The Effects of LPS With or Without Growth Factors on S16 Rat Schwann Cell Proliferation
As a preliminary study, the immortalized S16 rat Schwann cell line was cultured with different combinations of lipopolysaccharide (LPS, a cell wall immunostimulatory component found in Gram-negative bacteria) and growth factors (the neuronal mitogen heregulin and the artificial plant extract forskolin) to determine the effects of the different treatment combinations on cell proliferation. The cells received one of the following treatments: N2 control media, 12.5 ng/mL of heregulin (H), 2 uM of forskolin (F), or heregulin plus forskolin (H+F), and 0 ,5, 50, or 500 ng/mL of LPS, for 1, 3, 12, or 24 hours. The CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher Scientific) was used to perform the proliferation assay. Optical density was measured as an indicator of cell proliferation, with a high optical density representing high proliferation and a low optical density representing low proliferation. It was hypothesized that, for all time points, cells treated with LPS plus growth factors would experience more proliferation than cells treated with LPS only. The results only partially support the hypothesis because there were some cases where the LPS + growth factor treatments had less proliferation than the LPS only (N2) treatments (i.e., H + 5 or 50 ng/mL of LPS for 24 hours). One of the most important findings is that between the treatments used, it appears as though either 5 or 50 ng/mL of LPS combined with both heregulin and forskolin for 24 hours results in the highest optical density, and therefore the most Schwann cell proliferation. It also appears as though Schwann cells can tolerate higher concentrations of LPS for shorter periods of time and lower concentrations of LPS for longer periods of time. This indicates that, eventually, higher doses of LPS may become too toxic for the Schwann cells to handle, inhibiting proliferation, and causing cell death. Also, considering Schwann cells treated with LPS for 24 hours had generally higher optical densities than cells treated with LPS for 1, 3, and 12 hours, there may be some period of recovery between 12 and 24 hours. When looking at each individual growth factor treatment, the results suggest that during exposure to particular concentrations of LPS for 24 hours, Schwann cells grown in the presence of forskolin, with or without heregulin, experience an increase in proliferation, whereas Schwann cells grown in the presence of heregulin experience a decrease in proliferation. These findings suggest that, when Schwann cells are stimulated with LPS, heregulin and forskolin, alone, activate two very distinct pathways to initiate opposite responses, with heregulin hindering cell division and forskolin promoting cell division. However, when heregulin and forskolin are combined, the forskolin-activated cAMP pathway may overcompensate by promoting even more cell division to offset the decrease in cell division initiated by the heregulin pathway.