Eur. J. Immunol. zyxwvutsrqpo 1993. 23: 3003-3010 A soluble form of human CD58 3003 Jorg C. Hoffmann, Thomas J. Dengler, Percy A. Knolle, Marion Albert-Wolf, Matthias Roux, Reinhard Wallich and Stefan C. Meuer Applied Immunology, German Cancer Research Center A soluble form of the adhesion receptor CD58 (LFA-3) is present in human body fluids* The human adhesion receptor CD58 (LFA-3) is expressed on most human cell types. Here we report on a soluble form of CD58 (sCD58) in human serum, human urine, and culture supernatants of several cell lines. sCD58 partially purified from human serum, from supernatant of the Hodgkin cell line L428, and purified sCD58 from human urine were found to have a molecular mass of 40-70 kDa under denaturating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting). However, gel filtration of sCD58 purified from human urine gave a molecular mass of 118-166 kDa, suggesting a noncovalent homotrimer conformation or its association with other molecules. Using an enzyme-linked immunosorbent assay specific for CD58 we found that sera from patients suffering from different forms of hepatitis contained elevated sCD58 levels zyxwvu (n zyxwvut = 108). Accordingly, there was a fivefold increase of supernatant sCD58 when the hepatocellular carcinoma cell line Hep G2 was incubated with 25 ng/ml recombinant tumor necrosis factor-a in vitro. In contrast, sCD58 serum levels of 337 additional patients suffering from various other immunological disorders were not found to be raised. At high concentrations sCD58 binds to CDZpositive cells and inhibits rosette formation of human T cells to human erythrocytes. Thus, local release of large quantities of naturally occurring sCD58 may interfere with intercellular adhesion in vivo. 1 Introduction Soluble forms of membrane proteins such as cytokine receptors or cellular adhesion molecules (CD14, TNF receptor, CD25, IL-6 receptor, IFN-y-receptor, and CD54) have recently been detected in human body fluids [l-111. As some of them bind to their natural ligands, i.e. cytokines or cell surface molecules, they may have important func- tions in immune regulation by blocking receptorlligand interactions [3-5,8,11]. In addition, some of these soluble membrane proteins seem to be of clinical relevance since elevated serum levels have been observed in certain populations of patients [5, 6, 10, 111. The cellular adhesion molecule CD58 (formerly known as LFA-3), a heavily glycosylated protein, is expressed by most human tissues including epithelial cells, endothelial cells and virtually all hematopoetic cells [12]. Both CD58 and its natural ligand CD2 consist of two domains and both molecules belong to the immunoglobulin superfamily [13-181. Using a panel of CD58 mAb we have recently identified four discrete epitopes on the N-terminal domain (domain 1) and two overlapping epitopes on the mem- brane-proximal domain (domain 2) [ 17].The N terminus of the CD58 molecule represents the interaction site with CD2. In contrast, domain 2 links CD58 to the membrane anchor and does not participate in CD2 binding [17]. The interaction between CD58 and CD2 is important for antigen-specific T cell activation as well as activation processes in NK cells [14, 19-24]. In addition, the CD2/CD58 interaction is involved in adhesion between T lymphocytes and other CD58-positive cells. Thus, CD58 antibodies, purified soluble CD58, or recombinant soluble CD58 have been shown to block adhesion of T cells to thymic epithelium [25], cytotoxic T celvtarget cell interac- tion [12,13,16,23,26-301.T cell/B cell interaction [31], the mixed lymphocyte reaction, and rosette formation of human T lymphocytes to human or sheep red blood cells [22-351. As patients suffering from various diseases such as Hodgkin's disease or sarcoidosis were reported to have unknown serum factors that inhibit E rosette formation [36-381 we hypothesized that a circulating soluble form of CD58 (sCD58) could be responsible for this phenomenon. Here we have purified and molecularly characterized a naturally occurring soluble form of CD58. Moreover, sera of normal controls and patients suffering from various immunological disorders were analyzed for sCD58 concen- trations in a newly established ELISA. [I 120051 2 Materials and methods * Supported by a grant from Tumorzentrum Heidelberg-Mann- heim 2.1 mAb mAb directed at different epitopes of human CD58 [17] were AICD58.1 (epitope 4 of domain 1, IgGZ,), AICD58.5 (epitope 5 of domain 2), AICD58.6 (epitope 1 of Correspondence: Stefan C. Meuer, Applied Immunology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, FRG (Fax: 49/62 21/42 25 61) Key words: Lymphocyte activation / Intercellular adhesion / Immune disorder / CD2 / CD58 domain 1, IgGh), AICD58.16 (epitope 6 of domain 2, IgGI) and TS2/9 (epitope 1 of domain 1, IgGI) which was z 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1993 0014-2980/93/1111-3003$10.00 + zy .25/0