Regulate Neutrophilic Skin Inflammation Characterization Of

The Journal of Immunology

Characterization of Human T Cells That Regulate Neutrophilic Skin Inﬂammation1

Patrick Schaerli,2† Markus Britschgi,2* Monika Keller,* Urs C. Steiner,* Lisa S. Steinmann,‡ Bernhard Moser,† and Werner J. Pichler3*

It is unknown whether neutrophilic inflammations can be regulated by T cells. This question was analyzed by studying acute generalized exanthematous pustulosis (AGEP), which is a severe drug hypersensitivity resulting in intraepidermal or subcorneal sterile pustules. Recently, we found that drug-specific blood and skin T cells from AGEP patients secrete high levels of the potent neutrophil-attracting chemokine IL-8/CXCL8. In this study, we characterize the phenotype and function of CXCL8-producing T cells. Supernatants from CXCL8؉ T cells were strongly chemotactic for neutrophils, CXCR1, and CXCR2 transfectants, but not for transfectants expressing CXCR4, CX3CR1, human chemokine receptor, and RDC1. Neutralization experiments indicated that chemotaxis was mainly mediated by CXCL8, but not by granulocyte chemotactic protein-2/CXCL6, epithelial cell-derived neutrophil attractant-78/CXCL5, or growth-related oncogene-␣,␤,␥/CXCL1,2,3. Interestingly, ϳ2.5% of CD4؉ T cells in normal peripheral blood also produced CXCL8. In addition to CXCL8, AGEP T cells produced large amounts of the monocyte/neutrophil-activating cytokine GM-CSF, and the majority released IFN-␥ and the proinflammatory cytokine TNF-␣. Furthermore, ,apoptosis in neutrophils treated with conditioned medium from CXCL8؉ T cells could be reduced by 40%. In lesional skin -CXCL8؉ T cells consistently expressed the chemokine receptor CCR6, suggesting a prominent role for CCR6 in early inflam matory T cell recruitment. Finally, our data suggest that CXCL8-producing T cells facilitate skin inflammation by orchestrating neutrophilic infiltration and ensuring neutrophil survival, which leads to sterile pustular eruptions found in AGEP patients. This mechanism may be relevant for other T cell-mediated diseases with a neutrophilic inflammation such as Behçet’s disease and pustular psoriasis. The Journal of Immunology, 2004, 173: 2151Ð2158.

epending on their state of activation and polarization, eruption acute generalized exanthematous pustulosis (AGEP)4 as a effector T cells directly influence various cell types that model to better understand the interplay between T cells and may lead to a certain pathology. IFN-␥-producing Th1 neutrophils. D ϩ cells are involved in macrophage and CD8 T cell activation and Some 90% of AGEP cases are related to the intake of drugs, in promote proinflammatory conditions (TNF-␣, IL-12), and are particular antibacterials such as aminopenicillins. AGEP has three by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. therefore ideally designed for immunity against intracellular specific features: 1) an acute generalized formation of numerous, pathogens. Additionally, many autoimmune diseases depend on a mostly nonfollicular intraepidermal or subcorneal sterile pustules Ͻ Th1 response to autoantigens. In contrast, Th2 responses use the ( 5 mm) on a widespread edematous erythema in the absence of a cytokines IL-5, IL-4, and IL-13, which enhance eosinophil and bacterial inflammation (7Ð9); 2) neutrophils appear after T cell infiltration (10Ð12); and 3) the possibility to induce the reaction by patch mast cell mobilization and cause certain allergic diseases such as testing with the corresponding drug, whereby a massive release of asthma. In addition, Th1 responses may be counteracted by Th2 CXCL8/IL-8 by both keratinocytes and isolated drug-specific T cells responses (1Ð5). upon stimulation can be observed (10, 13). The onset of AGEP is Drug hypersensitivity reactions are known as imitators of many acute; resolution of pustules occurs spontaneously within 4Ð10 days https://www.jimmunol.org T cell-mediated diseases, and understanding their pathomechanism after cessation of the incriminated drug (14). In summary, AGEP might contribute substantially to elucidate basic immunological seems to be a drug-induced, T cell-mediated disease, whereby the mechanisms (6). In this study, we used the severe pustular drug effector function of T cells leads to a neutrophil-rich inflammation. In an initial study, we demonstrated that lymphocyte proliferation tests can be positive with the incriminated drug (15). Culture of drug-reactive T cells isolated from peripheral blood or from Downloaded from *Division of Allergology, Clinic of Rheumatology and Clinical Immunology/Aller- † positive skin patch test biopsy specimens of AGEP patients led to gology, Inselspital, Bern, Switzerland; Theodor-Kocher-Institute, University of ϩ ϩ Bern, Bern, Switzerland; and ‡Clinic of Dermatology, University Hospital Zu¬rich, the generation of drug-specific, HLA-restricted, ␣␤TCR CD4 Zu¬rich, Switzerland and CD8ϩ T cell lines (TCL) and clones (TCC) (10, 12). Most of Received for publication January 22, 2004. Accepted for publication May 25, 2004. these drug-specific T cells produced high levels of the potent neu- The costs of publication of this article were defrayed in part by the payment of page trophil-attracting chemokine CXCL8. charges. This article must therefore be hereby marked advertisement in accordance In this study, we describe phenotypic and functional features of with 18 U.S.C. Section 1734 solely to indicate this fact. drug-specific CXCL8-producing (CXCL8ϩ) CD4ϩ T cells ob- 1 This work was supported by Grant 3100AO-101509 of the Swiss National Science Foundation (to W.J.P.) and Grant 99.0471-5 from the Bundesamt fuer Bildung und tained from AGEP patients and healthy individuals. We show that Wissenschaft (to B.M.). 2 P.S. and M.B. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Werner J. Pichler, Division of 4 Abbreviations used in this paper: AGEP, acute generalized exanthematous pustu- Allergology, Clinic of Rheumatology and Clinical Immunology/Allergology, PKT2 losis; B-LCL, B-lymphoblastoid cell line; CM, culture medium; SN, supernatant; D572, Inselspital, CH-3010 Bern, Switzerland. E-mail address: [email protected] TCC, T cell clone; TCL, T cell line.

ϩ CXCL8 T cells bear a specific chemokine receptor profile and from AGEP patients that produced Ͼ0.1 ng/ml CXCL8 were called ϩ express mainly GM-CSF, IFN-␥, and TNF-␣, and occasionally CXCL8 T cells. To produce SN for chemotaxis and apoptosis assays, ϫ 6 IL-4 and IL-5. Furthermore, factors produced by CXCL8ϩ T cells 0.5 10 cells/well in 24-well plates were unspecifically stimulated in 500 ␮lofCMϩ 10% FCS for 48 h. enhance neutrophil survival. Our results suggest that chronic neutrophil-rich inflammatory diseases involve CXCL8-producing T Statistical analysis cells, which can orchestrate and boost neutrophil inflammation. Regression and correlation of cytokine and chemokine production were Our findings may be relevant for other T cell-mediated diseases determined by the ANOVA (SigmaPlot 5.0; SPSS, Chicago, IL). R2 Ն 0.5 with similar pathology such as Behçet’s disease or pustular for linear regression, and p Յ 0.05 were considered significant. psoriasis. Flow cytometric analysis Materials and Methods Fluorochrome-labeled mAb against CD3, CD4, CD8, and isotype controls Culture medium and Ags were supplied by BD Pharmingen. Monoclonality of TCC was shown by TCR V␤ chain staining using a panel of 22 mAbs recognizing different V␤ gene Culture medium (CM) consisted of RPMI 1640 (Sigma-Aldrich, St. Louis, products, which detect ϳ75% of all V␤ families (Beckman Coulter, Mar- MO) supplemented with 10% pooled heat-inactivated human AB serum seilles, France) (18). Flow cytometry was performed on a Coulter EPICS XL- (Swiss Red Cross, Bern, Switzerland), 25 mM HEPES buffer (Seromed, MCL flow cytometer (Beckman Coulter). Abs to human CCR and CXC were Basel, Germany), 2 mM L-glutamine (Biotest Diagnostics, Dreieich, Ger- purchased from the following sources: mAbs to CCR2 (48607.211) and many), 10 ␮g/ml streptomycin, and 100 U/ml penicillin (Amimed, CXCR6 (56811) from R&D Systems; mAbs to CCR3 (7B11) from Millenium BioConcept, Allschwil, Switzerland). For the culture of TCC, the CM was Pharmaceuticals (Cambridge, MA); and mAbs to CCR4 (1G1), CCR5 (2D7), enriched with 200 U/ml human rIL-2 (CMϩ). EBV-transformed B-lym- CCR6 (11A9), CCR7 (2H4), CXCR1 (5A12), CXCR2 (6C6), CXCR3 (1C6), phoblastoid cell lines (B-LCL) were generated, as described (16). Amoxi- and CXCR4 (12G5) from BD Pharmingen. Rabbit IgG to CXCR5 was from cillin (Sigma-Aldrich) and celecoxib (Pharmacia, St. Louis, MO) were a noncommercial source, as described elsewhere (19). used for proliferation assays. Tetanus toxoid (Serum and Vaccine Institute, Bern, Switzerland) was used as a control Ag. Stock solutions of each drug Analysis of CXCL8-producing T cells in healthy individuals were always freshly prepared in CM just before use. Celecoxib was dis- CD4ϩ T cells were isolated from PBMC of healthy individuals by negative solved in RPMI 1640 with 0.05 M NaOH. MACS selection (Miltenyi Biotec, Bergisch Gladbach, Germany). A total Generation and culture of drug-specific T cells of 2 ϫ 106 cells/ml/24 well was stimulated with 10 ng/ml PMA/1 ␮M ionomycin (Calbiochem, La Jolla, CA) or anti-CD3/anti-CD28 ϩ 20 U/ml Drug- and tetanus toxoid-specific TCL and TCC from blood (designated IL-2. At various time points, SN were harvested for cytokine/chemokine B#) of drug-allergic patients AP, JS, and EB were generated, as described detection. For detection of intracellular cytokines, 10 ␮g/ml brefeldin A elsewhere (10). Skin-derived T cells (designated S#) were obtained from was added together with PMA/ionomycin for 5 h. mAbs to IFN-␥, TNF-␣, 5-mm punch biopsy specimens of positive epicutaneous test reactions upon IL-4, IL-5, IL-6, IL-10, IL-13, and CXCL8 were from BD Pharmingen. amoxicillin application on the back of patients AP and JS. Skin reactions Stainings were performed on fixed (2% paraformaldehyde/PBS) and per- were observed after 48 h and scored as described (17). T cells were ex- meabilized (0.5% saponin/PBS) cells. panded in CMϩ and restimulated every 14 days with irradiated (45 Gy), allogeneic PBMC and 1 ␮g/ml PHA. Ag specificity of TCL and TCC was Chemotaxis assay tested by incubating 5 ϫ 104 T cells (day 10Ð16 after restimulation) with either 5 ϫ 104 of irradiated (45 Gy), autologous PBMC or 1 ϫ 104 irra- Neutrophils from fresh blood of healthy volunteers (20) or murine pre-B diated (60 Gy), autologous B-LCL as APCs in the presence or absence of 300-19 cells stably transfected with human CXCR1, CXCR2, CXCR4, ␮ ␮ CX3CR1, RDC1, or human chemokine receptor were used. Cell migration

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. Ag in 200 l of CM in U-bottom 96-well microplates. After 48 h, 0.5 Ci of [3H]thymidine was added for 8Ð14 h. Finally, cells were harvested and was measured in 48-well chemotaxis chambers (NeuroProbe, Cabin John, incorporated radioactivity was measured with a beta counter (Inotech Filter MD). Briefly, chemically synthesized CXCL8 (I. Clark-Lewis, Biomedical Counting System INB 384; Inotech, Dottikon, Switzerland). Research Centre, University of British Columbia, Vancouver, Canada) in HEPES-buffered RPMI 1640 supplemented with 1% pasteurized plasma Isolation of skin T cells protein (Swiss Red Cross Laboratory) or cell SN were added to the lower wells, and 105 cells in control medium to the upper wells. Polyvinylpyr- Healthy abdominal skin was excised using a dermatome (0.3Ð0.5 mm rolidone-free polycarbonate membranes (Poretics, Livermore, CA) coated 2 thick), and 100-cm sections were digested in 10 ml of RPMI 1640 con- with type IV collagen and with 5-␮m pores for neutrophils or 3-␮m pores taining 1 mg/ml collagenase D (1088866; Roche, Basel, Switzerland) for for transfectants were used. After incubation for 20 or 90 min, respectively, 30 min at 37¡C on a shaker. Digestion was stopped by adding 10 mM the membrane was removed, washed on the upper side with PBSϪ, fixed, EDTA and immediate cooling and processing on ice. Floating cells were and stained. Migrated cells were counted microscopically at ϫ1000 mag- https://www.jimmunol.org collected and pooled with cells obtained by washing the remaining tissue Ϫ nification in five randomly selected fields (high power fields) per well. The several times with ice-cold PBS /10 mM EDTA (minimum 10ϫ excess of assay was performed in triplicates. For inhibition of CXCL8-dependent digestion volume). Cells were passed through a 70-␮m-pore nylon mesh chemotaxis, chemokines or SN and cells were incubated with 10 ␮g/ml (BD Pharmingen, San Diego, CA), centrifuged (300 ϫ g) for 20 min at neutralizing Ab to CXCL8 (clone 2A2, azide free; BD Pharmingen) 15 min 4¡C. After resuspension in cold complete medium, cells were passed before the chemotaxis assay. through a 40-␮m-pore mesh and centrifuged on a Ficoll-Paque gradient (600 ϫ g; 20 min at 4¡C). Interphase cells consisted of Ͼ90% viable T Apoptosis assay ␣␤ Downloaded from cells (as judged by CD45, TCR, and propidium iodide staining). 5 ␮ T cells from a biopsy of a positive 48-h Ni-patch test were isolated by Fresh neutrophils were incubated at 10 cells/100 l in Eppendorf tubes in diluted SN from different CXCL8-producing TCC (see above). After 5 h, cutting of skin tissue into small pieces. First, released cells were collected ϩ Ϫ and remaining tissue was digested for 45 min in collagenase D (Roche) to apoptotic cells (annexin V /propidium iodide ) were determined by flow obtain the second fraction (ϳ10% of first fraction). Chemokine receptor cytometry. expression analysis was performed on both fractions separately and did not differ. Results The study was approved by the ethics committee of the University of T cell involvement in the formation of sterile pustules during Bern, and informed consent was obtained by all participants of the study. drug-induced AGEP Generation of supernatants (SN) AGEP patients described in this work had an acute onset of gen- For cytokine detection by ELISA, SN were used from T cell specificity eralized, sterile pustules after drug intake (AGEP validation score assays and from unspecific stimulation for 48 h with immobilized anti-CD3 Ն10 following the EuroSCAR study group) (9, 14) (Fig. 1A). (1 ␮g/ml; Okt3) and soluble anti-CD28 (1 ␮g/ml; BD Pharmingen) in Drug-specific TCC isolated from peripheral blood of AGEP pa- flat-bottom 96-well plates (Nalge Nunc International, Roskilde, Denmark; 5 ϫ 104 cells/well, 200 ␮lofCMϩ 40 U/ml IL-2). ELISA sets used: IL-5 tients (patients AP and JS, amoxicillin; patient EB, celecoxib) and and CXCL8 (BD Pharmingen); IFN-␥, IL-4, and TNF-␣ (Diaclone, from skin biopsy specimens of positive epicutaneous patch tests Besançon, France); and GM-CSF (R&D Systems, London, U.K.). TCC (patients AP and JS) (Fig. 1B) were found to recognize the drug in The Journal of Immunology 2153

