Astrocytoma cells upregulate expression of pro-inflammatory cytokines after co-culture with activated peripheral blood mononuclear cells

Abstract

In this study, we investigated the effect of CD3/CD28-activated peripheral blood mononuclear cells (PBMCs) on two human astrocytoma cell lines (1321N1 and CCF-STTG1), with respect to the expression of cytokines and chemokines. We used an in vitro co-culture system in which the astrocytoma cells and PBMCs were separated by a membrane in a transwell system. Proliferation of T cells was quantified by a [(3) H]-thymidine incorporation assay. Differential gene expression of astrocytoma cells cultured alone or with PBMCs was identified using gene arrays. Protein expression of CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1β was determined by Luminex. Co-culture of activated T cells and astrocytoma cells resulted in inhibition of T cell proliferation. Moreover, astrocytoma cells upregulated a number of pro-inflammatory genes including CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1β upon co-culture with activated PBMCs. These results were confirmed on the protein level. Astrocytoma cells inhibited the proliferation of T cells, indicating that astrocytes may play an important role in controlling T cell mediated immune responses in the brain. Contrary to this, soluble factors derived from CD3/CD28-activated PBMCs affected the transcriptome of astrocytoma cells, which then upregulated several pro-inflammatory genes and proteins. This could be part of neuroinflammatory events and may play a role in neurodegenerative diseases.

The Cancer Genome Atlas expression profiles of low-grade gliomas

“…6,36 The inflammatory markers upregulated in astrocytomas in the current analysis may reflect the increased number of microglia in astrocytic gliomas relative to oligodendrogliomas 7,26 or the inherent expression of cytokines by astrocytic gliomas. 12,19,35,36 Aberrant miRNA expression is a universal theme during carcinogenesis. 16 Moreover, the pattern of miRNA expression often marks the lineage of origin with regard to the tumor.…”

Immune Privilege as an Intrinsic CNS Property: Astrocytes Protect the CNS against T-Cell-Mediated Neuroinflammation

“…The effect was more pronounced when direct contact was allowed between astrocytes and T cells but remained strong when astrocytes and T cells were separated by cell culture inserts [123]. The finding that T-cell proliferation was still inhibited by astrocytes when astrocytes and T cells were separated by a cell culture insert or a transwell-membrane showed that a soluble factor produced by astrocytes is responsible for this inhibition [107, 123, 124]. However, astrocytes might conceivably have protruded cellular nanotubes through the cell culture inserts so as to contact the T cells.…”

“…However, astrocytes might conceivably have protruded cellular nanotubes through the cell culture inserts so as to contact the T cells. The separating membranes had pore sizes of 200 nm [123] or 400 nm [107, 124]. An electron-microscopical study of astrocytes growing on engineered surfaces showed that astrocytes extend nanotubes with a diameter below 100 nm to make contact with other cells and may even exchange substances via these nanotubes [125].…”

“…Blockade of TGF- β had no [124] or only a minor effect [107] on the inhibition of T-cell proliferation. Inhibition of nitric oxide production also did not reverse the inhibitory effect [123, 124]. Furthermore, inhibition of indoleamine-2,3 dioxygenase (IDO) by methyltryptophan did not affect astrocyte-mediated inhibition of T-cell proliferation [123].…”

“…Under the influence of IFN- γ , astrocytoma cells upregulate expression of chemokines including CCL3, CCL5, CXCL8, and CXCL10, as well as proinflammatory cytokines such as IL-6 and IL-1 β (but also an anti-inflammatory IL-1 receptor antagonist) [123]. Most of these chemokines attract Th1 cells more than Th2 cells, thus aggravating neuroinflammation.…”