1. Selective Upregulation of Fibroblast Fas Ligand Expression, and Prolongation of Fas/Fas Ligand-Mediated Skin Allograft Survival, by Retinoic Acid: the Skin as a Retinoide-Inducible Immune Privilege Site 
Atsushi Saitoh, Takeshi Kawanabe, He Weidong, Tatsuyoshi Kawamura, Shinji Shimada 
Journal of Investigative Dermatology 
Volume 115, Issue 2, Pages (August 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of Fas antigen and FasL on C57BL/6 mouse dermal fibroblasts and keratinocytes were investigated by reverse transcription–PCR. Representative reverse transcription–PCR results are shown here from three independent experiments. Total RNA was propagated from third passaged keratinocytes and fibroblasts. mRNA for Fas, FasL, and β-actin were amplified by reverse transcription–PCR.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Induction of FasL mRNA in fibroblasts by RA. Dermal fibroblasts were cultured by RA in various concentrations of RA (10-8– 10-2 M). After 24 h, RNA was harvested using RNAZol B and FasL and β-actin mRNA were amplified by reverse transcription–PCR.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Confirmation of Fas and FasL mRNA expression of fibroblasts by RA. (a) RA, M; (b) RA, M. RPA was performed using [32P]-labeled Fas and FasL probes. Hybridization was performed for overnight and hybridized products were digested by RNase A and T1. Digested products were resolved on denaturing 6% polyacrylamide gel. The size of protected fragments were 307 bp for Fas (b) and 277 bp for FasL (a). [32P]-labeled cyclophilin probe was used for internal control (103 bp). Results shown are representative data from three different assays.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Cell surface expression of FasL on RA-treated fibroblasts. Cultured cells were treated with RA (10-6 M) and metalloproteinase inhibitor (KBR8301); stained with anti-FasL antibodies and analyzed by flow cytometry. KBR8301 also did not induce FasL.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Cytotoxicity by FasL on RA-treated fibroblasts. (a) RA-treated fibroblasts (closed triangle) were able to kill Fas+ Jurkat T cells, which are susceptible to FasL. 51Cr-labeled Jurkat cells were mixed with RA-treated dermal fibroblasts in various E/T ratios. The cytotoxicity caused by FasL was measured by 51Cr release after 4 h of coincubation. Supernatants were collected and counted by γ-counter. L5178Y cells (□) that were stably transfected of FasL cDNA were used as positive control, RA-treated dermal fibroblasts (▴), DMSO-treated dermal fibroblasts (▪), and RA-treated keratinocytes (▵) were used as negative control. (b) The cytotoxicity by RA-treated fibroblasts was neutralized by anti-FasL antibodies. RA-treated effector fibroblasts were preincubated with anti-FasL antibodies for 1 h in various concentrations. After treatment with antibodies, cytotoxic assay was performed as described above. Results shown were the averages of three experiments performed on separate occasions. A representative experiment was shown in (b). L5178Y+ anti-FasL antibodies (□), RA-treated FB+ anti-FasL antibodies (▴), and DMSO-treated FB+ anti-FasL antibodies (▪).
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Alloskin graft survival by daily RA injection. (a) Alloskin graft survival was prolonged by the treatment of RA injection. Sized BALB/C donor skin (20 × 30 mm2) was grafted on C57BL/6 mouse trunk. RA or DMSO as control was injected under the grafted skin every day. The concentration of DMSO in RA is as high as DMSO only treatment. Its prolongation by RA was significant from day 3 (n = 5; p <0.01, Student's t test). RA injection (○), DMSO injection (□). (b) Specific antibodies for FasL was able to block the graft prolongation. Anti-FasL antibody injected skin, which was treated by RA, started to reject after injection of antibodies (•), but RA and control IgG injected skin showed its prolongation (○). DMSO-injected control group showed quick rejection of allograft skin (□). The rejection of allograft skin that was injected with RA and anti-FasL antibodies was significant from day 7 (n = 5; p <0.01, Student's t test).
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
FasL mutated gld/gld mouse donor skin did not show allograft prolongation by daily RA injection. (a) Graft experiment using C57BL/6 donor skin on to BALB/C trunk was also performed as described above. RA-injected allograft skin (○), DMSO-injected allograft skin (□). The concentration of DMSO in RA is as high as DMSO only treatment. The allograft skin prolongation by RA was significant from day 5 (n = 5; p <0.01, Student's t test). (b) FasL mutant gld/gld (C57BL/6 background) mouse skin was not able to induce allograft prolongation. These graft experiments were performed three times independently and representative data were shown. There were no significant differences between RA and DMSO treatment. RA injection (○), DMSO injection (□).
Journal of Investigative Dermatology  , DOI: ( /j x)
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9. Figure 8
Macroscopic and histopathologic presentation. Allografted skin that was injected with RA every day showed little lymphocyte and neutrophil infiltration in the upper and middle dermis (a, day 5; c, day 10). On the other hand, allografted skin that was injected with DMSO every day showed many lymphocytes and neutrophils infiltration at day 5 (b) and day 10 (d). Hematoxylin and eosin.
Journal of Investigative Dermatology  , DOI: ( /j x)
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10. Figure 9
Immunohistochemical staining of FasL+ fibroblasts. Newborn mouse was injected with RA or DMSO on the trunk area. After 24 h, injected skin area was removed and frozen by liquid nitrogen. Frozen tissue sections were stained by rabbit anti-mouse FasL antibodies and control antibodies. Positive cells are observed in dermis that was injected with RA. These cells looks like fibroblasts (a). On the other hand, no positive cells were seen in dermis that was injected with DMSO (b).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions