IFN-γ Activation Research Articles

Interferon-γ alters expression of endothelial cell-surface glycosphingolipids

Our previous work on human endothelial cell (EC) glycosphingolipids (GSLs) demonstrated that these cells contain a large diversity of GSLs, predominantly with lacto core structures. In order to evaluate the role of GSLs as EC antigens and receptors, we investigated their cell-surface expression on confluent EC monolayers and ECs activated by interferon-γ (IFN-γ) and interleukin-1 (IL-1). IFN-γ activation of endothelial cells resulted in a small change in GSL composition, but greatly increased surface expression of gangliosides and decreased surface expression of neutral GSLs. In particular, surface expression of the major neutral GSL, globoside, decreased three- to fourfold as measured both by galactose oxidase labeling and by binding of the anti-globoside monoclonal antibody 9G7. IFN-γ did not significantly alter the total cell content of globoside, as measured by metabolic labeling, but rather altered the ratio of accessible cell surface to intracellular globoside. Two mechanisms appear to contribute to the decreased cell-surface globoside expression. IFN-γ treatment increased the relative proportion of intracellular globoside which is associated with the cell cytoskeleton. IFN-γ treatment also caused more of the cell-surface globoside to be inaccessible to antibody, and both neuraminidase and trypsin treatment of the cells increased globoside accessibility. IL-1 treatment increased total cell GSL content, but did not alter GSL composition or cell-surface binding by six anti-carbohydrate antibodies. The specific modulation of cell-surface GSLs by IFN-γ suggests that GSLs may play a role in the altered adhesive and receptor activities of IFN-γ-activated ECS.

The functional properties of Fcγ RI, II and III on myeloid cells: A comparative study of killing of erythrocytes and tumor cells mediated through the different Fc receptors

Three distinct Fc receptors for IgG, Fcγ RI, Fcγ RII and Fcγ RIII are known to be associated with human myeloid cells. Using mAb specific for these receptors, and the hydridoma cells lines that produce these mAb, we have examined the ability of each of these receptors on different myeloid cells and cell lines to mediate killing of tumor and red cell targets. Hybridoma cells (HC) expressing anti-Fcγ RI, Fcγ RII or Fcγ RIII upon their surface were used as model self-directed tumor targets. Chicken erythrocytes (CE) were used as another type of target cell and in this case effector cell cytotoxicity was mediated by heteroantibodies (HA) composed of Fab fragments of anti-Fcγ R mAb covalently linked to Fab fragments of rabbit anti-CE antibodies. Monocytes, lymphocytes, polymorphonuclear cells (PMNs) and the myeloid cell lines U937, HL-60 and THP-l were used as effector cells either in their native state or after activation with rIFN-γ. Direct comparison of cytotoxicity by the same effector cell population against both tumor and erythroid targets has permitted definitive evaluation of the ability of the different Fcγ R to promote cytolysis under two different conditions. Monocytes were able to utilize Fcγ RI, Fcγ RII and Fcγ RIII in killing both CE and HC targets, and incubation with rIFN-γ augmented their ability to kill CE, particularly through Fcγ RI. Fcγ RII and Fcγ RIII mediated killing of CE by untreated neutrophils. rIFN-γ induced PMNs to express Fcγ RI and to mediate killing of CE through this receptor. Moreover, HC targets were not lyzed by untreated neutrophils, but rIFN-γ activated neutrophils killed HC bearing surface anti-Fcγ RI and anti-Fcγ RII, but not anti-Fcγ RIII. Myeloid cell lines HL-60 and U937 were unable to perform cytotoxicity without prior culture with rIFN-γ, following which they killed CE through Fcγ RI and Fcγ RII, but were still incapable of HC lysis. THP-l, another myeloid cell line, was cytotoxic to CE through Fcγ RI and Fcγ RII without activation. Following rIFN-γ treatment, cytotoxicity through these two Fcγ R increased and was also mediated by Fcγ RIII but these cells were still unable to kill HC. However, after culture under conditions which promoted a macrophage-like phenotype, THP-l cells acquired the ability to kill HC through Fcγ RI and Fcγ RII. When differentiated THP-l cells were also stimulated with rIFN-γ, killing of HC was augmented and was mediated by Fcγ RI, Fcγ RII and Fcγ RIII. Thus, untreated monocytes performed cytotoxicity of CE and HC through all three Fcγ R, whereas untreated neutrophils performed killing of CE only through Fcγ RII and Fcγ RIII and killed HC only after treatment with IFN-γ and only through Fcγ RI and Fcγ RII. Fcγ RIII on PMNs was unable to promote tumor cell killing even after IFN-γ activation of these cells. These studies indicate that the different Fcγ R on the various effector cell populations have distinct abilities to mediate cytotoxicity of tumor and red cell targets.

Single step screening of monoclonal antibodies against interferon-γ-induced surface molecules on human monocytes

Interferon-γ (IFN-γ) activation of human monocytes in vitro results in enhanced phagocytosis and cellular cytotoxicity. These enhanced effector functions are attributable, at least in part, to increased expression of recognition molecules on the plasma membrane. In this article we report a rapid screening procedure for the primary selection of monoclonal antibodies (mAbs) which bind to cell surface molecules, the expression of which is increased or decreased by IFN-γ. The procedure is based on flow cytometric analysis of mixed cell populations. Mouse mAbs were prepared using human monocytes cultured for 40 h with 400 U/ml of IFN-γ as the immunogen. The hybridoma supernatants were screened using mixtures of six cell populations, some of which were pretreated with IFN-γ for 40 h. Cells included in the mixture were chosen for their distinctive light scatter profiles. mAbs of interest were identified by preferential binding to monocytes and increased or decreased binding to monocytes treated with IFN-γ. This procedure allowed us to screen several hundred clones per day, and to immediately eliminate mAbs that bound to B cells, T cells, neutrophils, and several cell lines. We selected ten mAbs which bound to surface molecules on monocytes that were modulated by IFN-γ. Further characterization of five of the initial ten mAbs revealed that mAb γMθ 22.2 and mAb γMθ 197.1 bind to the high affinity Fc receptor for IgG (FcγRI). mAb γMθ 28.3 appears to bind to a class II histocompatibility antigen and mAb γMθ 150.3 and mAb γMθ 195.18 appear to have binding patterns to human leukocytes and cell lines which are distinct from previously described mAbs. This rapid and specific procedure for screening mAbs has broad application for selecting mAbs that are specific for any given cell type and/or for surface molecules that are modulated by any cytokine and other hormone.

Activation of Human Monocyte-Derived Macrophages Cultured on Teflon: Response to Interferon-γ during Terminal Maturation in vitro

Macrophages (M) are potential antitumor effector cells derived from circulating blood monocytes (mo). Most studies on human mo/M biology and function have been performed using immature mo precursor cells. However, the conclusions drawn may be questionable, as mo have to undergo terminal differentiation before they reach relevant tissue sites of inflammation and immune reaction. We have analyzed the ability of mo-derived, teflon-cultured M to respond to activating stimuli with an increased tumor cytotoxic effector cell function using recombinant interferon-gamma (IFN-γ), IFN-α2, granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin(IL) 2, IL 1α, and bacterial lipopolysaccharides (LPS) as mediator molecules. It could be shown that the response of M to the most potent activator molecule, IFN-γ, depends on the terminal differentiation from the mo stage to the mature M. Whereas adherent mo could be activated only moderately, M increased their cytotoxicity by a factor of up to 400. IFN-γ activation positively correlated with the effector cell number, the time of incubation and the dosage used. Activation did not depend on the presence of LPS, and was lost within 24 to 48 h. LPS itself activated cells only in the microgram range. IFN-α2 activated M only at a two log higher concentration than IFN-γ; GM-CSF was only slightly effective, whereas M incubation with IL 1α or IL 2 did not result in M activation. Thus, the ability of human M to become activated appears to be a function of cellular maturation and is acquired during the terminal step of M differentiation. Teflon-cultured M could facilitate studies of the activation of human M and may be more suitable cells for adoptive immunotherapy in cancer patients than blood monocytes.

Effects of interferon on Natural Killer (NK) cells assessed by fluorescent probes and flow cytometry

The effects of interferon-α and -γ (IFN) on natural killer (NK) cells was investigated by labelling cells with fluorescent membrane and intracellular probes and analysing these by flow cytometry. Peripheral blood mononuclear cells (PBMCs), sorted NK cells and non-NK cells were labelled with one of the fluorescent probes, 3,3′-dihexyloxacarbocyanine (DiOC 6(3)), N-phenyl-1-naphthylamine (NPN) or Quin 2, subsequent to incubation with IFN-α or IFN-γ and the change in their fluorescence was monitored by flow cytometry. NK activity after treatment with IFN-α or -γ was also monitored in parallel using a standard 51Cr-release assay. IFN-α treatment of PBMCs caused an apparent depolarisation and subsequent hyperpolarisation of the cell membranes. Such changes reflect movement of ions across the cell membrane and also the binding of IFN-α to the cell surface receptor. Molecular conformational changes in the cell membrane due to IFN-α and IFN-γ were monitored by labelling cell populations with NPN. Changes in NPN-labelled cell fluorescence intensity, indicating changes in membrane conformation, were greatest in NK cell populations activated by IFN-γ. IFN-α had a more profound effect on non-NK cell populations. The concentration of free intracellular Ca 2+ ions is also affected by IFN-α and IFN-γ activation, as monitored by the fluorescent probe, Quin 2. There is an apparent decrease in intracellular Ca 2+ ion concentration in the NK cell population when treated with IFNs, with the greatest effect being shown by IFN-γ. These data indicate that the effects of IFNs on NK cells can be monitores at a cellular level using fluorescent probes and flow cytometry. As analysed by these probes, IFN-α and IFN-γ appear to affect NK cells via different mechanisms.