1. A Ligand of Human Thy-1 is Localized on Polymorphonuclear Leukocytes and Monocytes and Mediates the Binding to Activated Thy-1-Positive Microvascular Endothelial Cells and Fibroblasts 
Anja Saalbach, Uwe F. Haustein, Ulf Anderegg 
Journal of Investigative Dermatology 
Volume 115, Issue 5, Pages (November 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Thy-1 ligand is expressed on inflammatory cells in situ. Cryostat sections of bullous pemphigoid were incubated with biotinylated bovine serum albumin (a) as negative control, MoAb AS02 (b, c) or purified and biotinylated human Thy-1 (d–f). The biotinylated albumin showed no reaction (a). The Thy-1 antigen detected by MoAb AS02 was expressed on EC and fibroblasts (b, c). The Thy-1 ligand was detected on inflammatory cells (red) using the biotinylated Thy-1. Keratinocytes, fibroblasts, and EC did not express the Thy-1 ligand. Scale bar: 25 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Thy-1 ligand is expressed on PNC and monocytes detected by flow cytometry. PNC, MNC, and monocytes were labeled with biotinylated Thy-1 and bovine serum albumin as negative control followed by streptavidin–allophycocyanin and analyzed by flow cytometry. PNC and monocytes could bind the biotinylated Thy-1 whereas lymphocytes did not. Biotinylated albumin did not react with any of these cell types. Preincubation of PNC with unlabeled Thy-1 was able to chase the signal of biotinylated Thy-1-binding.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Establishment of experimental cell models consisting of Thy-1-positive and Thy-1-negative HDMEC or fibroblasts. Phorbol 12-myristate 13-acetate-stimulated HDMEC, a fibroblast cell line (A2a), and subsequently Thy-1-positive and Thy-1-negative cell populations isolated by immunomagnetic separation using the MoAb AS02 as described in Materials and Methods were tested for the expression of Thy-1 by flow cytometry using MoAb AS02 and goat anti-mouse fluorescein isothiocyanate. A mouse isotype antibody was used as a control.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Adhesion of PNC to Thy-1-positive HDMEC is increased compared with Thy-1-negative HDMEC and is inhibited by blocking Thy-1. Confluent Thy-1-positive and Thy-1-negative HDMEC were incubated with biotinylated PNC, unbound cells were removed, and attached cells were visualized by streptavidin–CY3. Ten microscopic fields (magnification 200×) were counted. Thy-1-positive HDMEC bound about two times more PNC than Thy-1-negative HDMEC. Blocking of Thy-1 inhibited the adhesion of PNC to Thy-1-positive HDMEC up to about 50% whereas the binding of PNC to Thy-1-negative HDMEC was not effected. Preincubation of PNC with purified Thy-1 (1 μg per ml, 15 min, 4°C) also effectively inhibited cell adhesion to Thy-1-positive HDMEC. Results represent the mean ± SD of five independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Adhesion of monocytes to Thy-1-positive HDMEC is increased compared with Thy-1-negative HDMEC, and is inhibited by blocking Thy-1. Confluent Thy-1-positive and Thy-1-negative HDMEC were incubated either with biotinylated monocytes, unbound cells were removed and attached cells were visualized by streptavidin–CY3. Ten microscopic fields (magnification 200×) were counted. Thy-1-positive HDMEC bound about 2.5 times more monocytes than Thy-1-negative HDMEC. Preincubation of HDMEC with MoAb AS01/AS02 cross-linked by a goat anti-mouse F(ab)2 antibody lead to an decreased number of attached monocytes to Thy-1-positive HDMEC, whereas the adhesion to Thy-1-negative HDMEC was not changed by the antibodies. Preincubation of monocytes with purified Thy-1 (1 μg per ml, 15 min, 4°C) also effectively inhibited cell adhesion to Thy-1-positive HDMEC. Results represent the mean ± SD of five independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Adhesion of PNC to Thy-1-positive fibroblasts is increased compared with Thy-1-negative fibroblasts. Confluent Thy-1-positive and Thy-1-negative immortalized fibroblasts (A2a) were incubated with biotinylated PNC or monocytes, unbound cells were removed and attached cells were visualized by streptavidin–CY3 and counting 10 microscopic fields (magnification 200 ×). Thy-1-positive fibroblasts bound two times more PNC than Thy-1-negative fibroblasts. Adhesion of monocytes to Thy-1-positive and Thy-1-negative fibroblasts did not differ. Data represent the mean ± SD of seven independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 7
Blocking of Thy-1 decreased the adhesion of PNC and partially of monocytes to primary fibroblasts. Confluent human dermal primary fibroblasts were incubated either with MoAb AS01/AS02 cross-linked by a goat anti-mouse F(ab)2 antibody, with an anti-HLA-ABC antibody as control antibody or without antibody. Then, cells were incubated with biotinylated PNC or monocytes, unbound cells were removed and attached cells were visualized by streptavidin–CY3 and counting 10 microscopic fields (magnification 200×). Blocking of Thy-1 inhibited the adhesion of PNC to fibroblasts up to 79% and the adhesion of monocytes up to 20%. The anti-HLA-ABC antibody did not decrease the adherence of PNC to fibroblasts compared with the control without antibody incubation. Further, the adhesion of PNC to fibroblasts could be blocked very efficiently by preincubation of PNC with purified Thy-1. Data represent the mean ± SD of five independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions