Role of the Chemokine Receptor CCR4 and its Ligand Thymus- and Activation-Regulated Chemokine/CCL17 for Lymphocyte Recruitment in Cutaneous Lupus Erythematosus
2005; Elsevier BV; Volume: 124; Issue: 6 Linguagem: Inglês
10.1111/j.0022-202x.2005.23755.x
ISSN1523-1747
AutoresJoerg Wenzel, Stephanie Henze, Eva Wörenkämper, Etiena Basner‐Tschakarjan, Małgorzata Sokołowska‐Wojdyło, Julia Steitz, Thomas Bieber, Thomas Tüting,
Tópico(s)Chemokine receptors and signaling
ResumoSkin-infiltrating T lymphocytes are thought to play a major role in the pathogenesis of cutaneous lupus erythematosus (CLE). In this study, we investigated the role of the chemokine receptor 4 (CCR4) and its ligand thymus- and activation-regulated chemokine (TARC/CCL17) for the recruitment of T cells in inflamed skin of patients with CLE. We found significant numbers of CCR4+ T lymphocytes in the skin of all patients with CLE. Interestingly, a subset of patients with disseminated scarring skin involvement were characterized by both lesional and circulating CD8+ T cells expressing CCR4. Destruction of epidermal and adnexal structures was histomorphologically associated with CCR4+ cytotoxic T cells invading basal layers of the epidermis where keratinocytes showed apoptotic death. The CCR4 ligand TARC/CCL17 was strongly expressed in skin lesions and elevated in the serum of CLE patients. The functional relevance of lymphocytic CCR4 expression could be confirmed by TARC/CCL17-specific in vitro migration assays. Our investigations suggest that CCR4 and TARC/CCL17 play a role in the pathophysiology of CLE. In particular, cytotoxic CD8+ T cells expressing CCR4 appear to be involved in scarring subtypes of CLE. Skin-infiltrating T lymphocytes are thought to play a major role in the pathogenesis of cutaneous lupus erythematosus (CLE). In this study, we investigated the role of the chemokine receptor 4 (CCR4) and its ligand thymus- and activation-regulated chemokine (TARC/CCL17) for the recruitment of T cells in inflamed skin of patients with CLE. We found significant numbers of CCR4+ T lymphocytes in the skin of all patients with CLE. Interestingly, a subset of patients with disseminated scarring skin involvement were characterized by both lesional and circulating CD8+ T cells expressing CCR4. Destruction of epidermal and adnexal structures was histomorphologically associated with CCR4+ cytotoxic T cells invading basal layers of the epidermis where keratinocytes showed apoptotic death. The CCR4 ligand TARC/CCL17 was strongly expressed in skin lesions and elevated in the serum of CLE patients. The functional relevance of lymphocytic CCR4 expression could be confirmed by TARC/CCL17-specific in vitro migration assays. Our investigations suggest that CCR4 and TARC/CCL17 play a role in the pathophysiology of CLE. In particular, cytotoxic CD8+ T cells expressing CCR4 appear to be involved in scarring subtypes of CLE. chronic discoid lupus erythematosus cutaneous lymphocyte antigen cutaneous lupus erythematosus disseminated CDLE localized CDLE lupus erythematosus subacute cutaneous LE systemic LE thymus- and activation-regulated chemokine Lupus erythematosus (LE) is an autoimmune disorder with a broad clinical spectrum reaching from primarily cutaneous manifestations (CLE) up to systemic disease (SLE). Frequent cutaneous subtypes of LE are the scarring chronic discoid LE (CDLE) and the non-scarring subacute CLE (SCLE). Patients suffering from CDLE are characterized by scarring discoid plaques, ranging from localized to disseminated variants. SCLE patients are characterized by nummular or gyrated erythematosquamous lesions, predominantly in sun-exposed areas without scarring. Histological analyses of CLE lesions typically reveal dense CD3+ junctional and periadnexal infiltrates (Tebbe et al., 1995Tebbe B. Mazur L. Stadler R. Orfanos C.E. Immunohistochemical analysis of chronic discoid and subacute cutaneous lupus erythematosus—relation to immunopathological mechanisms.Br J Dermatol. 1995; 132: 25-31Crossref PubMed Scopus (52) Google Scholar). These supposedly autoaggressive T lymphocytes are thought to play a significant role in the pathogenesis of CLE lesions. A little more than 10 y ago, studies addressing the immunobiology of T cell trafficking provided the first insights into the molecular regulation of T cell homing to the skin (Robert and Kupper, 1999Robert C. Kupper T.S. Inflammatory skin diseases, T cells, and immune surveillance.N Engl J Med. 1999; 341: 1817-1828Crossref PubMed Scopus (431) Google Scholar). It could be shown that naïve T cells express adhesion molecules like L-selectin on their surface, which allow them to enter lymph nodes via high endothelial venules expressing appropriate ligands such as peripheral node adressin. Once T cells become activated by skin-derived antigen-presenting dendritic cells in draining lymph nodes, they proliferate, express activation and effector molecules, and undergo the transition to memory T cells (Sallusto et al., 2000Sallusto F. Mackay C.R. Lanzavecchia A. The role of chemokine receptors in primary, effector, and memory immune responses.Annu Rev Immunol. 2000; 18: 593-620Crossref PubMed Scopus (926) Google Scholar). During this transition, some effector and memory T cells acquire new molecular keys such as the cutaneous lymphocyte antigen (CLA), which allow them to preferentially enter inflamed skin where the appropriate ligands are expressed on cutaneous microvascular endothelial cells (Tietz et al., 1998Tietz W. Allemand Y. Borges E. et al.CD4+T cells migrate into inflamed skin only if they express ligands for E- and P-selectin.J Immunol. 1998; 161: 963-970PubMed Google Scholar). It could be shown that the majority of T cells in inflamed skin lesions such as atopic dermatitis, psoriasis vulgaris, lichen planus, discoid lupus erythematosus, and cutaneous T cell lymphoma indeed express CLA on their cell surface (Heald et al., 1993Heald P.W. Yan S.L. Edelson R.L. Tigelaar R. Picker L.J. Skin-selective lymphocyte homing mechanisms in the pathogenesis of leukemic cutaneous T-cell lymphoma.J Invest Dermatol. 1993; 101: 222-226Abstract Full Text PDF PubMed Google Scholar; Nakamura et al., 1998Nakamura K. Yasaka N. Asahina A. et al.Increased numbers of CD68 antigen positive dendritic epidermal cells and upregulation of CLA (cutaneous lymphocyte-associated antigen) expression on these cells in various skin diseases.J Dermatol Sci. 1998; 18: 170-180Abstract Full Text PDF PubMed Scopus (29) Google Scholar). The majority of CLA+ memory T cells express the chemokine receptor (CCR)4 (Campbell et al., 1999Campbell J.J. Haraldsen G. Pan J. et al.The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (737) Google Scholar). With regard to T cell trafficking into inflamed skin, recent studies have highlighted the role of CCR4 and its ligand thymus- and activation-regulated chemokine (TARC/CCL17) (Schon et al., 2003Schon M.P. Zollner T.M. Boehncke W.H. The molecular basis of lymphocyte recruitment to the skin: Clues for pathogenesis and selective therapies of inflammatory disorders.J Invest Dermatol. 2003; 121: 951-962Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Increased expression of CCR4 was demonstrated in atopic dermatitis (Wakugawa et al., 2001Wakugawa M. Nakamura K. Kakinuma T. et al.CC chemokine receptor 4 expression on peripheral blood CD4+T cells reflects disease activity of atopic dermatitis.J Invest Dermatol. 2001; 117: 188-196Crossref PubMed Scopus (91) Google Scholar), psoriasis (Inaoki et al., 2003Inaoki M. Sato S. Shirasaki F. Mukaida N. Takehara K. The frequency of type 2 CD8+T cells is increased in peripheral blood from patients with psoriasis vulgaris.J Clin Immunol. 2003; 23: 269-278Crossref PubMed Scopus (19) Google Scholar), and in cutaneous T cell lymphoma (Kallinich et al., 2003Kallinich T. Muche J.M. Qin S. et al.Chemokine receptor expression on neoplastic and reactive T cells in the skin at different stages of mycosis fungoides.J Invest Dermatol. 