FIGURE 1. Involvement of drug-speciﬁc CXCL8-producing T cells in AGEP. A, One of the main features of AGEP is the formation of numerous, intraepidermal or subcorneal sterile pustules (Ͻ5 mm in diameter) on an erythematous background. The onset of the disease is acute, and most patients re- quire hospitalization. Resolution of pustules occurs spontaneously within 15 days after drug withdrawal. B, Patch testing with the eliciting drug in patients with AGEP can elicit localized pustule formation between 72 and 96 h. C, Most drug-speciﬁc TCC from AGEP patients produce high levels of CXCL8. Drug-dependent proliferation and CXCL8 production of three representative TCC are shown. Duplicate cultures of 5 ϫ 104 T cells and 104 autologous B-LCL were incubated with indicated concentrations of the drug. CXCL8 production was detected in SN after 48 h by sandwich ELISA (SD is given; detection limit 0.003 ng/ml), and proliferation was measured after a further over- night incubation with [3H]thymidine.

an HLA-DR-, but not in an allele-restricted manner (data not their cytokine/chemokine profile. A significant correlation was shown) (21). Dose-response curves for three representative blood- found between the production of CXCL8 and the Th1-associated derived CD4ϩ TCC AP B10, EB B14, and EB B27 are shown (Fig. cytokine IFN-␥ (R2 ϭ 0.58, p ϭ 0.0038) and with GM-CSF (R2 ϭ 1C). More than 80% of the generated drug-specific TCC produced 0.59, p ϭ 0.01), but not with the Th2-associated cytokines IL-4 reproducibly high amounts of CXCL8 (10). In this study, we show and IL-5, or the proinflammatory cytokine TNF-␣ (Fig. 2). No that for most of the TCC induction of CXCL8 production is Ag correlation was found for IL-10, RANTES/CCL5, or eotaxin/ specific and drug concentration dependent (Fig. 1C). Interestingly, CCL11, and G-CSF could not be detected (data not shown) (10). several TCC secreted CXCL8 at low levels even before stimula-

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. tion (100Ð340 pg/ml; data not shown). In contrast to AGEP pa- Chemokine receptor expression in normal and lesional skin T tients, TCC derived from noninﬂamed skin of healthy individuals cells failed to produce CXCL8 (n ϭ 12; data not shown). Chemokine receptor expression of CXCL8-producing CD4ϩ TCC

