IFN-γ producing effector CD8 + T cells and IL-10 producing regulatory CD4 + T cells in fixed drug eruption

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1 IFN-γ producing effector CD8 + T cells and IL-10 producing regulatory CD4 + T cells in fixed drug eruption Yuichi Teraki, MD, and Tetsuo Shiohara, MD Tokyo, Japan Background: Although effector and regulatory T cells play roles in the progression and resolution of inflammatory diseases, respectively, little in vivo data exist regarding the T-cell dynamics in the pathogenesis of inflammatory skin diseases in humans. Objective: Our aim is to phenotypically and functionally characterize the T cells responsible for initiation and regulation of inflammatory events in fixed drug eruption (FDE) as a disease model to study the role of cytokines produced within the epidermis in the pathogenesis of inflammatory skin disease. Methods: By use of flow cytometry, we phenotypically and functionally characterized the intraepidermal T cells that persist as a stable population in resting (pigmented) FDE lesions and that are present in active FDE lesions. Results: In resting FDE lesions, most of the intraepidermal T cells were of the CD8 phenotype, most of which expressed cutaneous lymphocyte-associated antigen, α4β1, CD11a, αeβ7, and CD45RA but not CD27, CD62L, CCR4, or CCR7. This population selectively expressed CD122 but not CD25. Intracellular staining demonstrated that most intraepidermal CD8 + T cells were capable of producing IFN-γ and TNF-α but produced little IL-2 and IL-4. On the other hand, in the FDE lesions that arose after challenge, a significant number of CD4 + T cells capable of producing IL-10 migrated into the lesional epidermis. Moreover, nearly 70% of the CD4 + T cells migrating into the lesional epidermis expressed CD25. Conclusions: Effector IFN-γ producing CD8 + T cells and regulatory IL-10 producing CD4 + T cells might be responsible for the progression and resolution of FDE, respectively. (J Allergy Clin Immunol 2003;112: ) Key words: Fixed drug eruption, intraepidermal T cell, effectormemory T cell, regulatory T cell, IFN-γ, IL-10, homing receptor, chemokine receptor, skin inflammation Both the progression and resolution of skin inflammation require interactions between infiltrating inflammatory cells and resident skin cells. This communication is mainly mediated by means of the release of cytokines by both cell populations. T lymphocytes play a central role in the pathogenesis of a variety of diseases, including From the Department of Dermatology, Kyorin University School of Medicine, Tokyo. Received for publication February 10, 2003; revised April 23, 2003; accepted for publication May 6, Reprint requests: Yuichi Teraki, MD, Department of Dermatology, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo , Japan Mosby, Inc. All rights reserved /2003 $ doi: /mai Abbreviations used CLA: Cutaneous lymphocyte-associated antigen FDE: Fixed drug eruption PE: Phycoerythrin autoimmune diseases, allergic diseases, and numerous inflammatory skin diseases. 1-5 During the past several years, progress has been made ascertaining the roles of the subsets of T lymphocytes in inflammatory skin diseases. For example, in allergic contact dermatitis, haptenspecific CD8 + T cells (also CD4 + T cells) play a distinct role in the progression of disease, providing the relevant effector mechanisms for tissue damage. 3,4 On the other hand, the resolution of allergic contact dermatitis involves secretion of IL-10 by CD4 + T cells, the production of which impairs the functions of dendritic cells but also directly affects T cells. 5 Thus, although evidence is accumulating that the progression and resolution of inflammatory diseases are mediated at least in part by effector and regulatory T cells, respectively, there are little in vivo data regarding the T-cell dynamics in the pathogenesis of inflammatory skin diseases in humans. Fixed drug eruption (FDE) is a distinctive drug eruption that is characterized by recurrence at the same site on the skin or mucous membrane and by a cell-mediated cytotoxic reaction against epidermal keratinocytes. Although mechanisms and factors that contribute to the preferential localization of FDE lesions to certain skin sites are not fully understood, strong evidence exists that the intraepidermal CD8 + T cells in the lesional skin are the final effector cells in the epidermal injury of FDE, resulting in the selective destruction of the lesional epidermis. 6,7 In fact, after clinical resolution, CD8 + T cells persist in the resting (pigmented) FDE lesions and are found residing among the basal keratinocytes. 6-8 FDE offers a good disease model to study the role of local cytokines production in the pathogenesis of human inflammatory skin diseases. This is an important model for the following reasons: (1) the inflammatory reaction is confined to the lesional epidermis; (2) the reaction can be reproduced by a clinical challenge; and (3) the orchestrated series of events, which results in the destruction of the epidermal cells, can be examined in its entirety. In this study, to clarify the cellular events underlying the progression and resolution of FDE, we phenotypically and functionally characterized the cells that are present in the active FDE lesions and that persist as a stable population in the epidermis of resting FDE lesions. Here- 609

2 610 Teraki and Shiohara J ALLERGY CLIN IMMUNOL SEPTEMBER 2003 in, we demonstrated that effector IFN-γ producing CD8 + T cells and regulatory IL-10 producing CD4 + T cells might be responsible for the progression and resolution of FDE, respectively. METHODS Subjects Twelve patients with FDE (5 men and 7 women; age range, 23-80) were enrolled in this study after giving informed consent. The drugs responsible for FDE were as follows: allylisopropylacetylurea (n = 3), borraginol-n R (n = 1), ethenzamide (n = 2), allopurinol (n = 1), barbital (n = 1), ibuprofen (n = 1), mefenamic acid (n = 1), sulfamethoxazole (n = 1), and tipepidine hybenzate (n = 1). Biopsy specimens were obtained from resting FDE lesions at least 4 weeks after erythema had subsided. In 6 of 12 cases, biopsy specimens were obtained from the site of recurrence sites 24 hours after challenge with the causative drug (allylisopropylacetylurea [n = 3], etenzamide [n = 1], ibuprofen [n = 1], sulfamethoxazole [n = 1]). The doses used for the challenges were as follows: allylisopropylacetylurea (30 mg), ethenzamide (200 mg), ibuprofen (100 mg), and sulfamethoxazole (1 g). This study was approved by the ethics committee of Kyorin University School of Medicine. MAbs MAbs against cutaneous lymphocyte-associated antigen (CLA) (FITC-conjugated), CCR4 (phycoerythrin [PE]-conjugated), CCR7 (PE), IL-2 receptor β (CD122) (PE), and IL-10 (PE) were obtained from Pharmingen (San Diego, Calif). MAbs against αeβ7 (CD103) (PE) and CXCR3 (carboxyfluorescein succinimidylester) were obtained from Dako Cytomation (Glostrup, Denmark). MAbs against CD3 (allophycocyanin [APC]), CD4 (APC), CD8 (peridnin chlorophyll protein), IL-2 receptor α (CD25) (PE), CD27 (FITC), CD45RA (FITC), CD62L (L-selectin) (PE), CD11a (FITC), α4β1 (CD49d) (PE), IFN-γ (FITC, PE), TNF-α (FITC, PE), IL-2 (PE), IL-4 (PE), and isotype controls were obtained from Becton Dickinson (San Jose, Calif). Epidermal cell preparation and isolation of PBMC Epidermal single cell suspensions were prepared as described previously. 9 Epidermal sheets were separated in RPMI 1640 medium (supplemented with 10% FCS, 10 mmol/l HEPES buffer, 0.5 mmol/l 2-ME, 2 mmol/l glutamine, 1 mmol/l sodium pyruvate, 100 U/mL penicillin, and 100 µg/ml streptomycin) containing 10 mg/ml dispase for 2 hours at 37 C. To obtain single cell suspensions, the epidermal sheet was further treated with RPMI medium containing 10 mg/ml dispase for 1 hour at 37 C. Epidermal single cell suspensions were filtered thorough a nylon mesh, followed by 3 washes with HBSS. Cells were incubated for 12 hours at 37 C and then were prepared for flow-cytometric analysis. PBMCs were isolated from heparinized venous blood by density gradient sedimentation over Ficoll-Histopaque (Sigma Chemical Co., St Louis, Mo). Flow-cytometric assays Staining of cells for flow cytometry was performed as described previously. 10 After washing with PBS containing 0.1% BSA (PBS/BSA), cells were stained with cell surface markers for 25 minutes at 4 C. After staining, the cells were washed and resuspended in 0.5 ml of 1% paraformaldehyde in PBS before flowcytometric analysis. For intracellular staining, cells were stimulated for 4 hours at 37 C in RPMI 1640 medium containing 25 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma), 1 µmol/l of ionomycin (Sigma), and 10 µg/ml of Brefeldin A (Sigma). After harvesting, the cells were directly stained with cell-surface markers for 25 minutes at 4 C. After washing with PBS/BSA, the cells were incubated in 0.5 ml of lysing solution and then 0.5 ml of permeabilizing solution (Becton Dickinson) at room temperature, washed with PBS/BSA, and then incubated for 25 minutes with fluorochrome-conjugated mabs specific for IL-2, IL-4, IL-10, IFN-γ, or TNF-α. Samples were analyzed for 4-color staining with the FAC- SCalibur Flow Cytometer (Becton Dickinson). Multiparameter data files were analyzed with PAINT-A-GATE plus software (Becton Dickinson). Statistical analysis The results were expressed as the mean value ± SEM. Data were analyzed with the Mann-Whitney U test. P values less than.05 were considered significant. RESULTS Phenotypic analysis of intraepidermal T cells in the resting FDE lesion We previously demonstrated by immunohistochemistry that a significant number of CD8 + T cells can be seen among the basal keratinocytes for a considerable amount of time after clinical resolution of the FDE lesions. In this study, epidermal single-cell suspensions were prepared from skin biopsy specimens of resting FDE lesions, and the lymphocytes within them were characterized by flow cytometry. Because the preparation of single-cell suspensions involved enzymatic digestion, it was necessary to incubate the cells for at least 12 hours before staining to allow recovery of cell-surface markers (CD3, CD4, and CD8). To show that expression of surface markers does in fact return to normal after 12 hours of culture, PBMCs were treated with the same enzyme followed by culture for 12 hours. We found no significant differences in the percentages of CD3, CD4, CD8, CD25, CD27, CD45RA, CD122, CLA, α4β1, CD11a, CD62L, αeβ7, CCR4, CCR7, and CXCR3 positive cells between enzyme-treated and freshly separated PBMCs. As shown in Fig 1, a lymphocyte population consisting of primarily CD3 + T cells was identified by gating on the CD3 + population versus the side scatter population. Most of intraepidermal T cells in the FDE lesions consisted of CD8 + T cells (92.6% ± 1.7%), consistent with previous immunohistochemical studies. 6,8 We then examined the phenotypic properties of intraepidermal CD8 + T cells in the resting FDE lesions (Fig 2). Recent studies have shown that memory T cells can be divided into the central-memory and effectormemory phenotype on the basis of several surface markers, including CD45RA, CD62L, and CCR7. 11,12 Phenotypic analysis revealed that most of the intraepidermal CD8 + T cells were positive for CD45RA, but not CD27, CD62L, and CCR7, indicative of the effector-memory phenotype. Because effector-memory T cells possess diverse migratory specificities for nonlymphoid tissues such as skin and gut, homing properties of the intraepidermal CD8 + T cells were also examined. The intraepidermal CD8 + T cells expressed CLA, α4β1, and CD11a, indicating that these cells have a skin-specific migratory

3 J ALLERGY CLIN IMMUNOL VOLUME 112, NUMBER 3 Teraki and Shiohara 611 capacity. It has been reported that CCR4 is expressed at high levels by CLA + memory T cells in the peripheral blood 13,14 ; however, only a few intraepidermal CD8 + T cells expressed CCR4. In contrast, about 30% of the intraepidermal CD8 + T cells expressed CXCR3. Because recent evidence has suggested that αeβ7, a ligand for E- cadherin, might play a role in the localization of T cells within the epidermis, 15,16 we also examined αeβ7 integrin expression by intraepidermal CD8 + T cells. More than 95% of the intraepidermal CD8 + T cells in the FDE lesions were positive for αeβ7, indicating that intraepidermal CD8 + T cells are, in fact, effector-memory cells with epidermal localization capacity. To determine the factors involved in maintaining survival of the intraepidermal CD8 + T cells in the lesional skin, we subsequently examined IL-2 receptor α-and β- chain on the intraepidermal CD8 + T cells in the FDE lesions. Most intraepidermal CD8 + T cells selectively expressed the β-chain but not the α-chain, indicative of resting T cells. Although IL-2 is thought to be an important cytokine for T cell growth, intracellular IL-2 staining revealed that IL-2 was not expressed by the intraepidermal CD8 + T cells. These results suggest that the survival of intraepidermal CD8 + T cells might not be dependent on autocrine IL-2 production. Considering the fact that IL-2 receptor β-chain is common to both the IL-2 and IL- 15 receptors and the fact that IL-15 is produced by a wide variety of tissues including the skin, 20,21 the survival of intraepidermal CD8 + T cells in FDE lesions is likely maintained by IL-15. Capability of intraepidermal CD8 + T cells in the resting FDE lesion to produce cytokines To examine cytokine production at a single-cell level, intraepidermal CD8 + T cells in resting FDE lesions were stimulated for 4 hours with phorbol 12-myristate 13- acetate and ionomycin in the presence of Brefeldin A (Fig 3). Nearly 90% of the intraepidermal CD8 + T cells were capable of producing both IFN-γ and TNF-α, whereas few CD8 + T cells were capable of producing IL- 2 or IL-4. The proportions of CD8 + T cells capable of producing IFN-γ and TNF-α were significantly higher in the FDE lesions than in the peripheral blood counterparts from the same patients, whereas those capable of producing IL-2 and IL-4 were significantly lower in FDE lesions than peripheral blood CD8 + T cells. These results indicate that intraepidermal CD8 + T cells in resting FDE lesions have effector-memory functions with a high capacity to produce IFN-γ and TNF-α. CD25 + CD4 + T cells capable of producing IL-10 migrated into the epidermis in the active FDE lesion In active FDE lesions, numerous cells migrate into the epidermis of lesional skin. We therefore examined T cell subsets in the active FDE lesions 24 hours after challenge with the causative drug. Although few CD4 + T cells were observed in the resting FDE lesions, a number of CD4 + FIG 1. Flow cytometric analysis of intraepidermal T cells in fixed drug eruption (FDE) lesions. Epidermal cellular suspensions were prepared from skin biopsy specimens of resting (pigmented) FDE lesions. A lymphocyte population was isolated by gating on the CD3 + population versus the side scatter population. A substantial number of CD3 + T cells were detected within the epidermis of the resting FDE lesions, most of which were CD8 + T cells. T cells, as well as CD8 + T cells, migrated into the epidermis of active FDE lesions. The proportions of lymphocytes positive for CD4 and CD8 were 32.4% ± 5.3% and 65.3% ±5.1%, respectively (Fig 4). There is now compelling evidence that a subset of CD4 + T cells, named regulatory T cells, specialize in the suppression of immune responses and play an important role in the control of immune pathology. 2,22,23 Recent studies have shown that regulatory T cells control harmful immune responses through the production of suppressive cytokines, such as IL-10. In addition, evidence exists that a subset of regulatory T cells is enriched within the CD25 + CD4 + subset. In active FDE lesions, nearly 70% of CD4 + T cells within the epidermis expressed CD25. This proportion was significantly higher than that in the peripheral blood counterparts of the same patients (Fig 5, A). In addition, intracellular cytokine staining demonstrated that there were a significant number of CD4 + T cells capable of producing IL-10 in the epidermis of active FDE lesions. The proportion of IL-10 producing CD4 + T cells was also much higher in FDE lesions than in the peripheral blood counterparts of the same patients (Fig 5, B). The proportion of CD8 + T cells capable of producing IFN-γ decreased in the active FDE lesions compared with that in the resting FDE lesions (51.1% ± 2.9% vs 86.4% ± 3.7%). These results suggest that CD4 + T cells migrating into the epidermis of active FDE lesions might exert a regulatory function by means of the release of IL-10, resulting in spontaneous resolution of the lesion. DISCUSSION T cells are divided into naive and memory phenotypes on the basis of cell surface markers and functional differences. Recent work has further subdivided the memory population into 2 types with different functional activities and migration properties. 11,12 CCR7 + CD62L + cells are referred to as central-memory cells and are home to lym-

4 612 Teraki and Shiohara J ALLERGY CLIN IMMUNOL SEPTEMBER 2003 FIG 2. Phenotypic properties of intraepidermal CD8 + T cells in the resting fixed drug eruption lesions. Most of intraepidermal CD8 + T cells expressed CD45RA, but not CD27, CD62L, and CCR7, indicative of the effector-memory phenotype. Most of these cells expressed the skin-homing receptor cutaneous lymphocyteassociated antigen, α4β1 and CD11a. These cells also expressed αeβ7. Only a few intraepidermal CD8 + T cells expressed CCR4, whereas about 30% of these cells expressed CXCR3. Most intraepidermal CD8 + T cells selectively expressed IL-2Rβ (CD122) but not IL-2Rα (CD25). phoid organs, whereas the CCR7 CD62L cells are referred to as effector-memory cells and might possess diverse migratory specificities for nonlymphoid tissues and peripheral sites of inflammation. Although migration and differentiation of CD8 + T cells seem to be somewhat more complex, several lines of evidence have shown that the effector-memory CD8 + subset expresses CD27 CD28 CD45RA + CD62L CCR7 and has high levels of perforin and a high capacity to produce IFN-γ and TNFα. 12,24-27 In view of these data, the intraepidermal CD8 + T cells in the resting FDE lesion are consistent with the effector-memory phenotype. Our results indicate that the intraepidermal CD8 + T cells in the resting FDE lesion consist of an oligoclonal population with regard to phenotypic and cytokine profiles. This finding is also supported by a previous report that intraepidermal T cells in FDE lesions use a very limited T cell repertoire consisting of the Vα and Vβ gene families. 28 Thus, it is likely that epidermal injury in the active FDE lesion is at least in part mediated by IFN-γ and TNF-α, which are produced by intraepidermal CD8 + T cells in the lesional skin. It is interesting to consider the mechanisms underlying the persistence of CD8 + T cells in the epidermis of FDE lesions. One possibility is that CD8 + T cells are maintained by continual recruitment from the peripheral blood. Tissue-selective migration of memory and effector T cells is tightly regulated by a variety of adhesion molecules and molecular signaling. 29,30 In this study,almost all intraepidermal CD8 + T cells expressed CLA, α4β1, and CD11a. In addition, we previously demonstrated that E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, which are ligands for CLA, α4β1, and CD11a, respectively, were strongly upregulated on the vascular endothelium in the active FDE lesions. 6 These findings suggest that CD8 + T cells might be a population migrating into the lesional skin through the interaction of CLA/E-selectin, α4β1/vascular cell adhesion molecule-1, and CD11a/intercellular adhesion molecule-1. It has been

5 J ALLERGY CLIN IMMUNOL VOLUME 112, NUMBER 3 Teraki and Shiohara 613 FIG 3. Cytokine production capability of intraepidermal CD8 + T cells in the resting fixed drug eruption (FDE) lesion. Cytokine production capability of intraepidermal CD8 + T cells was measured at a single cell level after stimulation for 4 hours with phorbol 12-myristate 13-acetate and ionomycin in the presence of Brefeldin A. Nearly 90% of the intraepidermal CD8 + T cells were capable of producing both IFN-γ and TNF-α, whereas few CD8 + T cells were capable of producing IL-2 or IL-4. The proportions of CD8 + T cells capable of producing IFN-γ and TNF-α were significantly higher in the FDE lesions than in the peripheral blood counterparts from the same patients (P <.05), whereas the proportions of cells capable of producing IL-2 and IL-4 were significantly lower in FDE lesions (P <.05). FIG 4. The percent of CD4 + and CD8 + lymphocytes in the epidermis of active fixed drug eruption lesions 24 hours after challenge with the causative drug. A number of CD4 + T cells migrated into the lesional epidermis. The proportions of CD4 + and CD8 + T cells were 32.4% ± 5.3% and 65.3% ± 5.1%, respectively. shown that circulating CLA + CD4 + T cells selectively express high levels of CCR4 and that thymus and activation-regulated chemokine, a ligand for CCR4, is expressed on inflamed cutaneous endothelium. 13,14 Therefore, the interaction of CCR4/thymus and activation-regulated chemokine is thought to participate in the skin-specific lymphocyte recruitment. However, most of the intraepidermal CD8 + T cells in the resting FDE lesions did not express CCR4. This might be because CCR4 expression on CD8 + T cells differs from those on CD4 + T cells. Alternatively, it might be ascribed that the intraepidermal CD8 + T cells are of the Tc1 phenotype,

6 614 Teraki and Shiohara J ALLERGY CLIN IMMUNOL SEPTEMBER 2003 A B FIG 5. CD25 expression and IL-10 producing capability by the CD4 + T cells in the epidermis of active fixed drug eruption lesions. A, The proportion of CD25-expressing cells in the lesional skin was significantly higher than that in the peripheral blood counterparts from the same patient (P <.05). B, Also, the proportion of IL-10 producing cells in the lesional skin was much higher than that in peripheral blood counterparts from the same patient (P <.05). because CCR4 is preferentially expressed on T H 2 cells. 31 It is also possible that cells downregulate receptor expression on reaching their destination. Another possible explanation is that the intraepidermal CD8 + T cells are maintained by continual proliferation in the epidermal microenvironment. The potent T- cell growth factor IL-2 is produced mainly by T cells; however, intracellular IL-2 staining revealed a lack of IL- 2 expression by intraepidermal CD8 + T cells in the resting FDE lesions. In this regard, it should also be noted that the intraepidermal CD8 + T cells in FDE lesions expressed the IL-2 receptor β-chain but not the α-chain. The IL-2 receptor β-chain is shared between both the IL- 2 and IL-15 receptors, and IL-15 has been shown to stimulate lymphocytes by way of the IL-2 receptor βγ complex IL-15 is produced by a wide variety of tissues, including epidermal keratinocytes. 21 Taken together, these data suggest that survival of intraepidermal CD8 + T cells in FDE lesions might be maintained by IL-15. This possibility is similar to the recent finding that IL-15 plays a key role in maintaining the survival of intraepithelial lymphocytes. 32,33 There is accumulating evidence that CD4 + regulatory T cells play a key role in the control of immune pathology. Although many distinct types of regulatory T cells have been described in a number of experimental models, it has been established that regulatory T cells suppress immune responses by means of cell-to-cell interactions or the production of cytokines. 2,22,23 IL-10 is a potent anti-inflammatory and suppressive cytokine produced by various types of cells,including T lymphocytes. 34 Therefore, it is important that a significant number of CD4 + T cells capable of producing IL-10 were recruited into the active FDE lesion 24 hours after challenge. Moreover, it should be noted that nearly 70% of the CD4 + T cells migrating into the active FDE lesions expressed CD25, because the CD25 + CD4 + T cells have been found to produce high levels of IL Significantly fewer of the lesional CD8 + T cells in active FDE lesions were capable of expressing IL-10 compared with the lesional CD4 + T cells (CD8; 1.5% ± 0.3% vs CD4; 6.1% ± 1.0%), thus IL- 10 released by infiltrating CD25 + CD4 + T cells might suppress the effector-memory function of CD8 + T cell in the active FDE lesions. On the other hand, a recent study has demonstrated that IL-15 induces and IL-2 suppresses the division of memory CD8 + T cells. 36 In this context, Murakami et al 37 showed that CD25 + CD4 + T cells are involved in the IL-2 mediated inhibition of memory CD8 + T cell division. In any case, the migration of CD25 + CD4 + T cells into the lesional epidermis of FDE might result in spontaneous resolution on withdrawal of the causative drug.

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