2003; 121: 1045-1052Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Interaction between CCR4 and its ligand TARC/CCL17 on activated endothelial cells mediates T cell extravasation by stimulating integrin-dependent adhesion of CLA+ T cells to intercellular adhesion molecule-1 (Campbell et al., 1999Campbell J.J. Haraldsen G. Pan J. et al.The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (737) Google Scholar). In this study, we investigated the expression of CCR4 on T lymphocytes in both skin and peripheral blood of patients suffering from CLE. We show that significant numbers of lesional T lymphocytes express CLA and CCR4. Importantly, the coexpression CLA+ and CCR4+ on lesional and circulating CD8+ T lymphocytes defines a subset of CLE patients characterized by disseminated scarring lesions. Destruction of epidermal and adnexal structures was associated with infiltration of CD8+ T cells largely expressing the cytotoxic molecule granzyme B. Increased TARC/CCL17 levels were found in lesional skin and serum of CLE patients. Importantly, CCR4-expressing lymphocytes isolated from the blood of CDLE patients showed TARC/CCL17-specific in vitro migration, confirming the functional relevance of our findings. CLE lesions are immunohistologically characterized by a dense junctional and perivascular T cellular infiltrate, primarily composed of CD4+ and CD8+ cells (Figure 1a–c). In initial studies, we further investigated infiltrating lymphocytes by immunohistochemistry for the homing molecules CLA and CCR4. As expected, large numbers of CLA-expressing lymphocytes were present in skin lesions of CDLE, SCLE, and SLE when compared with healthy controls. Figure 1 shows a semiquantitative analysis with numbers of cells per high-power field of view. Furthermore, as detailed in Figure 1e, significantly increased expression of CCR4 was also found in lesions of CDLE when compared with healthy controls (p<0.01). We consistently noted a prominent infiltration with CD8+ T cells in lesions from patients with scarring subtypes of CLE. To assess the cytotoxic function of these CD8+ T cells, we stained for the cytotoxic molecule granzyme B. As shown in Figure 1f, patients with CDLE displayed significantly increased expression of granzyme B (p<0.01) when compared with other LE subtypes and healthy controls. Significant differences between patients suffering from localized and disseminated CDLE were not found. Thus, strong expression of granzyme B was closely associated with epidermal and adnexal destruction. Representative clinical, histomorphological, and immunohistochemical findings of a patient suffering from disseminated scaring CDLE are depicted in Figure 2.Figure 2Representative clinical and immunohistochemical findings in a patient suffering from scarring discoid lupus erythematosus (LE). Clinical investigation revealed scarring disseminated erythematosquamous plaques (a, b). Skin lesions were immunohistologically characterized by a dense junctional and perivascular lymphocytic infiltrate primarily composed of T cells, with a slight majority of CD4+ T cells (c) over CD8+ T cells (d). Furthermore, a high expression of cutaneous lymphocyte antigen (CLA) (e), CC receptor (CCR)4 (f), and granzyme B (g) was found (original magnification: × 200). Immunofluorescence double staining for CD8 (fluorescein isothiocyanate, green) and CCR4 (TRITC, red) revealed the presence of CCR4-expressing CD8+ T cells (yellow, →) in the upper dermis (h; original magnification: × 400).View Large Image Figure ViewerDownload (PPT) To confirm the expression of CCR4 on lesional CD8+ T cells, immunofluorescence double staining was performed. The analyses revealed expression of CCR4 on CD8+ infiltrating T lymphocytes in all of the four patients with disseminated CDLE (dCDLE). A representative result is shown in Figure 2h. Subsequently, we investigated the expression of skin homing molecules on circulating lymphocytes by flow cytometry. Significantly increased numbers of CD4+ T cells expressing CLA were present in peripheral blood mononuclear cells of patients with dCDLE and psoriasis when compared with healthy controls (Figure 3a). Furthermore, the percentage of circulating CD4+T lymphocytes expressing CCR4 was significantly higher in all investigated skin diseases when compared with healthy controls (Figure 3b). As a consequence, elevated numbers of CD4+ T lymphocytes expressing both CLA and CCR4 were found in patients with dCDLE and psoriasis (Figure 3c). Most importantly, we detected significantly increased numbers of circulating CD8+ T cells expressing CLA and CCR4 exclusively in patients with disseminated discoid LE (Figure 3d–f). This corresponds to the histopathological findings described above in this subgroup of patients. Representative flow cytometric results are depicted in Figure 4.Figure 4Representative findings of cutaneous lymphocyte antigen (CLA) and CC receptor (CCR)4 expression on CD8+ T cells. Typical flow cytometric findings of peripheral blood lymphocytes of a patient suffering from disseminated chronic discoid lupus erythematosus (dCDLE) and a healthy volunteer are depicted. Numbers represent the percentage of CD8+ T cells coexpressing CLA+ or CCR4+.View Large Image Figure ViewerDownload (PPT) As the expression of CCR4 on lesional and circulating lymphocytes in LE patients was increased, we analyzed the expression of the CCR4 ligand TARC/CCL17 in the skin by immunohistochemistry and in serum by ELISA. A high epidermal and perivascular expression of TARC/CCL17 was found in CDLE as well as in SCLE skin lesions (Figure 5a and b). Additionally, elevated TARC/CCL17 serum levels were found in all investigated LE subsets when compared with healthy controls (Figure 5c). To confirm the functional relevance of CCR4 on the surface of circulating T lymphocytes, we performed TARC/CCL17-directed chemokine migration assays in vitro. A TARC/CCL17 concentration of 40 ng per mL was found to be optimal for strong migration of CD4+ and CD8+ cells of patients with dCDLE (Figure 6). Migrated cells were exclusively CCR4 positive. Using the optimal TARC/CCL17 concentration, a mean chemotactic index of 11.1 for CD4+ cells and 12.7 for CD8+ cells was found. In healthy controls, the chemotactic index ranged between 0.7 and 1.7. Differences were highly significant (p<0.01). The relative TARC-directed migration of CD4+ CCR4+ lymphocytes (migrated cells/not migrated cells) ranged from 73% to 95% (mean 87.5±5 SEM, control without TARC: from 0% to 19%, mean 7.5±4.1 SEM); the TARC directed migration of CD8+ CCR4+ lymphocytes ranged from 88% to 94%, with a mean of 91.2%±2.1 SEM (control without TARC: from 0% to 23%, mean 8.25%±5.1 SEM). In this study, we found strong expression of the skin homing molecules CLA and CCR4+ on lesional and circulating T lymphocytes in CLE. CLA is well known to be involved in skin-directed inflammation (Rossiter et al., 1994Rossiter H. van Reijsen F. Mudde G.C. et al.Skin disease-related T cells bind to endothelial selectins: Expression of cutaneous lymphocyte antigen (CLA) predicts E-selectin but not P-selectin binding.Eur J Immunol. 1994; 24: 205-210Crossref PubMed Scopus (112) Google Scholar). Recently, a possible pathogenetic role for CCR4 in skin diseases has been considered.Nakatani et al., 2001Nakatani T. Kaburagi Y. Shimada Y. et al.CCR4 memory CD4+T lymphocytes are increased in peripheral blood and lesional skin from patients with atopic dermatitis.J Allergy Clin Immunol. 2001; 107: 353-358Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar described an increased expression of CCR4 on lesional and circulating CD4+ lymphocytes in patients suffering from atopic dermatitis.Wakugawa et al., 2001Wakugawa M. Nakamura K. Kakinuma T. et al.CC chemokine receptor 4 expression on peripheral blood CD4+T cells reflects disease activity of atopic dermatitis.J Invest Dermatol. 2001; 117: 188-196Crossref PubMed Scopus (91) Google Scholar found elevated expression of CCR4 on peripheral blood CD4+ lymphocytes in atopic dermatitis, which correlated with disease activity.Inaoki et al., 2003Inaoki M. Sato S. Shirasaki F. Mukaida N. Takehara K. The frequency of type 2 CD8+T cells is increased in peripheral blood from patients with psoriasis vulgaris.J Clin Immunol. 2003; 23: 269-278Crossref PubMed Scopus (19) Google Scholar published data of a slightly, but significantly elevated expression of CCR4 on peripheral blood CD8+ cells in psoriasis as compared with healthy volunteers.Ferenczi et al., 2002Ferenczi K. Fuhlbrigge R.C. Pinkus J. Pinkus G.S. Kupper T.S. Increased CCR4 expression in cutaneous T cell lymphoma.J Invest Dermatol. 