Enhanced Expression and Activity of Protein-tyrosine Kinases Establishes a Functional Signaling Pathway Only in FcεRIhigh Langerhans Cells from Atopic.

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Enhanced Expression and Activity of Protein-tyrosine Kinases Establishes a Functional Signaling Pathway Only in FcεRIhigh Langerhans Cells from Atopic Individuals Stefan Kraft, Jörg H.M. Weßendorf, Jörg Haberstok, N. Novak Journal of Investigative Dermatology Volume 119, Issue 4, Pages 804-811 (October 2002) DOI: 10.1046/j.1523-1747.2002.00282.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Langerhans cells express the PTK p53/56lyn, p59fyn, p72syk, p56/59hck, p55c-fgr, p60c-src, and p50csk, but not p56lck. Lysates from positive control cells (lane 1), if applicable, negative control cells (lane 2), Langerhans cell-enriched epidermal cells (lane 3) and crude epidermal cells were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with the antibodies indicated. Detection of bands was performed with a horseradish-peroxidase-conjugated secondary antibody followed by an enhanced chemiluminescence protocol according to the manufacturer's instructions. Equal amounts of protein were loaded on to each lane within each immunoblot. Journal of Investigative Dermatology 2002 119, 804-811DOI: (10.1046/j.1523-1747.2002.00282.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The semiquantitative PTK profile in Langerhans cells shows high levels of p53/56lyn, p59fyn, p72syk, p56/59hck, and p50csk, and low to moderate levels of p60c-src and p55c-fgr. Epidermal cell suspensions were fixed with 4% formaldehyde and permeabilized with 0.5% saponin. Intracellular staining was performed using polyclonal antibodies or MoAb against PTK with appropriate controls followed by a FITC-conjugated secondary antibody and a PE-conjugated anti-CD1a antibody. Data were acquired on a FACScan flow cytometer. Shown are histograms of Langerhans cells gated for their CD1a expression. Journal of Investigative Dermatology 2002 119, 804-811DOI: (10.1046/j.1523-1747.2002.00282.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 PTK expression profiles of freshly isolated FcεRIlow Langerhans cells compared with cultured Langerhans cells (A), FcεRIhigh Langerhans cells from AD (B), or FcεRIhigh IDEC from AD (C). Cells were stained and analyzed as described under Fig 2. (A) PTK expression of freshly isolated Langerhans cells is not significantly changed when compared with cultured Langerhans cells except for downregulation of p56 /59hck expression (*p <0.05) (data shown as mean ± SEM; n = 8). (B) FcεRIhigh Langerhans cells from AD (n = 6) show significantly higher expression (*p <0.05) than FcεRIlow Langerhans cells from normal skin (NS; n = 15) except p50csk. (C)FcεRIhigh IDEC from AD (n = 6) do not show significant differences in PTK expression when compared with FcεRIlow Langerhans cells from normal skin (NS, n = 15), except upregulation of p55c-fgr (*p <0.05). IDEC were discriminated from Langerhans cells by their lower CD1a expression. CD1alow cells were confirmed to be negative for Lag, but highly positive for FcεRI. Journal of Investigative Dermatology 2002 119, 804-811DOI: (10.1046/j.1523-1747.2002.00282.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 FcεRI-induced phosphorylation of PLC-γ1 is restricted to FcεRIhigh Langerhans cells. Langerhans cells were enriched from epidermal cells and FcεRI expression was determined by flow cytometry (upper histograms). FcεRIhigh Langerhans cells (left panel) and FcεRIlow Langerhans cells (right panel) were incubated with IgE, washed, and then stimulated with anti-IgE antibody for 1 min at 37°C (IgE/α-IgE) and compared with unstimulated cells (nil). After lysis, PLC-γ1 was immunoprecipitated and the samples were separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes followed by an incubation with antibody 4G10 (anti-ptyr) or anti-PLC-γ1. Detection of bands was performed with a horseradish-peroxidase-conjugated second-step antibody followed by an enhanced chemiluminescence protocol according to the manufacturer's instructions. Journal of Investigative Dermatology 2002 119, 804-811DOI: (10.1046/j.1523-1747.2002.00282.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions