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.

Tumor necrosis factor alpha (TNF-alpha) is a multifunctional cytokine that has an important role in the pathogenesis of inflammation, cachexia, and septic shock. Although TNF-alpha is mainly produced by macrophages, there is evidence regarding TNF-alpha production by cells that are not derived from bone marrow. TNF-alpha production by normal and inflamed human liver was assessed at both mRNA and protein levels. Using a wide panel of novel anti-TNF-alpha monoclonal antibodies and a specific polyclonal antiserum, TNF-alpha immunoreactivity was found in hepatocytes from patients chronically infected with either hepatitis B virus (HBV) or hepatitis C virus. Minimal TNF-alpha immunoreactivity was detected in the mononuclear cell infiltrate and Kupffer cells. In situ hybridization experiments using a TNF-alpha RNA probe showed a significant expression of TNF-alpha mRNA in hepatocytes, Kupffer cells, and some infiltrating mononuclear cells. By contrast, TNF-alpha was detected at low levels in liver biopsies from normal individuals or patients with alcoholic liver disease and low expression of TNF-alpha mRNA was observed in these specimens. Transfection of HepG2 hepatoblastoma cells with either HBV genome or HBV X gene resulted in induction of TNF-alpha expression. Our results demonstrate that viral infection induces, both in vivo and in vitro, TNF-alpha production in hepatocytes, and indicate that the HBV X protein may regulate the expression of this cytokine. These findings suggest that TNF-alpha may have an important role in human liver diseases induced by viruses.

Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation, which has been also involved in lymphocyte proliferation. Although AIM is absent from peripheral blood resting lymphocytes, it is expressed by in vivo activated lymphocytes infiltrating sites of chronic inflammation in several pathologies, as well as by lymphocytes after in vitro activation with different stimuli. We have investigated the possibility that tumor necrosis factor-alpha (TNF-alpha) gene expression and protein secretion could be induced in peripheral blood T cells through the AIM/CD69 molecule. Anti-AIM monoclonal antibodies (mAb) were able to induce TNF-alpha secretion in T cells when protein kinase C (PKC) was simultaneously activated by treatment with phorbol esters. TNF-alpha secretion was detected at 24 h and peaked at day 3 upon T lymphocyte activation with anti-AIM mAb. Immunoprecipitation studies with an anti-TNF-alpha mAb from surface iodinated T cells activated through AIM, demonstrated that TNF-alpha first appeared as a cell surface molecular form of 26 kDa, which is subsequently released to the extracellular medium as the 17-kDa molecular form of TNF-alpha. AIM stimulation dramatically increased TNF-alpha mRNA levels, and this mRNA induction and subsequent TNF-alpha secretion were virtually abrogated by the immunosuppressive drug cyclosporin A. Taken together these results indicate that AIM constitutes a novel molecular pathway in T lymphocytes for induction of TNF-alpha, and suggest a relevant pathologic role for AIM+ lymphocytes located at sites of tissue injury in the pathogenesis of different chronic inflammatory diseases.

The LFA-1 leucocyte integrin is known to participate in natural killer (NK) cytolytic activity, mediating effector target interactions. The possibility that LFA-1 may also play an active regulatory role in NK cells has been explored. To this end, we have employed a monoclonal antibody (HP1N) raised against recombinant interleukin-2 (rIL-2)-activated NK cells, which recognizes the alpha chain of the LFA-1 heterodimer (CD11a). In contrast to other anti-CD11a mAb the HP1N and its F(ab')2 fragment did not affect NK cell-mediated cytotoxicity and triggered a strong homotypic adhesion of NK cells and other LFA-1+ cells. Cellular aggregation was inhibited by anti-CD18 mAb, anti-ICAM-1 mAb, and other anti-CD11a mAb. Remarkably, the HP1N mAb was also shown to induce tumor necrosis factor-alpha (TNF-alpha) production from NK cells upon costimulation with anti-CD16 mAb. Such an effect appeared to be independent from homotypic adhesion since it took place in Mg(2+)-free medium, where NK cell aggregation was inhibited. Moreover, incubation with the HP1N mAb triggered a Ca2+ influx into the cytosol; this effect was clearly observed upon cross-linking of cell bound HP1N and was also substantiated with other anti LFA-1 (CD11a and CD18) mAb. Taken together these results indicate that the LFA-1 molecule is capable of transducing signals in NK cells, which regulate the intercellular interaction with its ligand, and enhance the activation via Fc gamma receptor type III.

Intercellular adhesions which occur during the mononuclear phagocyte differentiation are predominantly mediated by the lymphocyte-function-associated antigen-1 (LFA-1) family and the intercellular-adhesion molecule-1 (ICAM-1) which is a ligand for LFA-1. Thus, differentiation of U-937 promonocytic cells induced by phorbol esters occurs concomitantly with intercellular LFA-l/ICAM-1 -dependent cluster formation. Since these homotypic adhesions can be inhibited by monoclonal antibodies (mAb) directed to either LFA-1 or ICAM-1, we have analyzed whether the lack of cell -cell adhesions impairs the differentiation process. Treatment of U-937 cells with the phorbol ester 12-0-tetradecanoylphorbol 13-acetate in the presence of mAb to LFA-1 or ICAM-1 antigens yielded cells free from homotypic adhesions but differentiated as evidenced by their decreased proliferation and enhanced capacity for generation of superoxide anion. In addition, expression of the C D l l c antigen was increased, whereas the transferrin receptor disappeared from the cell surface. Vimentin gene transcription was also greatly augmented as opposed to a clear diminution in the levels of c-myc and ornithine decarboxylase transcripts. These results clearly demonstrate that phorbol esters can induce differentiation of monocytic cells independently of cell -cell adhesion.Intercellular adhesions play an important role in many immunological functions. Many of the adhesion phenoma of leucocytes are mediated by the lymphocyte-function-associated antigen 1 (LFA-1) family and the intercellular-adhesion molecule 1 (ICAM-1, CD54) molecule, which is a ligand for LFA-1 [I -61. These molecules are involved in antigen presentation [2, 7, 81, T cell effector functions [9-121, cooperation between B and T lymphocytes [I31 and chemotaxis of myeloid cells [I, 14, 151. The LFA-1 family of antigens is composed of three structurally related glycoproteins, LFA-1 (CD1 la), Mol (CDllb) and p150,95 (CDllc) [16]. These three antigens are noncovalently linked a : j heterodimers which have distinct a subunits of 170, 165 and 150 kDa, respectively, and share a common j subunit of 95 kDa (CD18) [16,17].In mononuclear phagocytes, the regulation of cell -cell adhesion is an important aspect of their function and development. Thus, monocytes initiate diapedesis by adherence to the endothelium. This adhesion to endothelial cells allows the monocytes to exit from blood vessels by passing between adjacent endothelial cells. At the same time that these adhesion processes take place, monocytes maturate into tissue macrophages. Also, upon antigen challenge, there is a massive infiltration of macrophages showing homotypic (macrophage -macrophage) and heterotypic (macrophage -T-cell) interactions. These cellular associations can lead to granuloma Several markers for monocytic differentiation of myeloid cell lines have been used, such as an arrest in their proliferative capacity [23 -25, 281, variations in the levels of specific gene transcripts [26, 29 -331, changes in the expression of specific...

The molecular nature of the structural changes on the T cell-CD6 glycoprotein upon cell activation has been investigated. Cell surface 125I labeling and immunoprecipitation studies from PBMC revealed that after stimulation by different activators of protein kinase C, or after exposure to either human or FCS, the anti-CD6 mAb precipitated an additional protein of 130 kDa, together with the 105-kDa protein present in resting cells. Cell surface expression of this 130-kDa CD6 protein form could be detected as early as 15 min after PKC activation, without requiring de novo protein synthesis. Pulse and chase activation experiments of radioiodinated cells suggested that the 130-kDa molecule is the result of a posttranslational modification of the 105-kDa protein and that this conversion is a reversible process. Studies of 32P-cell labeling and immunoprecipitation by anti-CD6 mAb revealed that only the 130-kDa form was phosphorylated, whereas the 105-kDa protein was unphosphorylated both in resting and activated cells. Moreover, the removal of phosphate groups from the 130-kDa CD6-form by enzymatic treatment with alkaline phosphatase resulted in its conversion to the 105-kDa form. Taken together, these results demonstrate the existence of two CD6 molecular forms that are in a dynamic equilibrium and differ only at their degree of phosphorylation: a 105-kDa unphosphorylated form present in resting T cells that changes very rapidly to a 130-kDa phosphorylated form by exposure of cells either to serum or to activators of PKC.

Functional domains on the recombinant interferon-alpha 2 (rIFN-alpha 2) molecule, which are involved in antiviral and NK enhancing activities, have been defined by immunochemical mapping with MAb, and their relationship with the IFN cellular receptor binding site has been studied. With 20 different anti-IFN-alpha 2 MAb selected by their binding to 125I-labeled IFN and by immunoprecipitation of the 20 Kd IFN molecule, we have defined three spatially separated epitopes (designated as sites A, B, and C) and two partially overlapping antigenic determinants on the IFN-alpha 2 molecule. Functional relation of IFN-alpha 2 A, B, and C epitopes have been determined by assaying the effect of various anti-IFN MAb on IFN-mediated biologic activities. MAb directed to sites A and B neutralized the antiviral activity of IFN. Furthermore, the MAb specific for site B displayed a neutralizing potency threefold higher than MAb directed to site A. Site B was also involved in the enhancing activity of IFN on NK-mediated cell cytotoxicity, whereas site A was not. MAb directed to site C partially affected the IFN-boosted NK activity but did not neutralize the IFN antiviral activity. Inhibition studies of 125I-IFN binding to human U-937 myelomonocytic cells by anti-IFN MAb demonstrated that MAb directed to site B blocked different IFN biologic functions by preventing its binding to the cellular receptor, whereas MAb directed to sites A and C caused no inhibition and partial inhibition of this binding, respectively.