Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-beta (TGF-beta) and whose expression is up-regulated on myeloid cells upon differentiation to macrophages. We have isolated full-length cDNA clones from a lambda gt 10 library, prepared from phorbol 12-myristate 13-acetate-differentiated HL60 cells by screening with an endoglin-specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL-60 and U-937, and placenta. Independent cDNA constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF-beta 1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide-linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.

In this report, we describe a novel activation antigen that appears very early after T cell activation and is absent in resting lymphocytes, through which agonistic proliferative signals can be triggered by mAb binding. It has been designated as activation inducer molecule (AIM) and is a disulphide-linked heterodimeric structure containing two polypeptide chains of Mr 33,000 and 27,000. The expression of AIM can be induced by different activation stimuli such as PMA, PHA, or anti-CD3 mAb, but not by the Ca2+ ionophore A23187, and it precedes the expression of other activation molecules such as 4F2 or the IL-2-R. Once AIM antigens are expressed on lymphocytes after stimulation with submitogenic doses of PMA, the binding of anti-AIM mAbs triggers a strong proliferative response. Furthermore, a comitogenic effect of the anti-AIM mAbs is exerted in the presence of either PHA or anti-CD3 mAb. The activation of lymphocytes through AIM antigens induces both IL-2 and IL-2-R receptor synthesis and is inhibited by anti-IL-2-R mAbs.

The family of human T cell activation-associated proteins, named VLA complex, is formed by the molecular association of cell surface glycoproteins of 210, 165, 130 and 80 kDa. In this report, we describe eight different monoclonal antibodies (HP mAb) specific for the 80-kDa polypeptide which preferentially associates with the 165-kDa member. Comparative immunoprecipitation and cell-binding studies demonstrated that the HP mAb recognize an epitope(s) on the 165/80 kDa subset different from those recognized by other anti-VLA mAb previously described. Furthermore, cellular and tissue distribution studies by flow cytometry and peroxidase staining showed that the HP reactivity pattern is different from other VLA specificities. The 165/80-kDa complex defined by HP mAb is present on human thymocytes, peripheral blood lymphocytes as well as on T, B and myelomonocytic cell lines. However, only the 80-kDa subunit was precipitated by HP mAb from activated T lymphocytes. These results suggest that the association between the 165- and 80-kDa subunits diminishes during the activation process, and that the epitopes recognized by the HP mAb are located on the 80-kDa protein. The novel polypeptide association of 165/80 kDa has been termed VLA-3.

AIM is an activation inducer molecule selectively expressed by activated lymphocytes through which agonistic proliferative signals can be triggered. The relationship between the expression of AIM with the activation of protein kinase C (PKC) has been studied. Different activators of PKC such as the active phorbol esters, phorbol myristate acetate and phorbol dibutyrate, or the phorbol-related ester mezerein were able to induce AIM expression on peripheral blood lymphocytes as assessed by immunofluorescence flow cytometry. Moreover, the expression of this activation antigen was also induced by treatment of peripheral blood lymphocytes either with dioctanoyl-rac-glycerol, a synthetic analogue of diacylglycerol, the physiological mediator of PKC activation. Further indirect evidence that AIM expression was dependent on the activation of PKC was obtained by blockade of the induction of its expression in cells treated with H7, an inhibitor of PKC. The AIM expression can be detected as early as 3 h after addition of phorbol esters and it requires active RNA and protein synthesis. The activation of PKC appears to be also required in the proliferative response induced by anti-AIM monoclonal antibody (mAb) in conjunction with phorbol esters. Agents such as phorbol myristate acetate, phorbol dibutyrate or mezerein but not the inactive phorbol ester methyl-phorbol myristate acetate induced a high proliferation of peripheral blood lymphocytes in the presence of anti-AIM mAb. In addition, we have demonstrated that the anti-AIM mAb is not sufficient by itself to induce cellular proliferation once the AIM antigen is expressed at the cell surface, requiring the simultaneous stimulation of the PKC to trigger high proliferative responses. Furthermore, the anti-AIM mAb did not appear to exert its effect on proliferation by rapidly increasing the intracytoplasmic Ca2+ levels. Taken together all these results indicate that the expression and function of AIM antigen is dependent on the activation of PKC.