Dermatology," University Hospital of Cleveland, ... recognize the 9-0-acetylated disialosyl group on ... contrast, 9-O-GD3 is found in basal cell carcinomas.
American Journal of Pathology, Vol. 150, No. 2, February 1997 Copyright s American Society for Investigative Pathology
Lesional Psoriatic T Cells Contain the Capacity to Induce a T Cell Activation Molecule CDw6O on Normal Keratinocytes
Lone Skov,* Lawrence S. Chan,t David A. Fox,* J0rgen K. Larsen,§ John J. Voorhees,11 Kevin D. Cooper,mT and Ole Baadsgaard* From the Department ofDermatology,* Gentofte Hospital, University of Copenhagen, Denmark; the Department of
Dermatology,t Northwestern University Medical School, Chicago, Illinois; the Departments of Rheumatolog and
Dermatology,)1
University of Michigan, Ann Arbor, Michigan; the Finsen Laboratory,§ Finsen Center, Rigshospitalet, Denmark; and the Department of Dermatology," University Hospital of Cleveland,
Cleveland, Ohio
In this report we demonstrate, that in psoriatic skin, basal and suprabasal keratinocytes express CDw6O. The CDw60-specwi'c monoclonal antibody, UM4D4, has recently been shown to recognize the 9-0-acetylated disialosyl group on ganglioside GD3. The CDw6O antigen on cultured keratinocytes also seems to be identical with the 9-0-acetylated disialosyl group, because the anti-UM4D4 binding was markedly reduced after neuraminidase treatment of keratinocytes. To examine whether factors from T ceUs in psoriatic lesions are responsible for the overexpression of CDw6O on keratinocytes, T ceU lines obtained from lesional skin were initiated and cloned by limiting dilution. Factors releasedfrom 19 of 19 activated T ceU clones up-regulated CDw60 expression on cultured normal keratinocytes. T-cell-secreted cytokines, including interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, transforming growth factor- (3, granulocyte/macrophage colony-stimulating factor, and interferon-y were tested for their capacity to modulate keratinocyte CDw6O expression. IL-4 and IL-13 strongly up-regulated the expression of CDw6O; by contrast, interferon-y down-regulated keratinocyte CDw6O expression. Interestingly, IL-13 may in part be responsible for the T-cell-induced up-regulation of CDw6O, because anti-IL-13 partly neu-
tralized this effect of the T ceU supernatant. In conclusion, CDw6O expression on psoriatic epidernal keratinocytes is likely induced by intralesionaly activated T ceUls and may in part be due to IL-13. These findings would represent a novel mechanism by wbicb T cells participate in the pathogenesis ofpsoriasis. (Am JPathol 1997,
150:675-683) Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of basal and suprabasal keratinocytes. The mechanism through which the keratinocytes attain their hyperproliferative state is unknown. The immune system has long been suggested to play an essential role in the pathogenesis of psoriasis.1 This hypothesis is supported by the observations that T cell clones initiated from lesional psoriatic skin have been shown to release factors that have the capacity to induce nonpsoriatic keratinocytes to express the surface molecules that are normally expressed on keratinocytes in lesional psoriatic skin2 and, even more important, to enhance proliferation of cultured keratinocytes.35 A T cell activation pathway that is triggered by a monoclonal antibody termed anti-UM4D4 has been described.6 The UM4D4 antibody, assigned to the CDw6O cluster of differentiation, identifies a surface molecule that is expressed on approximately 25% of peripheral blood T cells, resting or activated. The molecule is also expressed on approximately 75% of T cells in psoriatic lesions,2 on a subset of thymoSupported by grants from The Danish Medical Research Council 12-9676, The Novo Nordisk Foundation, The Danish Hospital Foundation for Medical Research (Region of Copenhagen, The Faroe Islands, and Greenland), The Leo Foundation, The Danish Psoriasis Research Foundation, The Danish Cancer Society, and The Babcock Dermatological Foundation. Accepted for publication October 19, 1996. Address reprint requests to Dr. Lone Skov, Department of Dermatology, Gentofte Hospital, University of Copenhagen, Niels Andersens Vej 65, 2900 Hellerup, Denmark.
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cytes,7 and on malignant T cells in patients with cutaneous T cell lymphoma.8 The UM4D4 antibody, which binds to CDw60, is mitogenic for resting T cells in the presence of other mitogenic stimuli such as phorbol myristate acetate, interleukin (IL)-2, or monocytes and is, in soluble form, directly mitogenic as a single stimulus for preactivated T cells such as T cell clones.6 The molecule is distinct from other known T cell activation molecules including CD2, CD3, and CD28. Recently, anti-UM4D4 has been shown to bind to the 0-acetylated form of ganglioside GD3, especially the 9-0-acetylated form.9 Gangliosides are sialylated glycosphingolipids primarily located in the plasma membrane, and it is suggested that they play a role in the control of cell proliferation and differentiation10-12 and in cell surface recognition.13 The 9-0-acetylated form of ganglioside GD3 has not been detected in normal human epidermis, where the major ganglioside is GM3.14 In contrast, 9-O-GD3 is found in basal cell carcinomas that also are shown to express CDw6O.15 In this paper we report the expression of CDw60 on a subset of keratinocytes in hyperproliferative lesions of psoriasis. CDw60 is likely induced on the keratinocytes by factors released from activated local T cells, because various lesional psoriatic T cell clones contain the capacity to up-regulate the expression of CDw60 on cultured normal keratinocytes. This up-regulation of CDw60 expression may partly be due to the T cell cytokine IL-13 as indicated by the finding that IL-13 also up-regulates keratinocyte CDw60 expression and that T cell induction of CDw60 in vitro was partly neutralized by anti-IL-13.
Materials and Methods Antibodies and Cytokines Primary antibodies used were anti-UM4D4 (murine IgM) provided by D. A. Fox, IgM isotype control (Becton Dickinson, Mountain View, CA), antiHLA-DR (IgG, Becton Dickinson), and anti-epidermal growth factor (anti-EGF) receptor, and anti-CD2 and anti-CD3 (IgG, DAKO, Copenhagen, Denmark). Secondary antibodies used were rhodamine-conjugated goat anti-mouse IgM (Tago, Burlingame, CA), fluorescein-conjugated goat anti-mouse IgG (Kirkegaard & Perry, Gaithersburg, MD), phycoerythrin-conjugated and fluorescein-conjugated goat anti-mouse IgM (Immunotech, Marseille, France), and rhodamine-conjugated goat antimouse IgG (Immunotech). The recombinant human cytokines IL-2, IL-3, IL-4, IL-6, transforming growth factor (TGF)-3, granulo-
cyte/macrophage colony-stimulating factor (GMCSF), and interferon (IFN)-y were obtained from Genzyme (Cambridge, MA), and IL-13 was from R&D systems (Abingdon, UK). Neutralizing polyclonal rabbit anti-human IL-4 (anti-IL-4) was obtained from Genzyme, and neutralizing rabbit antihuman IL-13 was kindly provided by K. Bendtzen, Department of Immunology, Rigshospitalet, Copenhagen, Denmark.
Keratinocyte Cultures Primary cultures of keratinocytes were initiated from epidermis of healthy volunteers obtained by keratome biopsy and were cultured in keratinocyte growth medium (KGM; Clonetics, San Diego, CA) using standard methods that eliminate cell types other than keratinocytes.16 Cells were used between two and five passages.
Immunohistochemistry Punch biopsies from 5 normal controls, 12 untreated patients with active expanding plaques-stage psoriasis, and 5 untreated patients with acute atopic dermatitis were embedded and frozen in Tissue TeK 11 OCT compound (Miles Laboratories, Naperville, IL) and stored at -700C. Six-micron sections were cut and fixed in 100% methanol at -200C for 10 minutes, followed by equilibration with phosphate-buffered saline (pH 7.4) at room temperature for 5 minutes. Indirect immunofluorescence staining was performed with anti-UM4D4, a mixture of anti-CD2 and anti-CD3, or isotype controls, followed by rhodamine-conjugated second antibodies, as previously described.2
Initiation and Stimulation of Lesional Psoriatic T Cell Clones In five patients, punch biopsies were obtained from active expanding psoriatic plaque lesions that had been treated with emollients alone for at least 1 week. The biopsies were cut horizontally, as close to the epidermis as possible, and then vertically into several 1-mm2 pieces. These specimens were incubated in RPMI 1640 containing 10% human AB serum, recombinant human IL-2 (50 U/ml; Cellular Products, Buffalo, NY), glutamine, and antibiotics in a 24-microwell plate at 370C. After 12 hours of incubation, T cells were seen migrating out from the biopsy fragments. The fragments of epidermis were removed, and after less than 1 week, the T cell lines
Lesional Psoriatic T Cells
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were cloned using limiting dilution. Briefly, the T cells were plated at 0.3 cells per well in 96-well V-bottom microtiter plates (Flow Laboratories, MacLean, VA), along with 50,000 autologous r-irradiated (5000 rad) mononuclear cells, 50 U/ml IL-2, and 1 ,ug/ml phytohemagglutinin (Burroughs Wellcome, Greenville, NC). When growth was observed, the cells were subsequently transferred to flat-bottom plates and restimulated with anti-CD3 (Coulter Clone, Hialeah, FL) or phytohemagglutinin and autologous mononuclear cells every 1 to 4 weeks. To activate resting T cell clones, the cells were incubated in serum-free medium AIM-V (GIBCO, Paisley, Scotland) with anti-UM4D4.6 After 4 hours of incubation, the T cells were washed three times and reincubated in fresh AIM-V. After 24 hours of incubation the cell-free supernatant from both antiUM4D4 activated and nonactivated T cells were harvested and frozen at -70°C.
Staining of Keratinocyte Cultures Keratinocytes
were
cultured in KGM with
were washed and incubated with anti-UM4D4, antiEGF receptor, or isotype control IgM and IgG antibodies as described above. Cells were washed and stained with phycoerythrin-conjugated goat antimouse IgM or rhodamine-conjugated goat antimouse IgG. Analysis was performed on a Becton Dickinson FACS IV or determined using a fluorescence microscope.
Statistics CDw6O expression on keratinocytes incubated with supernatant from activated or nonactivated T cells, with or without the tested cytokines or pretreated with neuraminidase, were compared with the Wilcoxon test for paired data.
Results Binding ofAnti-UM4D4 on Normal Cultured Keratinocytes
superna-
tant from T cell clones or cytokines for 72 hours and
washed. Keratinocyte suspensions were obtained by brief trypsinization and incubated with anti-UM4D4, anti-HLA, or isotype control IgM or IgG antibodies for 45 minutes at 40C. Cells were washed and then stained with fluorescein-conjugated or phycoerythrin-conjugated goat anti-mouse IgM and rhodamine-conjugated goat anti-mouse IgG for 30 minutes at 40C. The stained cells were fixed with 1% paraformaldehyde and filtered through 51-,um monofilament nylon mesh. Analyses were performed in a Becton Dickinson FACS IV or a FACS Vantage or determined using a fluorescence microscope. In the case of flow cytometry, cell debris and clumps were excluded by gating in forward and orthogonal light scatter. In experiments with cell cycle analysis, the cells, after incubating with anti-UM4D4, were exposed to acetone to make the cell membrane permeable for
propidium iodide. Thereafter, the cells were stained with propidium iodide and analysis of cell surface antigen and propidium iodide staining was performed on a Coulter model EPICS C.
Neuraminidase Treatment of Keratinocytes Keratinocytes obtained by brief trypsinization were incubated in 0.1 mol/L sodium acetate buffer, pH 5.5, with or without 0.2 U/ml Clostridium perfringens neuraminidase type VI (Sigma Chemical Co., St. Louis, MO) for 2 hours at 37°C. The keratinocytes
To study whether normal keratinocytes cultured under standard conditions (low Ca' and serum-free medium) express the CDw6O antigen, keratinocytes were stained with anti-UM4D4. Keratinocytes in culture express CDw60; however, the percentage of CDw60+ keratinocytes was variable from 3 to 68% (n = 20). Recently, by immunostaining of thin-layer chromatograms of structurally characterized gangliosides, anti-UM4D4 was shown to bind to the 9-0acetylated form of GD39 (H. Clausen, E Nudelman, and S. Hakomori, unpublished observations). To test whether anti-UM4D4 also binds to the 9-0-acetylated form of ganglioside GD3 on keratinocytes, cultured keratinocytes were incubated with or without neuraminidase and stained with anti-UM4D4 and, as control, anti-EGF receptor. Preincubation with neuraminidase decreased the percentage of CDw60+ keratinocytes from 41 ± 8% to 17 ± 12% (mean ± SD, n = 5, P < 0.05; Figure 1, representative experiment). By contrast, neuraminidase treatment of keratinocytes did not change the keratinocyte EGF receptor expression. These data indicate that sialic acid is involved in the binding epitope on keratinocytes in accordance with the ganglioside specificity of the antibody.
