A novel thymosin peptide stimulates interleukin-6 release from rat C6 glioma cells in vitro

Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus possessing immunopotentiating properties. TF5 also stimulates the hypothalamic-pituitary-adrenal axis in vivo and anterior pituitary hormone release in vitro. Interleukin-6 (IL-6) is an inflammatory, pyrogenic cytokine that also stimulates hypothalamic and anterior pituitary hormone release. We hypothesized that TF5 may activate the neuroendocrine system in part via the stimulation of central cytokine production. Therefore, we determined the effects of TF5 on IL-6 release from rat C6 glioma cells in vitro. Glioma cells (25-100 x 10(3)) were exposed to vehicle (RPMI-1640) or TF5 (10-1,000 micrograms/ml) in 96-well plates (200 microliters incubation volume) for 4-24 h to determine optimal cell number and incubation period conditions. TF5 (1,000 micrograms/ml) stimulated IL-6 release from 100 x 10(3) C6 cells/well by 9-fold following a 24-hour incubation (p < 0.01). Reducing the number of cultured C6 cells to either 50 or 25 x 10(3) cells/well resulted in diminished IL-6 responses to TF5. TF5 stimulated C6 cell IL-6 release in a time-dependent manner (4-24 h) at all concentrations tested. A 24-hour incubation period provided the largest TF5-stimulated increases in IL-6 release compared with shorter time intervals (i.e., 4-8 h). Pretreatment of C6 glioma cells with 1 microM phorbol myristate acetate (PMA) for 24 h completely blocked the subsequent stimulation of IL-6 release by PMA (20-250 nM) and partially blocked by 50% the TF5 stimulation of this cytokine. Peptides previously purified from TF5 had no effect on IL-6 release at 50-1,000 nM [i.e., thymosin alpha 1 (T alpha 1), thymosin beta 4 (T beta 4), MB35, MB40]. Therefore, TF5 was further fractionated into 7 pools by preparative reverse phase high performance liquid chromatography (HPLC). HPLC pools P1 (fractions 1-8) and P2 (fractions 9-12) significantly increased C6 cell IL-6 release (p < 0.01) to the same extent as 250 micrograms/ml TF5. Other HPLC pooled fractions (P3-P7) had no effect on IL-6 release from C6 glioma cells. P1 and P2 stimulated a 50- and 10-fold increase in IL-6 release, respectively, at a protein concentration of 1.0 microgram/ml. Therefore, P1 was more potent and displayed a greater efficacy for the stimulation of IL-6 release compared to P2. Analysis of individual fractions of P1 and P2 revealed that 1 microgram/ml of fraction 6 was as efficacious as 250 micrograms/ml TF5 for the stimulation of IL-6 release. These data indicate that one or more peptide components of TF5 enhance glial cell production of IL-6. In addition, the thymosin-stimulated production of extracellular IL-6 is mediated partially by one or more isoforms of protein kinase C. We hypothesize that a peptide product of the thymus transported across the CNS blood-brain barrier may stimulate the glial cell production of IL-6 and affect neuronal, neuroendocrine and/or inflammatory processes.