Age-related influences of clinical features of the lumber disc herniation – Immunological evaluation on co-cultured rat nucleus pulposus cells and macrophages – *
There have been only a few number of reports on the effects of aging on the immuno-inflammatory response in intervertebral disc herniation. The purpose of this study was to evaluate the effects of aging on nitric oxide（NO）and cytokine production in an in vitro model in which rat nucleus pulposus cells and macrophages were co-cultured. Male Sprague Dawley rats（n＝45）, including 15 animals from three different age groups（3-, 12-and32-weeks of age）, were used in this study. Coccygeal nucleus pulposus（NP）cells（4x105cells/well）and exudated peritoneal macrophages（4x106cells/well）were co-cultured in serum-free medium. The controls consisted of NP cells or macrophages cultured alone. NO levels in the culture medium were measured at2, 24, 48, and 72 hours by the Griess method. A cytokine array was done on the culture medium after seven days to evaluate the production of interferon-gamma（IFN-gamma）and interleukin-10（IL-10）and tissue inhibitor metalloproteinase-1（TIMP-1）． At24hours, NO levels had statistically increased in co-cultures of cells derived from the 32-week-old rats compared with younger rats（3-week-old:9.02μmol/l, 12-week-old:10.18μmol/l, 32-week-old:12.13μmol/l）（p＜0.001). After seven days of co-culture, TIMP-1 levels of the 3-week-old rats were statistically higher than those from the 12-and 32-week-old rats（3-week-old:302.83×103/control, 12-week-old:121.51×103/control, 32-week-old:107.15×103/control）（p＜0.001）. IFN-gamma levels in media from cells from the 3-week-old rats were statistically higher than those from the 12-and 32-week-old rats（3-week-old:75.01×103/control, 12-week-old:62.49×103/control, 32-week-old:41.21×103/control）（p＜0.01）. IL-10 levels from the 3-week-old rats were statistically lower than those from the 12-and 32-week-old rats（3-week-old:45.12×103/control, 12-week-old:89.46×103/control, 32-week-old:94.34×103/control）（p＜0.001）. In this study, age-related NO levels of the co-culture increased. The results show that inflammation increased along with aging. The outcomes of several diseases, such as aging, infections, and allergic and autoimmune disorders, have been linked to the balance between T helper（Th）1cells and Th2 cytokine production by Th cell-subsets. Th1 cells produce IFN-gamma, which activates cell-mediated immunity, while Th2 cells secrete IL-10, which activates humoral immunity. The results of this study show that the of aging effects on cytokine secretion related to the Th-type on co-cultures of rat NP cells and macrophages differ. In this study, an age-related imbalance between the levels of the cytokines, IFN-gamma and IL-10, was seen. In the younger rats, IFN-gamma levels were statistically higher while IL-10 levels were statistically lower. This suggests that this process may be caused by external stress and aging. Aging and stress are thought to affect the extracellular matrix and change the immunologic response. The younger rat NP cells had higher cell-mediated immunity activity, while the older rat NP cells had higher humoral immunity activity. These results show that aging has an effect of age on the immunologic responses attributable to NP cells. Further studies are needed to elucidate the mechanism of this newly observed phenomenon and ultimately to apply these findings clinically. (Accepted on March７，2006)