Expression of CDw6O on Keratinocytes in Psoriatic Skin In normal epidermis, CDw6O is expressed only on basal dendritic epidermal cells (Figure 2A). These
678 Skov et al AJP February 1997, Vol. 150, No. 2
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Log fluorescence intensity Figure 1. Histograms represent flow cytometnic analyses of keratinocytes pretreated with or without neuraminidase and stained with anti-UM4D4 or anti-EGF receptor ( ) or isotype controls (- -- ). Left boxes: keratinocytes stained with anti-UM4D4 (top) and keratinocytes stained with anti-EGF receptor (bottom). Right boxes: keratinocytes pretreated with neuraminidase and stained with anti-UM4D4 (top) or anti-EGF receptor (bottom). With FACS IV, CDw60 was measured by phycoerythrin fluorescence and EGF receptor by rhodamine fluorescence (excitation at 515 nm, emission at 563 to 588 nm). Cell debris and clumps were excluded by gating in forward and side scatter. Neuraminidase treatment of keratinocvtes reduces the CDw6O expression.
cells are melanocytes, as determined by doublestaining experiments in which these cells were tyrosinase positive. Rarely, a few scattered basal keratinocytes may be CDw60+ in normal skin. A few dermal CDw60+ cells are present in normal skin (Figure 2A). In contrast, psoriatic skin exhibits focal and confluent basal and suprabasal keratinocyte CDw6O expression (Figure 2, B-D). Dermal T cells express CDw6O as presented in Figure 2C. In each patient with plaque psoriasis the degree of CDw6O expression was uniform over large areas of epidermis; in contrast, among the different patients the expression of CDw6O was highly variable (Figure 2, E and F). In five of the patients with psoriasis, consecutive skin sections were also stained for T cells. However, we did not find a direct correlation between the number of T cells in the skin and the degree of CDw6O expression on keratinocytes. In another skin disease with T cell infiltration, namely, atopic dermatitis, basal keratinocytes in four out five patients also expressed CDw6O. The CDw6O expression on keratinocytes in atopic epidermis was mainly in the rete ridges.
Supernatant from Activated Psoriatic T Cell Clones Up-Regulates Keratinocyte CDw6O Expression Psoriatic skin is characterized by infiltration with activated T cells. Because lesional psoriatic T cells
contain the capacity to induce keratinocyte HLA-DR and ICAM-1 expression2 we also determined their capacity to regulate CDw60 expression. Supernatant from activated and nonactivated psoriatic T cell clones were therefore added to semiconfluent normal keratinocytes cultured in serum-free medium at a 10% dilution. After 72 hours of culture, the keratinocytes were harvested and stained with antiUM4D4 and the number of positive cells determined by flow cytometry. Supernatant from activated T cells increased the percentage of keratinocytes that express CDw6O from 22 ± 12% (mean ± SD, nonactivated T cells) to 42 ± 18% (mean ± SD, activated T cells; n = 19, P < 0.01).
Induced CDw6O Was Expressed in All Stages of the Cell Cycle At 72 hours after addition of supernatant from activated and nonactivated T cells or control medium with anti-UM4D4, the keratinocytes were stained with anti-UM4D4 and propidium iodide for cell cycle determination by two-color flow cytometric analysis. Keratinocytes cultured in KGM, KGM with antiUM4D4, or KGM supplemented with supernatant from nonactivated T cells exhibited CDw6O expression mainly within the GO/G1 population (Figure 3, upper left and right and lower left panel). By contrast, CDw6O expression was strongly induced by supernatant from activated T cells on the majority of the keratinocytes (Figure 3, lower right panel). The Tcell-induced CDw6O expression occurred in all stages of the cell cycle, but the proportion of S/G2/M cells that express CDw6O were higher than that of cells in GO/G1 (Figure 3, lower right panel). Furthermore, the total fraction of cells in S/G2/M increased in agreement with previously published data concerning the capacity of soluble factors from activated T cells to induce keratinocyte proliferation.34 Because supernatant from activated T cells clearly up-regulates CDw6O expression, we tested the capacity of T-cell-secreted cytokine to regulate CDw6O expression on keratinocytes.
T Cell Cytokines IL-4 and IL- 13 Up-Regulate Keratinocyte CDw6O Expression Various cytokines, including IL-2, IL-3, IL-4, IL-6, IL-13, TGF-,B, IFN-y, and GM-CSF, were tested for their capacity to regulate CDw6O expression on the keratinocytes. Keratinocytes were incubated for 72 hours with increasing concentrations of the cytokines, harvested, and stained for CDw6O expression.
Lesional Psoriatic T Cells 679 AJP February 1997, Vol. 150, No. 2
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Figure 2. Immunohistochemical staining for CDw60 expression in normal and psoriatic lesional skin sections. A: Basal dendritic epidermal CDw60+ cells (solid arrows) as well as rare dermal CDw6O0 cells (open arrow) are present in normal skin. Original magnification, x 100. B: Psoriatic lesional skin demonstrated focally confluent basal and suprabasal keratinocyte membrane expression of CDw6O as well as bright CDw6O T dermal cells (curved arrows). Original magnification, x 50. C: Similar basal and suprabasal keratinocyte membrane expression of CDw6O is seen in lesional skin ofanotherpatient with psoriasis. Original magnification, x 100. D: Some distinct, brigbt CDw60+ mid-epidermal mononuclear cells (straight arrows) are present in lesionalpsoriatic skin. Keratinocyte membrane CDw6O is expressedfocally on basal cells in this third patient. Original magnification, X50. E and F: Low-power photomicrographs from two different patients with plaque psoriasis showing the the variable expression of CDw6O among the patients. Original magnification, X 16.
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Antibodies Against IL- 13, but Not IL-4 Neutralize the Capacity of Supernatant from Activated T Cell Clones to Up-Regulate CDw6O Expression
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Linear red fluorescence Figure 3. Flow cytometric two-color analysis of keratinocytes incubatedfor 3 days in KGM (upper left), KGM plus anti-UM4D4 (upper right ), KGM plus 1096 mediumfrom nonactivated Tcells (lower left), and KGM plus 10% medium from activated T cells (lower right). The vertical axis shows log green fluorescence intensity (CDw60) and the horizontal axis denotes linear red fluorescence intensity (produmiodide-stained DNA). G, and G21M denote different stages of the cell cycle. Supernatant from activated psoriatic T cell clone up-regulates keratinocyte CDw6O expression in all stages of the cell cycle.
Interestingly, IL-4 (n = 8) and IL-13 (n = 9) demonstrated capacity to up-regulate CDw60 expression on keratinocytes (Table 1). Although IFN-,y induces keratinocyte HLA-DR expression, increasing concentrations of IFN--y from 10 to 1000 U/ml resulted in decreased expression of CDw6O (Figure 4). Concomitant with CDw60 down-regulation by IFN-,y, the HLA-DR expression on keratinocytes increased (Figure 4, representative experiment). IFN-,y (1000 U/ml) decreased the expression of CDw6O from 32 ± 21% to 16 ± 20% (mean ± SD, n = 6, P < 0.05). The expression of CDw60 and HLA-DR seems to be mutually exclusive as virtually none of the cells expressed the two molecules together (Figure 4). None of the other tested cytokines demonstrated the capacity to regulate keratinocyte CDw6O expression. Table 1. IL-4 and IL-13 Up-Regulate CDw60 Expression on Normal Keratinocytes n
Control IL-4 (5 ng/ml) Control IL-13 (2.5 ng/ml)
8 9
Percent CDw6O+ keratinocytes 14 ± 10% 48 ± 13% 46 ± 11% 76 ± 16%
ence Figure 4. Flow cytometrc two-color analysis of keratinocytes incubatedfor3 days in KGM(Ieft), KGMplus 100 U/ml IFN-y (middle), and KGM plus 1000 U/ml IFN-y (right). The vtical axis sbows log red fluorescence (HLA-DR) and the horizontal axis shows log green fluorescence (CDw60). IFN-y down-regulates CDw6O expression on normal keratinocytes.
P value
