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Introduction
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Psoriasis represents one of the most common T cellmediated autoimmune diseases (15). The initial onset of skin lesions is followed by chronic relapses of disease, not unlike the clinical course for multiple sclerosis or rheumatoid arthritis (6). Based on various animal models (79) and indirect clinical observations (1, 6), T cells responsible for psoriatic skin lesions are thought to be confined to the circulating pool of immune cells. However, the role of resident T cells in the development of psoriatic lesions has not been investigated yet, mainly due to the lack of an appropriate animal model. Current psoriasis mouse models using SCID mice necessitate the injection of activated PBMCs with or without superantigen to induce a psoriatic phenotype in the human prepsoriatic skin graft (7, 8). SCID (and recombinase activating gene (RAG)-/-) mice lack T and B cells but show mature NK cells with normal NK cell activity (1012). Since NK cells are involved in rejection of xenogeneic tissue (13), SCID mice are not ideal acceptors of human skin grafts. To improve graft acceptance, we used mice deficient in type I (A) and type II (G) IFN receptors in addition to being RAG-2-/- (AGR129 mice). AGR129 mice show immature NK cells with severely impaired cytotoxic (cytolytic) activity in vitro and in vivo (unpublished data) due to a deficiency in type I (A) and type II (G) IFN receptors, in addition to lacking T and B cells (RAG-2-/-) (14). Disruption of both IFN receptors has been previously shown to lead to decreased NK cytotoxic (cytolytic) activity in vitro and in vivo (15), whereas type I IFN receptor or type II IFN receptordeficient mice demonstrated reduced but fully functional NK cell activity (12, 16). In the current study, we use AGR129 mice as excellent acceptors of human prepsoriatic skin grafts to show spontaneous onset of bona fide psoriatic lesions that do not require any exogenous cells or factor except for those contained within the engrafted skin itself. Our observations establish proliferation of local T cells, dependent on TNF-
production, as essential elements of psoriatic lesion formation.
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Materials and Methods
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Animals, Patients, and Transplantation Procedure.
AGR129, mice, mice deficient in type I (A) and type II (G) IFN receptors in addition to being RAG-2-/-; AR129 mice, mice deficient in type I (A) IFN receptors in addition to being RAG-2-/-; and GR129 mice, mice deficient in type II (G) IFN receptors in addition to being RAG-2-/- were either on a 129Sv/Ev or C57BL/6 background. Mice were kept pathogen free throughout the study. Animal studies were approved by the Kantonale Veterinaeramt of Zurich and human studies by the Institutional Review Boards of the University Hospital of Zurich. Keratome biopsies (6 x 2 x 0.04 cm) of symptomless prepsoriatic skin (PN skin) were taken from the lower back or buttock of patients with confirmed plaque-type psoriasis after informed consent was obtained. No topical or systemic medication was administered for at least 4 wk before the study. Skin grafts were transplanted to the back of mice using an absorbable tissue seal (Vet-Seal; B. Braun Medical AG).
Immunohistochemistry, Confocal Laser Scanning Microscopy (CLSM) Analysis, and Flow Cytometry Analysis.
Acetone-fixed cryostat sections were stained using standard staining techniques. Unspecific Fc receptor binding of antibodies was measured with isotype-matched controls. Unconjugated antibodies used were monoclonals to CD1c (Immunotech), CD1d (51.1.3, provided by Dr. S.P. Balk, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA), CD3 (Dako), CD4 (Dako), CD8 (Dako), CD31 (Dako), CD54 (BD Biosciences), HLA-DR (BD Biosciences), IFN-
(BD Biosciences), IL-12 (R&D Systems), keratin 16 (Sigma-Aldrich), Ki-67 (Dako), TNF-
(R&D Systems), polyclonals to involucrin (Anawa Trading SA), and conjugated CD3-PE (BD Biosciences), CD69-FITC (BD Biosciences), CD83-PE (Immunotech), and TNF-
FITC (BD Biosciences). Sensitivity of the flow cytometry analyses was between 1:1,000 and 1:3,000 human to mouse PBMCs for the antihuman CD3-PE mAb (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20031482/DC1).
Histologic Assessment and Quantification Experiments.
Histologic quantification experiments represent the mean of three random fields with a 400-fold magnification. The acanthosis and papillomatosis index was defined as published (17). The indicated values of both indices represent the mean of 10 random areas of each sample.
Quantitative Real-time RT-PCR.
Total RNA was extracted from organs (High Pure RNA Isolation kit; Roche) followed by reverse transcription (AMV First Strand cDNA Synthesis kit; Roche). Real-time PCR was performed in a LightCycler (Roche). Primer sequences were for human GAPDH, 5'-ATTGCCCTCAACGACCACTTTG-3' and 5'-TTGATGGTACATGACAAGGTGCGG-3' and mouse GAPDH, 5'-CATCAAGAAGGTGGTGAAGC-3' and 5'-CCTGTTGCTGT-AGCCGTATT-3'. Sensitivity of the RT-PCR method was between 1:100,000 and 1:1,000,000 human PBMCs to mouse splenocytes for the human GAPDH primer (Fig. S1 B), and its specificity for human cDNA was demonstrated by the lack of amplification of specific cDNA sequences when applied on RNA isolated from spleens of normal C57BL/6 mice.
Neutralization Studies.
Dosage and route of administration of the reagents applied was deduced from therapeutic trials in humans, calculated using the allometric approach, and administered as follows: 17 µg muromonab-CD3 (neutralizing antihuman CD3 mAb, clone OKT3) twice weekly, i.v.; 1,000 µg infliximab (antihuman TNF-
mAb) on days 0, 14, 28, 42, and 56, i.v.; 90 µg etanercept (TNF receptor fusion protein) twice weekly, s.c. Control mice received isotype-matched antibodies or PBS.
Online Supplemental Material.
Fig. S1 shows the detection threshold for flow cytometry and RT-PCR analyses, and Fig. S2 shows TNF-
production by few T cells. Figs. S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20031482/DC1.
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Results and Discussion
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Spontaneous Development of a Psoriatic Phenotype upon Transplantation of Symptomless Prepsoriatic Skin onto AGR129 Mice.
PN skin from 12 different patients with confirmed plaque-type psoriasis was transplanted onto AGR129 mice. The skin grafts developed a psoriatic phenotype in 28 out of 31 (90%) grafted mice. Phenotypic conversion started at week 4 and was fully developed at 68 wk after engraftment. Appearance of PN skin on the day of transplantation (Fig. 1, A and B) was comparable to normal human skin. In contrast, 8 wk after transplantation onto AGR129 mice, PN skin grafts showed typical features of psoriasis (Fig. 1, C and D). Histology of PN skin after development of a psoriatic phenotype (Fig. 1 C) was comparable to a biopsy of a psoriasis lesion from the same patient donating the PN skin graft (Fig. 1 E). Several different combinations of human skin and mouse strains served to demonstrate that development of a psoriatic phenotype was unique to PN skin transplanted onto AGR129 mice (Fig. 1, FH). Quantification of psoriatic features in a time-course experiment using papillomatosis and acanthosis indices (17) reflected significant (P = 0.0002) changes of PN skin transplants on AGR129 mice, whereas changes in phenotype were clearly absent from control skin grafts (Fig. 1 H).