ϩ generated from blood or lesional skin of two AGEP patients was Cytokine/chemokine profile of CXCL8 TCC analyzed by flow cytometry and compared with CD4ϩ TCC from Drug-specific CD4ϩ TCC from AGEP patients could be grouped healthy skin (Fig. 3A). Resting CD4ϩ TCC from AGEP expressed into low (0.2Ð0.6 ng/ml), medium (1.0Ð1.6 ng/ml), or high (2.9Ð the Th1-associated chemokine receptors CCR5 and CXCR6 more 4.9 ng/ml) CXCL8 producers. Tetanus toxoid-specific TCC from frequently than control skin CD4ϩ TCC, although the frequency healthy individuals and drug-specific TCC from patients with ei- was below 25%. In contrast, CCR6 was expressed by the majority https://www.jimmunol.org ther maculopapular exanthema or bullouse exanthema secreted of T cells from AGEP clones (Ͼ85%), whereas normal skin TCC Ͻ0.1 ng/ml CXCL8 and were defined as non-CXCL8 producers showed significantly less CCR6 positivity. In addition, the level of (data not shown). Twelve TCC with low, medium, or high CXCL8 CCR6 expression was higher on AGEP clones than on normal skin secretion from three different donors were analyzed in detail for clones (data not shown). Interestingly, CCR6 was only detected on Downloaded from

FIGURE 2. Cytokine/chemokine secretion pattern of CXCL8-producing T cell clones. Lin- ear correlation of CXCL8 production with IFN-␥, IL-4, IL-5, GM-CSF, and TNF-␣. Concentrations of cytokines/chemokines are in ng/ml. Statistics were done by the ANOVA on 12 TCC from three donors; R2 Ն 0.5 for linear correlation, and p Յ 0.05 was considered signiﬁcant. 2154 T CELL-ORCHESTRATED NEUTROPHILIC INFLAMMATIONS

FIGURE 3. Chemokine receptor expression on healthy and lesional skin T cells. A, Blood- and skin-derived CXCL8-producing CD4ϩ T cell clones from two AGEP patients (circles, patient AP amoxicillin reactive, n ϭ 5; rectangles, patient US prednisolone reactive, n ϭ 8Ð11) were analyzed for chemokine receptor expression and compared with CD4ϩ T cell clones from healthy skin (squares, n ϭ 10). The mean values are indicated as horizontal bars. The p Ͻ 0.005; t test). B, Chemokine receptors were analyzed on CD4ϩ and CD8ϩ ,ءءء) highly signiﬁcant difference between AGEP and healthy skin is marked T cells isolated from an early (48-h) patch test reaction to nickel and compared with healthy skin T cells. This patient had developed AGEP after implantation of a nickel containing osteosynthesis material and developed the typical pustular skin reaction 72Ð96 h after patch testing. The data for healthy skin are representative for ﬁve individuals. The fraction of CD4ϩ T cells among CD3ϩ T cells from lesional skin was 85%, which is 1.6 times higher than in normal skin.

skin-derived, but not blood-derived AGEP TCC. CCR2, CCR3, suggests an obvious involvement of CCR6 and its ligand CCL20/ CCR7, CCR9, CXCR1, CXCR2, and CXCR5 were completely liver and activation-regulated chemokine in the recruitment of

