrtion of Helsinki Principles. All prticipnts gve their written informed consent. Monocytes were depleted by dherence nd CD4 þ T cells were purified from nondherent cells by using negtive selection kit (Miltenyi Biotech, Bergisch Gldbch, Germny). Purity of the resulting CD4 þ T cells ws consistently X95%, s verified by flow cytometry nlysis of CD4 þ T cells (mouse nti-humn CD4 mab; Beckmn-Coulter, Krefeld, Germny). After seprtion, cells were resuspended in Iscove medium (Biochrom, Berlin, Germny) supplemented with 4% humn AB serum. Primry cultures of norml humn kertinocytes were prepred from foreskin of children undergoing surgery s described previously (Wittmnn et l., 25). In brief, the single-cell suspension of kertinocytes ws cultured in serum-free kertinocytes growth medium (Kertinocyte Growth ium 2 Kit; PromoCell GmbH, Heidelberg, Germny). All cell cultures were incubted in humidified tmosphere contining 5% CO 2 t 371C nd were used t the pssge 2 5. Hydrocortisone-free medium ws used for ll experiments. The purity of HPKs ws verified by the expression of the epithelil mrker cytokertin (nti-humn cytokertin ntibody, clone: MNF-116; DkoCytomtion, Hmburg, Germny). All cells (more thn 95%) were found to be uniformly positive for cytokertin. Flow cytometric nlysis of intrcellulr nd membrne molecules Expression of surfce ntigens ws ssessed s described previously (Wittmnn et l., 22). The following phycoerythrin- or FITClbelled mab were used: CXCR3 (clone 4981; R&D Systems, Wiesbden, Heidelberg, Germny), CCR4, (clone 1G1; BD Biosciences), nd CRTH2 (clone BM 16; Miltenyi Biotech, Bergisch Gldbch, Germny). Cytokertin (DkoCytomtion) ws detected by intrcellulr stining using the Cytofix/Cytoperm kit (BD Biosciences). Stined cells were mesured by flow cytometry (FACSClibur) nd nlyzed using CELLQuestPro TM softwre (BD Biosciences). Migrtion ssy Purified CD4 þ T cells (1 1 6 /15 ml) from either helthy or AD donors were dded to the top chmber of 5-mm pore size polycrbonte 24-well Trnswell culture insert (Costr, NY) contining undiluted superntnts from either unstimulted or - stimulted HPKs (bout 7% confluent) in the bottom chmber. Migrtion ssys were crried out for 3 hours t 371C inco 2 incubtor. The numbers of migrted cells were counted by BD Truecount tubes (BD Biosciences) nd bsolute numbers were determined ccording to the mnufcturer s instructions. To determine the Th1 or Th2 phenotype, migrted cells were stined with fluorochrome-conjugted CXCR3, CCR4, or CRTH2 mabs, nd the number of cells migrted were clculted with the following formul (totl number of cells migrted % ge of subtype of cells/ 1). Chemotxis indices were clculted s the rtio of the bsolute numbers of cells migrted towrds stimulted cells divided by the numbers of cells migrted towrds unstimulted HPKs (negtive control). For the blocking experiments, superntnts derived from IL- 13-ctivted HPKs were pre-incubted with 1 mg/ml nti-humn CCL22 ntibody (clone ; R&D Systems) nd nti-humn CCL2 ntibody (clone 24822; R&D Systems) for 2 hours, before the setup of chemotxis ssy. mrna isoltion, reverse trnscription, nd LightCycler PCR mrna isoltion nd reverse trnscription were performed s described previously (Wittmnn et l., 24) by using the High Pure mrna Isoltion Kit (Roche Moleculr Biochemicls, Mnnheim, Germny) nd First Strnd cdna Synthesis Kit (MBI Ferments, St Leon-Rot, Germny). The quntittive rel-time PCR 149

8 -Stimulted Kertinocytes Attrct Th2-cells ws performed on LightCycler (Roche Moleculr Biochemicls) by using LightCycler s FstStrt DNA Mster PLUS SYBR Green I (Roche Moleculr Biochemicls). CCL22, CCL17, CCL2, CCL5, nd glycerldehyde-3-phosphte dehydrogense (GAPDH) were run in touchdown PCR progrm (trget temperture, 681C; secondry trget temperture, 581C; step size,.5; nd step dely, 1). The following primers were used for PCR mplifiction: CCL22 sense 5 -ACC TAT GTG TCG TGT CT-3 nd CCL22 ntisense 5 -CGC TAC GCT ACC ACA G-3 ; CCL17 sense 5 -ATG GCC CCA CTG AAG ATG CT-3 nd CCL17 ntisense 5 -TGA ACA CCA ACG GTG GAG GT-3 ; CCL2 sense 5 -AGA TGC AAT CAA TGC CC-3 nd CCL2 ntisense 5 -TGG AAT CCT GAA CCC AC-3 ; CCL5 sense 5 -CCA TGA AGG TCT CCG C-3 nd CCL5 ntisense 5 -CAT CCT AGC TCA TCT CCA A-3 ; nd GAPDH sense 5 -CCA CAT CGC TCA GAC-3 nd GAPDH ntisense 5 -GGC AAC AAT ATC CAC TTT ACC AGA GT-3. For quntittive nlysis, stndrd curves for CCL22, CCL17, CCL2, nd CCL5 were creted covering rnge of six orders of mgnitude by dilution series (dilutions from these stndrd curves were used s clibrtors in further experiments). These stndrd curves describing the PCR efficiencies of the trget CCL22, CCL17, CCL2, or CCL5 nd the reference gene (GAPDH) llow n efficiency-corrected quntifiction using the Reltive Quntifiction Softwre (Roche Moleculr Biochemicls). The clibrtor-normlized reltive quntifiction results in trget concentrtion expressed reltive to the concentrtion of the reference gene in the sme smple mteril. Quntifiction of chemokines In ll experiments, HPKs were grown in 24-well plte (Nunc Inc., Wiesbden, Germny) in 6 9% cell density in.5 ml of kertinocytes medium. After stimultion with pproprite cytokines for hours, superntnts were collected. Concentrtions of CCL22, CCL5, CCL2, nd CCL2 in the cell-free superntnts of cytokine(s)-stimulted HPKs were mesured by DuoSet ELISA of CCL22, CCL5, nd CCL2 (R&D Systems, Wiesbden, Germny) nd CCL2 ELISA development kit (Peprotech, Germny) ccording to the mnufcturer s instructions. Sttisticl nlysis Comprtive dt were nlyzed by using the Mnn Whitney rnksum test (Figure 2), t-test (Figure 6), nd pired t-test (pired dt re depicted). The softwre used to perform the sttisticl nlysis ws Prism 3.3. Men vlues7stndrd error of men re depicted. In the figures, *stnds for P-vlue o.5, Po.2, nd *Po.1. CONFLICT OF INTEREST The uthors stte no conflict of interest. ACKNOWLEDGMENTS Specil thnks to Dr med Kolb for continuous support to our kertinocytes project. We lso thnk Christin Hrtmnn for excellent technicl ssistnce. This study ws supported by DFG Grnt SFB 566, A6. REFERENCES Albnesi C, Scrponi C, Sebstini S, Cvni A, Federici M, De Pit O et l. 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