A Reducing Microenvironment Leads to the Generation of FcεRIhigh Inflammatory Dendritic Epidermal Cells (IDEC) Natalija Novak, MD, Stefan Kraft, Jörg.

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A Reducing Microenvironment Leads to the Generation of FcεRIhigh Inflammatory Dendritic Epidermal Cells (IDEC) Natalija Novak, MD, Stefan Kraft, Jörg Haberstok, Elisabeth Geiger, Pierre Allam, Thomas Bieber Journal of Investigative Dermatology Volume 119, Issue 4, Pages 842-849 (October 2002) DOI: 10.1046/j.1523-1747.2002.00102.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Enhanced FcεRI surface expression of dendritic cells in vitro could be achieved by incubation with 50 µM 2-ME. (A) The surface expression of FcεRI of monocytes (day 0) and dendritic cells on day 8 of the IL-4/GM-CSF driven culture of atopic and nonatopic donors generated in the presence of 50 µM 2-ME (black boxes) or in the absence of 50 µM 2-ME (gray triangles) is shown. The percentage of FcεRI positive CD14 (on day 0 of culture in the presence of 2-ME) or CD1a positive cells (on day 8 of culture) is indicated on the y-axis, the time of culture (days) is indicated on the x-axis. Taken together, dendritic cells generated from monocytes of atopic donors display a higher surface expression of the high-affinity receptor for IgE (FcεRI) on day 8 of culture in the presence of 2-ME, than dendritic cells generated from monocytes of nonatopic donors; n = 20 atopic donors and n = 15 nonatopic donors; **p <0.001. (B/C) The FcεRI surface expression of monocytes on day 0 and day 8 of culture without (Contr.) and with 2-ME of an atopic (B) and nonatopic (C) donor is shown as overlay histogram; this is one representative experiment of n = 20 for atopic and n = 15 for nonatopic donors. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reducing and oxidative agents influence exclusively FcεRI expression of MoDC from atopic donors in vitro. MoDC from atopic donors cultured for 8 d with IL-4 and GM-CSF (black bar; Contr.) or additionally with 50 µM, 10 µM or 5 µM 2-ME (gray bars), 0.5 mM DTT (hatched bar) or H2O2 (white bar). The percentage of the FcεRI surface expression of CD1a-positive cells is indicated on the y-axis; n = 5. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The amount of FεRIγ is higher in IDEC.(A) Semiquantitative reverse transcription–PCR revealed an increased transcription of the FcεRIγ mRNA detectable in lysates obtained from IDEC, whereas no differences in the signal for the FcεRIα mRNA transcripts of MoDC and IDEC could be detected. This is one representative experiment of n = 4. (B) The extracellular expression of the FcεRI α-chain (white bars), the intracellular amount of FcεRIα (gray bars), and the amount of the FcεRI γ-chains (black bars) of MoDC and IDEC on day 8 of culture is indicated as relative fluorescence index (RFI) on the y-axis. These data demonstrate that the amount of FcεRIγ is significantly enhanced in IDEC, n = 6. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IDEC from lesional skin of AEDS and IDEC generated from monocytes of atopic donors are phenotypically similar. The expression of surface markers of IDEC isolated from lesional skin of AEDS is indicated as fluorescence intensity (log) on the x-axis and CD1a expression as fluorescence intensity on the y-axis (upper panel). In comparison, the expression of these surface molecules detectable on IDEC generated in vitro are shown (lower panel). This is a representative experiment of n = 10. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The most striking difference of MoDC and IDEC generated in vitro is the surface expression of FcεRI. MoDC and IDEC did not differ in the surface expression of the costimulatory molecules CD80, CD86, MHC class I, MHC class II, or CD83. The most striking difference of MoDC and IDEC generated in vitro of monocytes of the same atopic donor is the elevated FcεRI surface expression of IDEC. The surface marker profile of MoDC and IDEC on day 8 of culture is shown as overlay histogram (gray curve) in correlation to the respective isotype control (black curve). The fluorescence intensity (log) is indicated on the x-axis, the number of CD1a positive MoDC or IDEC is indicated on the y-axis. CLA, cutaneous lymphocyte antigen; MR, mannose receptor; E-cadh., E-cadherin. This is a representative experiment of n = 15. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 FcεRI on IDEC binds monomeric IgE in high amounts. The FcεRI surface expression of IDEC on day 8 of culture is shown as overlay histogram (A). On day 8 of culture, no constitutive IgE binding of IDEC could be detected by staining with FITC-labeled anti-IgE MoAb (B). In blocking experiments, IDEC were preincubated with blocking antibody to CD23 (9P.25; 5 µg per ml) and 0.5 lactose. Then IDEC were loaded with 0.5 lactose µg per ml human myeloma IgE and stained with FITC-labeled anti-IgE MoAb (C). Additionally, preincubation of IDEC with blocking antibody to FcεRI and lactose completely abrogates IgE binding (D). Taken together, IDEC bind IgE via FcεRI in high amounts on their cell surface. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 FcεRI stimulation of IDEC induces the production of pro-inflammatory cytokines. Stimulation of FcεRI with MoAb against FcεRI induces an enhanced production of pro-inflammatory cytokines and chemokines. The percentage of the intracellular cytokine production of CD1a+ IDEC is indicated on the y-axis. White bars indicate unstimulated IDEC on day 8 of culture, black bars indicate IDEC, which have been stimulated via FcεRI for a time period of 18 h, gray bars indicate IDEC stimulated via mIgG and GaM-IgG, hatched bars indicate IDEC stimulated with GaM-IgG alone; n = 8. From these data we conclude that FcεRI on IDEC generated in vitro is functional as cross-linking of FcεRI induces an increased cytokine production. Journal of Investigative Dermatology 2002 119, 842-849DOI: (10.1046/j.1523-1747.2002.00102.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions