Interferon-γ-Stimulated Human Keratinocytes Express the Genes Necessary for the Production of Peptide-Loaded MHC Class II Molecules Cristina Albanesi.

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Interferon-γ-Stimulated Human Keratinocytes Express the Genes Necessary for the Production of Peptide-Loaded MHC Class II Molecules Cristina Albanesi andrea Cavani, Giampiero Girolomoni Journal of Investigative Dermatology Volume 110, Issue 2, Pages 138-142 (February 1998) DOI: 10.1046/j.1523-1747.1998.00098.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 IFN-γ-treated keratinocytes express the class II transcriptional activators RFX5 and CIITA, as well as HLA-DR, HLA-DM, and Ii genes (RT-PCR analysis). Normal human keratinocytes express constitutively low amounts of RFX5 (470 bp), DMα (835 bp), and Ii-p33 (384 bp). Weak signals corresponding to RFX5 and DMα are also detectable in NCTC cells whereas A431 cells express constitutively low levels of all genes studied. Treatment with IFN-γ (500 U per ml) for 24h induces the transcripts for CIITA (370 bp), DRα (645 bp), DMβ (300 bp), and Ii-p41 (560 bp). All amplificates are identical to those present in Raji B cells. Journal of Investigative Dermatology 1998 110, 138-142DOI: (10.1046/j.1523-1747.1998.00098.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 IFN-γ-stimulated, but not unstimulated, keratinocytes express Ii-p33/p41 and HLA-DMα genes (northern blot analysis). Cultured epithelial cells do not express constitutively appreciable amounts of Ii and DM genes as evaluated by northern blot analysis. Transcripts become evident in normal keratinocytes and A431 cells upon activation with IFN-γ (500 U per ml, 24 h), although the levels are lower than those present in Raji cells. In contrast, IFN-γ-treated NCTC cells do not show detectable amounts of Ii-p41 mRNA. Journal of Investigative Dermatology 1998 110, 138-142DOI: (10.1046/j.1523-1747.1998.00098.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 RFX5 and CIITA precede HLA-DR, Ii, and HLA-DM gene expression in IFN-γ-activated human keratinocytes. Upon treatment with IFN-γ (500 U per ml), CIITA transcript becomes detectable after 3h whereas DRα, DMβ, and Ii-p41 mRNA appear after 6h. RFX5 is constitutively expressed as are DMα and Ii-p33 genes (RT-PCR analysis). Journal of Investigative Dermatology 1998 110, 138-142DOI: (10.1046/j.1523-1747.1998.00098.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IFN-γ-treated keratinocytes display Ii-p33/p41 and HLA-DM proteins. Extracts prepared from Raji cells, keratinocytes, and A431 and NCTC cells were evaluated by western blot analysis. Ii isoforms and DM are present in Raji cells, and, upon IFN-γ-stimulation (500 U per ml, 48 h), also become detectable in epithelial cells. NCTC cells express Ii-p33 but not Ii-p41 or DM. Filters were probed with LN2 and PS/JG DMβ MoAb that recognize Ii and DMβ subunit, respectively. Journal of Investigative Dermatology 1998 110, 138-142DOI: (10.1046/j.1523-1747.1998.00098.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 IFN-γ-activated keratinocytes express membrane SDS-stable class II heterodimers. (a) Flow cytometry analysis: keratinocytes activated with IFN-γ (500 U per ml, 48 h, shaded profiles), but not untreated keratinocytes (–––––), express DR (L243 MoAb) on their surface; (·····) represent staining with FITC-conjugated mouse IgG2a. Raji cells express higher amounts of membrane DR than IFN-γ-activated keratinocytes. (b) Immunoblot analysis: NP-40 lysates from IFN-γ-treated keratinocytes and Raji cells prepared in the presence of 2% SDS contain DR protein dimer, which dissociates into the α and β chains following boiling. The filters was stained with the TAL.1B5 and DK22 MoAb, which recognize the α and the β monomers of DR, respectively. Similar results were obtained using the MoAb DA6.147 (anti-DRα) and HB10.A (anti-DRβ) together. Journal of Investigative Dermatology 1998 110, 138-142DOI: (10.1046/j.1523-1747.1998.00098.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions