Transient CD44 Variant Isoform Expression and Reduction in CD4+/CD25+ Regulatory T Cells in C3H/HeJ Mice with Alopecia Areata
2002; Elsevier BV; Volume: 118; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2002.01745.x
ISSN1523-1747
AutoresMargot Zöller, Kevin J. McElwee, Peter A. Engel, Rolf Hoffmann,
Tópico(s)T-cell and B-cell Immunology
ResumoAlopecia areata, an autoimmune disease affecting anagen stage hair follicles, can be induced by grafting spontaneous alopecia areata affected skin to normal-haired C3H/HeJ mice. As the onset of alopecia areata can be significantly retarded by anti-CD44 variant isoform 10 treatment, it was interesting to explore the underlying disease mechanism. Two weeks after transplanting alopecia areata affected skin, expression of CD44 variant isoforms 3, 6, 7, and 10 was strikingly upregulated as compared with sham-grafted mice. By 6 wk after grafting, CD44 variant isoform levels had returned to normal, whereas in draining lymph nodes, CD44 variant isoform expression was slightly decreased. Leukocytes in the skin of mice with chronic alopecia areata expressed a hematopoietic isoform of CD44 and CD44 variant isoform 6 at an elevated level, but CD44 variant isoform 3 expression was reduced. Cytokine expression in leukocytes of chronic alopecia areata affected skin was higher than in normal-haired controls. Cytokine expression also increased postsurgery in sham and alopecia areata grafted mice, but remained elevated only in mice receiving alopecia areata affected skin. Finally, from the skin of mice with chronic alopecia areata and of mice transplanted with alopecia areata affected skin, an increased number of CD4+ and CD8+ cells, but a strongly decreased number of CD4+/CD25+ regulatory T cells was recovered. Thus, expression of CD44 variant isoforms is important for the migration of leukocytes during the initial period of alopecia areata. CD44, however, is apparently not involved in the maintenance of the disease state, which is characterized by high cytokine expression levels, an increased number of CD4+ and CD8+ cells, but a low level of CD4+/CD25+ suppressor cells. Alopecia areata, an autoimmune disease affecting anagen stage hair follicles, can be induced by grafting spontaneous alopecia areata affected skin to normal-haired C3H/HeJ mice. As the onset of alopecia areata can be significantly retarded by anti-CD44 variant isoform 10 treatment, it was interesting to explore the underlying disease mechanism. Two weeks after transplanting alopecia areata affected skin, expression of CD44 variant isoforms 3, 6, 7, and 10 was strikingly upregulated as compared with sham-grafted mice. By 6 wk after grafting, CD44 variant isoform levels had returned to normal, whereas in draining lymph nodes, CD44 variant isoform expression was slightly decreased. Leukocytes in the skin of mice with chronic alopecia areata expressed a hematopoietic isoform of CD44 and CD44 variant isoform 6 at an elevated level, but CD44 variant isoform 3 expression was reduced. Cytokine expression in leukocytes of chronic alopecia areata affected skin was higher than in normal-haired controls. Cytokine expression also increased postsurgery in sham and alopecia areata grafted mice, but remained elevated only in mice receiving alopecia areata affected skin. Finally, from the skin of mice with chronic alopecia areata and of mice transplanted with alopecia areata affected skin, an increased number of CD4+ and CD8+ cells, but a strongly decreased number of CD4+/CD25+ regulatory T cells was recovered. Thus, expression of CD44 variant isoforms is important for the migration of leukocytes during the initial period of alopecia areata. CD44, however, is apparently not involved in the maintenance of the disease state, which is characterized by high cytokine expression levels, an increased number of CD4+ and CD8+ cells, but a low level of CD4+/CD25+ suppressor cells. alopecia areata hematopoietic isoform of CD44 CD44 variant isoforms delayed type hypersensitivity lymph node cells CD44s and CD44v isoforms phycoerythrin skin infiltrating leukocytes Alopecia areata (AA) is an autoimmune disease involving focal perifollicular and intrafollicular inflammation of anagen stage hair follicles. C3H/HeJ mice have been previously investigated as an appropriate animal model for AA (Sundberg et al., 1994Sundberg J.P. Cordy W.R. King Jr, L.E. Alopecia areata in aging C3H/HeJ mice.J Invest Dermatol. 1994; 102: 847-856Abstract Full Text PDF PubMed Google Scholar;McElwee et al., 1998aMcElwee K.J. Boggess D. 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Besides costimulatory molecules, the activity of regulatory T cells is of major importance in the progression of autoimmune diseases. Autoreactive T cells can be readily identified in the pool of peripheral lymphocytes, their activation being prevented by regulatory T cells (Shevach et al., 1998Shevach E.M. Thornton A. Suri-Payer E. T lymphocyte-mediated control of autoimmunity.Novartis Found Symp. 1998; 215: 200-211PubMed Google Scholar). Recently, it has been noted that a population of CD4+/CD25+ T cells is essential for maintaining homeostasis (Annacker et al., 2001Annacker O. Pimenta-Araujo R. Burlen-Defranoux O. Barbosa T.C. Cumano A. Bandeira A. CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10.J Immunol. 2001; 166: 3008-3018Crossref PubMed Scopus (487) Google Scholar). 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The chronic state of disease was characterized by high-level cytokine expression and a marked reduction in regulatory T cells. All C3H/HeJ mice were supplied from stocks at the Jackson Laboratory (Bar Harbor, ME) specific pathogen-free production facility. Mice received conventional low soy oil diet (altromin 1434, Altromin GmBH, Lage, Germany) and acidified water (pH 2.8–3.0) ad libitum. Forty female mice in age-matched groups were grafted at a mean age of 18 wk with AA-affected skin or normal skin using procedures as described previously (McElwee et al., 1998bMcElwee K.J. Boggess D. King Jr, L.E. Sundberg J.P. Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.J Invest Dermatol. 1998; 111: 797-803Crossref PubMed Scopus (124) Google Scholar). Circular sections of full thickness skin were pinch cut and one each grafted to normal-haired C3H/HeJ mice. Immediately after the surgical procedure all mice were housed in separate cages in isolation in a single room at the University of Marburg for the duration of the study. Mice were observed until necropsy and any changes in pelage coat quality were recorded. All mice used in this study were confirmed to have accepted their skin graft. Mice were killed at 2, 6, and 12 wk after grafting and skin was excised from AA-affected and sham-grafted control mice. Mice with chronic AA evident for 23 wk, along with nonsurgically manipulated, normal-haired mice, were also necropsied. Dorsal skin samples at the site of grafting and distant from the graft were fixed in Fekete's acid/alcohol/formalin solution and paraffin embedded. Skin samples were also embedded in OCT compound (Tissue Tek, Sakura, Zoeterwoude, the Netherlands) and snap frozen in liquid nitrogen for subsequent immunohistology. For flow cytometry analyses, the entire dorsal and ventral skin, draining lymph nodes, and spleen were collected. Single leukocyte cell suspensions were prepared by pressing lymph nodes and spleens through fine gauze. Skin was carefully freed of fat and subcutaneous muscle tissue. The skin was then put epidermis uppermost on sterile gauze in Petri dishes and was covered with 25 ml of a solution of 1% trypsin, 50 U collagenase per ml and 27 U DNase per ml. After 30 min the skin was pressed towards the sterile gauze and isolated leukocytes were collected in RPMI 1640 supplemented with 10% fetal bovine serum. The procedure was repeated three times. The collected cells were washed, resuspended in RPMI 1640 containing 10% fetal bovine serum plus 10-3 M HEPES buffer and were incubated for 2–3 h at 37°C, 5% CO2 in air in a humidified atmosphere to allow for re-expression of digested surface proteins. As in the flow cytometry analysis keratinocytes could be separated from leukocytes by size and granulation, preparations of SkIL were directly used for flow cytometry analysis after the recovery period. Lymphocytes were enriched by Ficoll gradient centrifugation in selected experiments. Fluorescence-activated cell sorter analysis of cells collected from the interphase provided staining profiles that did not differ from those of unseparated, but gated populations of leukocytes. Lymph node cells (LNC), spleen cells, and SkIL from two to three animals were pooled. Viability was determined by Trypan blue exclusion and was in the range of over 90% in LNC preparations and in the range of 70–80% in SkIL preparations. The following monoclonal antibodies (MoAb) were used: anti-mouse CD4 (clone RM4-5), biotinylated anti-mouse interleukin (IL)-2 (clone JES6-5H4), IL-4 (clone BVD6-24G2), IL-6 (clone MP5-32C11), IL-10 (clone JES3-16E3), IL-12 (clone C17.8), interferon (IFN)-γ (clone XMG1.2), and tumor necrosis factor (TNF)-α (clone MP6-XT3); fluorescein isothiocyanate-conjugated anti-mouse CD25 (clone PC61), phycoerythrin (PE)-conjugated anti-CD28 (clone 37.51), CD40 (clone 3/23), CD80 (clone 16-10A1), CD86 (clone GL1), and surface IgM (clone R6-60.2) (all Pharmingen, Hamburg, Germany); anti-CD8 (clone 53-6.7; Southern Biotechnology, Birmingham, AL); anti-mouse CD4 (clone YTA 3.2.1), CD8 (clone YTS 169.4.2.1), macrophage (clone YBM 6.6.10), ICAM-1 (clone YN1/1.7.4) (all European Animal Cell Culture Collection, Porton Down, U.K.); LFA-1 (clone M17/5.2), dendritic cell (clone 33D1), and panCD44 (CD44s and CD44v isoforms; clone IM7) (all American Type Culture Collection, Manassus, VA); unconjugated or biotinylated anti-CD44v3 (clone PTS33;Seiter et al., 1999Seiter S. Engel P. Föhr N. Zöller M. Mitigation of delayed-type hypersensitivity reactions by a CD44 variant isoform v3-specific antibody: blockade of leukocyte egress.J Invest Dermatol. 1999; 113: 11-21Crossref PubMed Scopus (17) Google Scholar), CD44v6 (clone 11A6;Seiter et al., 2000Seiter S. Schmidt D.S. Zöller M. The CD44 variant isoforms CD44v6 and CD44v7 are expressed by distinct leukocyte subpopulations and exert non-overlapping functional activities.Int Immunol. 2000; 12: 37-49Crossref PubMed Scopus (26) Google Scholar), CD44v7 (clone LN7.2;Wittig et al., 1998Wittig B. Schwärzler C. Föhr N. Günthert U. Zöller M. Curative treatment of an experimentally induced colitis by a CD44 variant V7-specific antibody.J Immunol. 1998; 161: 1069-1073PubMed Google Scholar), CD44v10 (clone K926;Rösel et al., 1997Rösel M. Seiter S. Zöller M. CD44v10 expression in the mouse and functional activity in delayed type hypersensitivity.J Cell Physiol. 1997; 171: 305-317Crossref PubMed Scopus (35) Google Scholar), and VLA-4 (clone PS/2) kindly provided by K. Miyake (Hession et al., 1992Hession C. Moy P. Tizard R. et al.Cloning of murine and rat vascular cell adhesion molecule-1.Biochem Biophys Res Commun. 1992; 183: 163-169Crossref PubMed Scopus (85) Google Scholar); secondary reagents were biotinylated, PE-labeled and fluorescein isothiocyanate-labeled anti-rat IgG, anti-mouse IgG, and streptavidin (all Dianova, Hamburg, Germany). Fixed tissue samples were sectioned at 6 µm and stained with hematoxylin and eosin for histologic evaluation. Immunohistology was performed on cryostat tissue sections as described elsewhere (Freyschmidt-Paul et al., 2000Freyschmidt-Paul P. Seiter S. Zöller M. et al.Treatment with an anti-CD44v10-specific antibody inhibits the onset of alopecia areata in C3H/HeJ mice.J Invest Dermatol. 2000; 115: 653-657https://doi.org/10.1046/j.1523-1747.2000.00113.xCrossref PubMed Scopus (42) Google Scholar). Briefly, tissue alkaline phosphatase activity was ablated with levamisole solution and nonspecific binding blocked using an avidin–biotin blocking kit (Vector Laboratories, Burlingame, CA) and 2% normal goat serum. Cryostat sections were exposed to the primary antibodies, biotinylated secondary antibodies and alkaline phosphatase conjugated avidin–biotin complex (Vector) solutions in sequence. Tissue sections were counter-stained with Mayer's hematoxylin. The primary antibody was replaced with normal rat IgG for negative controls. Digitized images were generated using a Leica DMRBE Microscope equipped with a SPOT CCD camera from Diagnostic Instruments and Software SPOT2.1.2 (Leica, Bensheim, Germany). Each cell sample was aliquoted and incubated with 10 μg of the primary antibody per ml. Cells incubated with unconjugated MoAb were subsequently incubated with the appropriate anti-mouse or anti-rat PE-labeled secondary antibody. Cells labeled with biotinylated MoAb were detected with streptavidin-PE. Negative controls were incubated with a nonbinding primary antibody and the same secondary reagents
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