Taizo Nitta

Human Glioma-derived Interleukin-10 Inhibits Antitumor Immune Responses In Vitro

Intracranial malignant gliomas are sequestered from the immune system yet are associated with broad suppression of host immunocompetence. Immune system dysfunction in patients with gliomas seems to be related to inhibitory mediators produced by glioma cells. We investigated the physiological roles of glioma-derived interleukin (IL)-10 in Class II expression of monocytes, cytokine secretion from lymphocytes, and T cell proliferation in vitro. We could detect the messenger ribonucleic acid transcript of IL-10 in four gliomas by the reverse-transcribed polymerase chain reaction. Glioma-derived IL-10 greatly down-regulated human lymphocyte antigens-DR expression on monocytes. The inhibitory effect of IL-10 on interferon-gamma and tumor necrosis factor-alpha was neutralized by the anti-IL-10 monoclonal antibody; however, the inhibitory effect on IL-2 was not neutralized. Next, supernatants of glioma cells remarkably suppressed T cell proliferation in a dose-dependent fashion; however, this inhibitory effect was not restored by adding anti-IL-10 monoclonal antibodies. The supernatant also inhibited the allocytolytic activity of lymphocytes that were not neutralized by anti-IL-10 monoclonal antibody. IL-10 plays an important role in cytokine synthesis; nevertheless, impaired T cell responsiveness cannot be solely explained by glioma-derived IL-10.

Expression of granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor genes in human astrocytoma cell lines and in glioma specimens

Expression of granulocyte (G) and granulocyte-macrophage (GM) colony stimulating factor (CSF) genes in human cells of astroglial lineage was studied. Primers for CSFs were used to analyze RNA transcripts in 5 cultured human astrocytoma cell lines and 8 fresh brain specimens by polymerase chain reaction. Constitutive expression of mRNA transcripts of GM-CSF could be detected in all astrocytoma and one neuroblastoma cell lines, and two out of 5 unstimulated astrocytomas, U87MG and U138 MG, expressed G-CSF genes. After stimulation with interleukin (IL)-1 beta + tumor necrosis factor (TNF)-alpha, all cell lines expressed G-CSF. In addition to the cultured cells, we examined gene expression within human malignant astrocytoma, peritumoral brain and autopsied normal brains. The results show that some of the tumor and its surrounding reactive lesions express G- and GM-CSF genes but normal brains do not. The concentration of G- and GM-CSF in supernatants of cultured cells was assessed at the protein level by ELISA. A low level of GM-CSF activity was constitutively present in all astrocytomas. G-CSF was detected in unstimulated U87MG and U138MG and other cell lines could synthesize G-CSF after the stimulation of IL-1 beta and TNF-alpha at the level of mRNA. Furthermore, the concentration of CSFs increased markedly upon stimulation with IL-1 beta and/or TNF-alpha in both a time- and dose-dependent fashion. From these results, it is suspected that astroglial cell-derived CSFs may participate in local immune reactions accompanying infection, degeneration and malignancies in the brain.

Neoplastic and reactive human astrocytes express interleukin-8 gene

Expression of lymphokine genes in the human astroglial cell lineage was studied. Primers for 9 different human lymphokines, from IL-1α to IL-8, were used to analyze RNA transcripts in 5 cultured human astrocytoma cell lines and fresh brain specimens by PCR. mRNA transcripts for IL-8 were detected in all neuroglial cells. In addition to the cultured cells, we examined IL-8 gene expression within human malignant astrocytoma, peritumoral brain and autopsied normal brains. The result shows that tumor and cells of the surrounding reactive lesion express IL-8 genes, but it is not expressed in normal brains. Next, the concentration of IL-8 in supernatants of cultured cells was measured quantitatively by a solid phase ELISA assay. IL-8 activity was produced constitutively in all astrocytomas and increased markedly upon stimulation with IL-1β or TNF α, in both a time- and dose-dependent fashion. From these results, it is supected that astroglial cell-derived IL-8 may take part in neutrophil-mediated inflammation which accompanies infection, degeneration and malignancy in the brain.

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

SummaryTo study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4+ and CD8+ T cell subsets. To analyze this reactivity, sorter-purified CD4+ and CD8+ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4+ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8+ grew vigorously and 29 cytolytic CD8+ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.

Lysosomal Enzymic Activity of Astroglial Cells

Using human and rat Glioma cell lines and mouse astrocytes prepared from newborn mice, lysosomal enzymatic activity, which is closely related with phagocytic activity, was assayed by spectrophotometric measurement of p-nitrophenol liberated from the relevant substrate. Lysosomal N-acetyl-beta-D-glucosaminidase and acid phosphatase activity was detected in neoplastic astrocytes at a level of enzymatic activity equal to that of monocyte/macrophage cell lines in certain glioma cells. From the implication of this data, it is suspected that astrocytes may function as phagocytic cells at inflammatory brain lesions.

Surrogate Receptor-Mediated Cellular Cytotoxicity against Malignant Human Glioma Cells

A method for incorporating palmitate-derivatized antibody molecules into effector cells and the influence of these incorporated antibodies (surrogate receptors) on the lytic potential of peripheral blood lymphocytes is described. This surrogate receptor-mediated cellular cytotoxicity provides a means by which killer cells are directed specifically to the desired target glioma cells. The cytotoxic activity does not require the Fc region of the incorporated antibody molecules and shows more lytic potential than ordinary antibody-dependent cellular cytotoxicity. By complement-mediated cell depletion and Percoll gradient separation, the effector cell populations for surrogate receptor-mediated cellular cytotoxicity are found to be natural killer cells, as in the murine system. This strategy of surrogate receptor incorporation into effector cells may provide not only valuable insights into the mechanism of cell-mediated cytotoxicity, but also a means of generating target-specific effector cells for clinical applications in neurosurgery.