Altered eicosanoid production and phospholipid remodeling during cell culture

Abstract

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B₂, PGE₂, and PGD₂, and the 5-lipoxygenase pathway, leukotriene (LT)B₄, LTC₄, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB₃, all trans-LTB₃, LTC₃, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.

Keywords: Mead acid; polyunsaturated fatty acid; volcano plot.

Fig. 1.

Cyclooxygenase products of AA that appear in the supernatant of RAW 264.7 cells carried in culture for the indicated number of days and then stimulated (or not) with calcium ionophore A23187. A: Prostaglandin D₂ production. B: Prostaglandin E₂ production. C: TxB₂ production. Data are expressed as average ± SEM, n = 3. Significance comparisons, *P < 0.01, **P < 0.001.

Fig. 2.

Levels of 20:4 and 20:3 fatty acids and their 5-lipoxygenase products that appear in the supernatant of RAW 264.7 cells carried in culture for the indicated number of days and then stimulated (or not) with calcium ionophore A23187. A: LTB₄ production. B: LTB₃ production. C: LTC₄ production. D: LTC₃ production. E: Free 20:4 accumulation. F: Free 20:3 accumulation. Data are expressed as average ± SEM, n = 3. Significance comparisons, *P < 0.01, **P < 0.001.

Fig. 3.

Phospholipid molecular species containing either 20:4 (orange bars) or 20:3 (red bars) esterified to PI (A), PS (B), PA (C), or BMP (D). Data are expressed as average ± SEM, n = 3.

Fig. 4.

A: Volcano plot comparing the relative abundances of phospholipid molecular species in RAW 264.7 cells cultured for 1 or 3 days. Those species significantly more than 2-fold more abundant at day 1 are in the upper left sector with a P-value (t-test) greater than 0.05. Those species more abundant at day 3 are in the right upper sector. The size of the circle is related to abundance as well as the color (as per the color scale) in units of abundance of ion divided by the abundance of the internal standard for that class of phospholipid (abundance ratio). B, C: Box plots of the abundance ratio of PC(16e_20:4) and PC(16e_20:3) in RAW cells after 1, 2, or 3 days of culture. The bar in the quartile-indicating box is the median value, n = 3 for each day in culture.

Fig. 5.

Production of TNFα by LPS (100 ng/ml) stimulation for 0, 1, 2, 4, 6, and 8 h in RAW cells cultured for 24 h (day 1) and 72 h (day 3) without medium change. Data are expressed as average ± SD, n = 6. ***P < 0.001 (paired t-test).