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. absent on resting AGEP clones, whereas CCR4 was expressed by these T cells into affected skin (see Discussion). Ͻ8% of T cells (data not shown). To confirm that the phenotype of cloned AGEP T cells reflects CXCL8-producing T cells in healthy individuals the in vivo situation of an ongoing skin inflammation, chemokine It is not known whether CXCL8-producing T cells are present in receptor profiles of skin T cells directly isolated from an epicuta- peripheral blood of healthy individuals. To address this question, neous patch test reaction were determined (Fig. 3B). Lesional skin secretion of CXCL8, IFN-␥, and IL-4 was followed over time in was strongly enriched by CD4ϩ T cells (85% of total CD3ϩ T PMA/ionomycin-stimulated CD4ϩ T cells. Surprisingly, substan- cells) as compared with healthy skin (50%; data not shown). In tial amounts of CXCL8 were produced by CD4ϩ T cells after 3 addition, neutrophils were absent at this time point of analysis, days (Ͼ20 ng/ml) (Fig. 4A). In agreement with previous data, https://www.jimmunol.org which supports the idea that early immigrating T cells may be able IFN-␥ levels were higher than IL-4 levels among CD4ϩ T cells to influence the subsequent neutrophil infiltration by production of (22). Interestingly, CXCL8 production was delayed compared with CXCL8 (data not shown). Most strikingly and in agreement with IFN-␥ and resembled IL-5 and TNF-␣ production (data not T cell clones, CCR6 expression was found on the majority (Ͼ75%) shown). Comparable results were obtained after anti-CD3/anti- of lesional skin CD4ϩ T cells, in contrast to the low frequency CD28 stimulation, but with lower cytokine and CXCL8 secretion (Ͻ25%) among healthy skin CD4ϩ T cells. Therefore, CCR6 ex- levels (data not shown). Intracellular stainings of CD4ϩ T cells ϩ Downloaded from pression seems to be a specific and stable marker on CD4 T cells were performed to determine the frequency and phenotype of from lesional skin as well as AGEP clones. The low frequency CXCL8-producing T cells in peripheral blood. CXCL8 was pro- (Ͻ20%) of T cells positive for CCR4, CCR5, and CXCR6 sug- duced by 2.4 Ϯ 0.8% of CD4ϩ T cells (mean Ϯ SE; n ϭ 9), and gests that these chemokine receptors may play minor roles in T cell double stainings revealed that the majority of CXCL8ϩ T cells recruitment. In addition, the lack of CCR7 on most T cells indi- (Ͼ75%) also released TNF-␣ (Fig. 4B). In contrast, CXCL8ϩ T cates that infiltrating T cells belong to the effector memory T cell cells completely failed to secrete IFN-␥, IL-4, IL-5, IL-10, and subset. The high frequency of CXCR3ϩ T cells among AGEP IL-13. TCC (Fig. 3A), which contrasts the minor expression of CXCR3 on lesional skin CD4ϩ T cells (Fig. 3B), may be simply due to an CXCL8-producing T cells induce chemotaxis of neutrophils up-regulation during activation and culturing of T cells, as has In vitro chemotaxis assays revealed that SN generated from been previously described for CXCR3. Interestingly, CXCR3 was CXCL8ϩ T cells induced strong chemotactic activity in freshly expressed by all lesional skin CD4ϩ T cells that have down-mod- isolated neutrophils and in transfectants bearing either CXCR1 or ulated CD3 receptor (ϳ8%), again indicating that CXCR3 expres- CXCR2 (Fig. 5, lower panel). Synthetical CXCL8 was used as sion correlates with T cell activation (data not shown). In conclu- control (Fig. 5, upper panel). No migration was observed for trans- sion, the prominent expression of CCR6 on CXCL8ϩ skin T cells fectants stably expressing chemokine receptor CXCR4 or The Journal of Immunology 2155

FIGURE 4. Detection of CXCL8-producing T cells in the peripheral blood of healthy individuals. A, CD4ϩ T cells were isolated by MACS (purity Ͼ98%) from the peripheral blood of a healthy individual and stimulated with PMA/ionomycin. SN were harvested at the time points indicated, and IFN-␥, IL-4, and CXCL8 were determined by ELISA. B, CD4ϩ T cells were activated for 5 h with PMA/ionomycin, and intracellular cytokines were detected in by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. fixed and permeabilized cells by flow cytometry. Dot plots show a representative analysis of CXCL8 production in combination with TNF-␣, IFN-␥, IL-4, IL-5, IL-10, or IL-13. Specificity was confirmed by isotype control stainings, and a representative control staining is shown for CXCL8 (IgG2b) and IL-4 (IgG1).

CX3CR1, and orphan receptors human chemokine receptor or whether drug-speciﬁc CXCL8ϩ T cells from AGEP patients can RDC1, which are all expressed on neutrophils (data not shown) promote neutrophil survival in vitro. Initially, we found that SN (23Ð27). This indicates that chemotaxis of neutrophils was medi- from CXCL8ϩ T cells not only attracted neutrophils (Fig. 5, lower ated exclusively by CXCR1 and CXCR2. panel), but also strongly increased adherence (data not shown), https://www.jimmunol.org Furthermore, CXCL8-neutralizing Abs completely blocked mi- suggesting that neutrophils can beneﬁt from promoted interactions gration of CXCR1 transfectants, but had only moderate effects on with extracellular matrix, a mechanism that has been shown to CXCR2 transfectants and neutrophils (Fig. 5, open symbols). This prevent apoptosis (29). Indeed, apoptosis was strongly reduced in suggests that besides CXCL8, SN contained factors with minor neutrophils treated with conditioned medium from CXCL8ϩ T chemotactic activity for CXCR2, seen only at low SN dilutions cells (Fig. 6). A maximal reduction of apoptotic cells of 40 Ϯ 4% (Fig. 5, lower panel). Because SN lacked granulocyte chemotactic was observed. Increased survival was most likely due to T cell-

Downloaded from protein-2/CXCL6, epithelial cell-derived neutrophil attractant-78/ released GM-CSF and/or IFN-␥ (Fig. 2), two cytokines that are CXCL5, and growth-related oncogene-␣,␤,␥/CXCL1,2,3 (data not known to efficiently suppress neutrophil apoptosis (28Ð30). Nei- shown), the other major chemokines acting via CXCR1 and ther CXCL8 nor other CXCR1/2 ligands alone were sufficient for CXCR2, we suggest that CXCL8ϩ T cells produce an additional, these effects (data not shown). Our findings clearly demonstrate as yet unidentified factor acting predominantly via CXCR2. that CXCL8-producing drug-specific T cells can provide the

ϩ proper cytokine microenvironment that enhances neutrophil CXCL8 T cells enhance survival of neutrophils survival. Neutrophils are cleared fast under noninﬂammatory conditions, and also pustular eruptions disappear soon after drug withdrawal in Discussion AGEP patients (14, 28). Interestingly, local pustule formation with Neutrophil recruitment in response to bacterial or fungal infection accumulation of neutrophils can readily be reinduced and main- is a rapid and T cell-independent process, whereby the activated tained by a patch test (Fig. 1B) (13). This suggests that not only resident tissue cells release neutrophil-activating cytokines and recruitment of neutrophils, but also suppression of their apoptosis chemokines (28, 31Ð35). Thus, neutrophil accumulation generally may be controlled by drug-speciﬁc CXCL8-producing T cells, precedes T cells, which may be recruited at later stages of the which results in a longer lasting lesion. Therefore, we tested infection and contribute to chronicity (36, 37). In contrast, the 2156 T CELL-ORCHESTRATED NEUTROPHILIC INFLAMMATIONS

FIGURE 5. Drug-specific AGEP T cells are strongly chemotactic for neutrophils. Upper panel, Control experiment showing that CXCR1 and CXCR2 transfectants and human neutrophils migrate toward synthetic CXCL8 (filled symbols). This migration is completely blocked by the addition of 10 ␮g/ml anti-CXCL8 neutralizing Ab (open symbols). Lower panel, Chemotactic migration of CXCR1 and CXCR2 transfectants and human neutrophils to T cell-derived SN at different dilutions. SN was harvested after activation by anti-CD3/CD28 Abs after 48 h (filled symbols). Complete inhibition by the addition of anti-CXCL8 Abs was observed for CXCR1 transfectants (open symbols), while migration of CXCR2 transfectants and neutrophils was only partially blocked. Experiments with SN of one representative TCC is shown, and SD of triplicates are given.

neutrophil-rich inflammation AGEP differs from this concept in Chemotaxis experiments revealed that CXCL8 is the dominant three ways: 1) epidermal vesicle formation by drug-specific CTLs chemokine, which efficiently attracted neutrophils and CXCR1/ expressing perforin/granzyme and Fas ligand precedes pustule for- CXCR2 transfectants. In addition, SN of stimulated AGEP clones mation (10Ð12); 2) formation of Ͼ100 sterile pustules in the dis- were devoid of other major chemokines acting via CXCR1 and eased skin occurs in absence of a bacterial inflammation (Fig. 1A); CXCR2, namely granulocyte chemotactic protein-2/CXCL6, epithe-

by guest on October 1, 2021. Copyright 2004 Pageant Media Ltd. and 3) a pustular reaction can be elicited by patch testing in a lial cell-derived neutrophil attractant-78/CXCL5, growth-related sensitized patient with the responsible drug, whereby a massive oncogene-␣,␤,␥/CXCL1,2,3, and neutrophil-activating peptide-2 release of CXCL8 by both keratinocytes and drug-specific T cells (data not shown). Because blocking of CXCL8 showed only a can be observed (Fig. 1B) (10, 13). Hence, AGEP seems to rep- partial inhibition of neutrophil migration, we hypothesize that resent an interesting model for diseases, wherein the effector func- CXCL8ϩ T cells produce an additional chemotactic factor that acts tions of involved T cells lead to a neutrophil-rich sterile inflam- most likely via CXCR2. The responsible chemoattractant has not mation. In this study, we have described these drug-specific been identified yet. In addition to chemotaxis, the SN also CXCL8ϩ T cells with regard to their phenotype and their effects on increased adherence and had an antiapoptotic effect, enhancing the recruitment and survival of neutrophils. the survival of neutrophils. Reduction of apoptosis was most https://www.jimmunol.org The phenotypic analysis of drug-specific CXCL8ϩ CD4ϩ TCC likely due to CXCL8ϩ T cell-derived GM-CSF and/or IFN-␥, from AGEP patients revealed a predominant Th1-type cytokine profile with high production of GM-CSF and IFN-␥, and various levels of TNF-␣. However, rare CXCL8ϩ TCC displayed a Th2- type cytokine profile with high IL-4 and IL-5 secretion. In combination with eotaxin/CCL11 and RANTES/CCL5 released occa-

Downloaded from sionally by perivascular cells, such TCC may contribute to the eosinophilia observed in ϳ30% of AGEP cases, and which was also detected in patient JS (9, 10). Interestingly, CXCL8ϩ TCC showed, independently of high IFN-␥/GM-CSF or IL-4/IL-5 production, a chemokine receptor proﬁle associated with Th1 effector memory T cells that preferentially express CCR5, CXCR3, and CXCR6 (38Ð40). The prominent expression of CCR6 on CD4ϩ T cells derived from AGEP skin as well as on T cells directly isolated from patch test-reactive skin may be explained by an en- hanced recruitment of CCR6ϩ T cells. A similar mechansim has FIGURE 6. CXCL8-producing T cells enhance survival of neutrophils. been previously suggested for psoriasis, which shows high expres- Neutrophils were cultured at different dilutions of SN (f) or control me- sion of the CCR6 ligand liver and activation-regulated chemokine/ dium (Ⅺ) for 5 h. Apoptotic cells were determined by quantifying per- CCL20 (41). An involvement of CCR6 in the pathology of AGEP centage of annexin V-positive cells among propidium iodide-negative is further supported by the minimal expression of this receptor and cells. Experiments with SN of one representative TCC are shown, and SD its ligand in healthy skin. of triplicates are given. The Journal of Immunology 2157

which have been previously shown to prolong neutrophil pression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eo- survival (28, 42). sinophils. Curr. Biol. 7:836. 3. Romagnani, S. 1999. Th1/Th2 cells. Inflamm. Bowel Dis. 5:285. In psoriasis, activation of keratinocytes by T cells was suggested 4. Albanesi, C., C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, S. Sozzani, and to lead to a neutrophil inflammation-boosting loop that may ex- G. Girolomoni. 2001. A cytokine-to-chemokine axis between T lymphocytes and keratinocytes can favor Th1 cell accumulation in chronic inflammatory skin dis- plain the local acute inflammatory changes of pustular psoriasis eases. J. Leukocyte Biol. 70:617. (43). In AGEP, tissue cells such as keratinocytes also produce 5. Agostini, C., M. Facco, M. Chilosi, and G. Semenzato. 2001. Alveolar macro- CXCL8 and may contribute to the recruitment of neutrophils as phage-T cell interactions during Th1-type sarcoid inflammation. Microsc. Res. 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Thus, this tissue cell-derived 10. Britschgi, M., U. C. Steiner, S. Schmid, J. P. Depta, G. Senti, A. Bircher, CXCL8 production is clearly a secondary response, as it is depen- C. Burkhart, N. Yawalkar, and W. J. Pichler. 2001. T-cell involvement in drug- induced acute generalized exanthematous pustulosis. J. Clin. Invest. 107:1433. dent on the persistent stimulation of specific T cells by the relevant 11. Britschgi, M., and W. J. Pichler. 2002. Acute generalized exanthematous pustu- Ag, which in AGEP is often a drug. The important role of an losis, a clue to neutrophil-mediated inflammatory processes orchestrated by T antigenic stimulus is also well documented by the natural course of cells. Curr. Opin. Allergy Clin. Immunol. 2:325. 12. Schmid, S., P. C. Kuechler, M. Britschgi, U. C. Steiner, N. Yawalkar, A. Limat, AGEP, as cessation of the incriminated drug leads to a rapid dis- K. Baltensperger, L. Braathen, and W. J. Pichler. 2002. 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Skin and laboratory tests in amoxicillin- ␣ ␥ and penicillin-induced morbilliform skin eruption. Clin. Exp. Allergy 30:590. were TNF- producers, but failed to secrete IFN- , IL-4, IL-5, 16. Wyss-Coray, T., H. Gallati, I. Pracht, A. Limat, D. Mauri, K. Frutig, and IL-10, and IL-13. The relevance and rather unique function of such W. J. Pichler. 1993. Antigen-presenting human T cells and antigen-presenting B CXCL8ϩ T cells are also underlined by an impressive report of a cells induce a similar cytokine profile in specific T cell clones. Eur. J. Immunol. 23:3350. patient who developed disseminated pustulosis in the frame of an 17. Wilkinson, D. S., S. Fregert, B. Magnusson, H. J. Bandmann, C. D. Calnan, erythrodermic cutaneous T cell lymphoma. These tumor cells pro- E. Cronin, N. Hjorth, H. J. Maibach, K. E. Malalten, C. L. Meneghini, and duced high amounts of CXCL8, which was seen as reason for the V. Pirila. 1970. Terminology of contact dermatitis. Acta Derm. Venereol. 50:287. 18. 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