Detection of human soluble Thy-1 in serum by ELISA - Fibroblasts and activated endothelial cells are a possible source of soluble Thy-1 in serum

The functions of Thy-1, a 35-kDa cell-surface glycoprotein, and its natural ligand are still unknown. Anchoring to the membrane via linkage to phospha tidylinositol (PI) raises the possibility of cleavage off the membrane by P I-specific phospholipases. Soluble Thy-1 (sThy-1) could interfere with the binding of the unknown natural ligand followed by regulation of different c ell functions. In this study we established an enzyme-linked immunosorbent assay (ELISA) to measure and quantify sThy-1 in serum and wound fluid. Reco mbinant human Thy-1 (rhThy-1) was expressed in Drosophila S2 cells, purifie d from culture supernatant and used as standard for quantitation of sThy-1 by the ELISA technique. There were no differences in sThy-1 levels in serum of healthy donors and patients with systemic sclerosis, leg ulcers, or rhe umatoid arthritis, respectively, detected by ELISA. In contrast, at the loc al site of inflammation, in wound fluid of venous leg ulcers and in synovia l fluid from joint puncture, we found strongly elevated levels of sThy-1 co mpared with sThy-1 in the serum of the same patient. Thy-1 is expressed in humans on brain cells, fibroblasts, a subpopulation of CD34+ blood stem cel ls, and possibly activated human dermal microvascular endothelial cells. In this study, we never found Thy-1 mRNA or protein expression in resting end othelial cells as shown by reverse transcriptase polymerase chain reaction (RT-PCR) and flow-cytometry. Thy-1 expression could be induced on endotheli al cells by phorbol myristate acetate and to a lesser extent by tumor necro sis factor-a (TNF-a). In situ, monoclonal antibodies to Thy-1 did not stain endothelial cells in normal skin, whereas endothelial cells in the synovia l membrane of rheumatoid arthritis patients and endothelial cells surroundi ng melanoma express Thy-1. In summary, our data indicate that Thy-1 is pres ent in soluble form in serum. Furthermore, Thy-1 seems to be a marker for e ndothelial cell activation. Therefore, activated endothelial cells as well as fibroblasts might be a possible source of sThy-1.