2002; 119: 1405-1410Crossref PubMed Scopus (221) Google Scholar andKallinich et al., 2003Kallinich T. Muche J.M. Qin S. et al.Chemokine receptor expression on neoplastic and reactive T cells in the skin at different stages of mycosis fungoides.J Invest Dermatol. 2003; 121: 1045-1052Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar found circulating and lesional T lymphocytes in cutaneous T cell lymphoma mainly to be CCR4+. CCR4 expression in CLE has not been analyzed so far.Hase et al., 2001Hase K. Tani K. Shimizu T. et al.Increased CCR4 expression in active systemic lupus erythematosus.J Leukoc Biol. 2001; 70: 749-755PubMed Google Scholar, however, found an increased CCR4 expression on CD4+ T lymphocytes in active SLE. Furthermore, in lupus nephritis, CCR4+ CD4+ T lymphocytes accumulate in renal tissues (Yamada et al., 2002Yamada M. Yagita H. Inoue H. et al.Selective accumulation of CCR4+T lymphocytes into renal tissue of patients with lupus nephritis.Arthritis Rheum. 2002; 46: 735-740Crossref PubMed Scopus (38) Google Scholar). Skin lesions from patients with localized and disseminated CDLE were characterized by a high number of infiltrating CD8+ CCR4+ T lymphocytes. Histomorphologically, lesional CD8+ T cells expressing the cytotoxic molecule granzyme B were associated with destruction of epidermal and adnexal structures in this subgroup of patients. Interestingly, in the blood we found CD8+ T cells expressing both CLA and CCR4 exclusively in patients with disseminated scarring CDLE. This novel finding has not been described to date. We believe that the increased numbers of circulating CCR4+ CD8+ T cells found in patients with disseminated CDLE reflect the expansion of cytotoxic T lymphocytes that recognize epidermal antigens and therefore are able to destroy keratinocytes. To confirm the pathogenetic relevance of lymphocytic CCR4 expression in CLE, we subsequently analyzed the presence of the CCR4 ligand TARC/CCL17 in the skin and serum of LE patients. TARC/CCL17 had previously been shown to be elevated in several other skin disorders. For example, in atopic dermatitis increased serum TARC/CCL17 levels were associated with disease severity (Kakinuma et al., 2003aKakinuma T. Sugaya M. Nakamura K. et al.Thymus and activation-regulated chemokine (TARC/CCL17) in mycosis fungoides: Serum TARC levels reflect the disease activity of mycosis fungoides.J Am Acad Dermatol. 2003; 48: 23-30Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Kakinuma et al., 2003bKakinuma T. Wakugawa M. Nakamura K. et al.High level of thymus and activation-regulated chemokine in blister fluid and sera of patients with bullous pemphigoid.Br J Dermatol. 2003; 148: 203-210Crossref PubMed Scopus (62) Google Scholar; Hijnen et al., 2004Hijnen D. De Bruin-Weller M. Oosting B. et al.Serum thymus and activation-regulated chemokine (TARC) and cutaneous T cell- attracting chemokine (CTACK) levels in allergic diseases: TARC and CTACK are disease-specific markers for atopic dermatitis.J Allergy Clin Immunol. 2004; 113: 334-340Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Serum TARC/CCL17 levels also reflected the disease activity in mycosis fungoides (Kakinuma et al., 2003aKakinuma T. Sugaya M. Nakamura K. et al.Thymus and activation-regulated chemokine (TARC/CCL17) in mycosis fungoides: Serum TARC levels reflect the disease activity of mycosis fungoides.J Am Acad Dermatol. 2003; 48: 23-30Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Kakinuma et al., 2003bKakinuma T. Wakugawa M. Nakamura K. et al.High level of thymus and activation-regulated chemokine in blister fluid and sera of patients with bullous pemphigoid.Br J Dermatol. 2003; 148: 203-210Crossref PubMed Scopus (62) Google Scholar). Furthermore, in SLE the plasma level of TARC/CCL17 was elevated in active disease and correlated with disease severity (Kallinich et al., 2003Kallinich T. Muche J.M. Qin S. et al.Chemokine receptor expression on neoplastic and reactive T cells in the skin at different stages of mycosis fungoides.J Invest Dermatol. 2003; 121: 1045-1052Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Additionally, we revealed strong migration of CCR4-expressing T lymphocytes from CDLE patients toward the CCR4 ligand TARC/CCL17 in chemokine migration assays, ascertaining the functional relevance of lymphocytic CCR4 expression. We hypothesize that the production of TARC/CCL17 and the expression of CCR4 on T lymphocytes might represent an important biological amplification mechanism to promote inflammatory responses in skin lesions of CLE. This has been described in other inflammatory skin diseases such as psoriasis and atopic dermatitis. At present, little is known about the critical factors in the generation of skin-homing T lymphocytes in CLE as well as in other cutaneous immune responses mediated by T lymphocytes. In an experimental mouse model for contact hypersensitivity,Dudda et al., 2004Dudda J.C. Simon J.C. Martin S. Dendritic cell immunization route determines CD8+T cell trafficking to inflamed skin: Role for tissue microenvironment and dendritic cells in establishment of T cell-homing subsets.J Immunol. 2004; 172: 857-863Crossref PubMed Scopus (175) Google Scholar recently reported that intracutaneous but not intravenous injections of haptenized bone marrow-derived dendritic cells were able to induce skin-homing CD8+ T cells that express E-selectin-binding molecules (the murine equivalent of CLA) as well as CCR4 and were responsible for effector functions in cutaneous contact allergy. The authors assumed a crucial role for the tissue microenvironment and the quality of the antigen-presenting dendritic cells in the instruction of T cells for tissue-selective homing. Based on these observations, we envision the following pathophysiologic scenario for CLE: skin-derived dendritic cells loaded with apoptotic keratinocytes induced by UV irradiation stimulate antigen-specific T lymphocytes expressing homing molecules such as CLA and CCR4 that confer the capacity to enter inflamed skin. Local expression of adhesion molecules and chemokines such as E-selectin and TARC/CCR17 subsequently help to attract these activated T lymphocytes into lesional skin of CLE. Taken together, our findings suggest that CLA and CCR4 expression on lesional and circulating T lymphocytes plays a role in the pathogenesis of CLE. Importantly, our results suggest that CLA+ and CCR4+ cytotoxic T cells participate in specific destruction of epidermal and adnexal structures in scarring CDLE. Twenty-five LE patients (17 female, eight male) who had an acute exacerbation of their LE with recent inflammatory skin lesions were included in the study. Five patients suffered from scarring localized CDLE (lCDLE), four from scarring dCDLE, 11 from SCLE, and five from SLE, with acute skin lesions (macular erythema or malar rash). Detailed clinical information of the investigated patients is provided in a supplementary table published on the web site (Table S1). Additionally, samples from five healthy donors, five patients with exacerbated psoriasis, and five patients with herpes zoster were analyzed for control purposes. A diagnosis of LE was established based on standard criteria including clinical symptoms, histological and immunohistochemical findings as well as on detection of antinuclear antibodies. Localized and disseminated CDLE were differentiated by the extent of skin involvement. lCDLE is restricted to one body region only; dCDLE involves more that one region (e.g., head and shoulders). dCDLE was separated from SCLE by the development of scarring skin lesions. All skin samples included were taken for diagnostic purposes immediately when the patients presented at our department with acute skin lesions prior to any treatment. Blood samples were taken at the same time. Skin lesions selected for biopsy had developed recently according to the patients' history. The age of patients and donors ranged between 18 and 72 y, respectively. All of them were Caucasian. Informed consent for participating in this study was obtained from all donors. The study was performed according to local ethical guidelines as well as the Declaration of Helsinki and was approved by the local regulatory committee. Serial sections were prepared from formalin-fixed, paraffin-embedded skin biopsies. Monoclonal antibodies (mAb) specific for CCR4 (1G1, biotin), CLA (HECA452) (both from BD-Pharmingen, San Diego, California), TARC/CCL17 (ETR01, from R&D Systems, Minneapolis, Minnesota), CD4 (1F6, Novocastra, Newcastle, UK), CD3 (F7238), CD8 (C8/144B), CD20 (L26), and granzyme B (GrB7) (all from DAKO, Hamburg, Germany) were used for primary staining. Appropriate isotype-matched controls were included. The specimens were incubated with the first antibody for 2 h. Secondary staining was performed using the LSAB2 staining kit (DAKO). Tetramethylrhodamineisothiocyanate (TRITC)-conjugated anti-mouse and fluorescein isothiocyanate (FITC)-labeled rabbit-streptavidin were used for fluorescence double staining (both from DAKO). Results were evaluated on blinded specimens by two experienced dermatopathologists independently. Cells were counted per high-power field (× 200), TARC/CCL17 expression was detected semiquantitatively (0,+,++,+++). PBMC were isolated from heparinized venous blood using density gradient centrifugation over Lymphoprep (Axis-Shield PoC AS, Norway). The following mAb were used in flow cytometric analyses: CCR4 phycoerythrin (PE) and biotin (1G1), CLA FITC (HECA452), CD3 PE/PerCP (SK7), CD4 PerCP (SK3), CD8 FITC (SK1), CD8 PerCP (SK3), all from BD Pharmingen (Franklin Lakes, New Jersey). Isotype matched controls were included. Analyses were performed on freshly isolated and on frozen material, stored at -80°C in RPMI medium containing 10% fetal calf serum and 10% dimethyl sulfoxide. Results of frozen and freshly isolated material were controlled carefully; significant differences were not found. Three-color flow cytometry was performed by incubation of 5 × 105 PBMC with the indicated mAb. After staining, cells were fixed with Cytofix Buffer (BD Pharmingen) and analyzed using a FACScan flow cytometer and CellQuest software (BD Pharmingen). Further evaluations were performed with WinMDI 2.8. Serum levels of TARC/CCL17 were determined by ELISA (Quantikine, R&D Systems), following the manufacturers' operation protocol. Chemotaxis assays were performed with PBMC in 24-well plates with transwell inserts of 5 μm pore size (all material from Costar, Cambridge, Masachussetts). 5 × 105 cells were plated on the cover membrane in RPMI-1640 medium containing 0.4% fetal bovine serum and incubated at 37°C. Serial dilutions of recombinant TARC/CCL17 (R&D Systems) were placed in the bottom chamber to determine the optimal concentration for migration. For determination of the chemotatic index, 20 μL of the cells that had migrated to the lower well after 2 h at 37°C and 20 μL of the sample in the upper well were collected in TruCOUNT tubes (BD Pharmingen) and analyzed by flow cytometry. These TruCOUNT tubes include a lyophilized pellet with a defined number of fluorescent beads. The absolute number of investigated cells in both samples was determined by comparing the relative count of investigated CD4+ CCR4+ and CD8+ CCR4+ cells by the relative count of fluorescence beads, using the following equation: (number of events in region containing cells/number of events in absolute count bead region) × (absolute number of beads per test/test volume)=absolute count of cells per volume. Basal migration and TARC/CCL17-directed migration were evaluated by determining the chemotactic index (specifically migrated cells divided by unspecifically migrated cells). Experiments were performed in triplicate and repeated at least three times. The expression of CCR4, CD4, CD8, and Granzyme B in flow cytometry and immunohistochemistry was evaluated by nonparametric tests. The Kruskal–Wallis test was used to analyze differences among the groups, followed by the unpaired Mann–Whitney test for comparisons between any two of the groups. An identical statistical process was performed to compare TARC/CCL17 serum levels. Probabilities <0.05 were considered to be significant (*), p-values<0.01 as highly significant (**). Results are given in mean±SEM. The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/JID/JID23755/JID23755.htm Table S1. Clinical information of cutaneous LE patients
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