Arachidonoyl-phospholipid remodeling in proliferating murine T cells

Abstract

Background: Previous studies have shown that the functional capacity of T cells may be modulated by the composition of fatty acids within, and the release of fatty acids from membrane phospholipids, particularly containing arachidonic acid (AA). The remodeling of AA within membrane phospholipids of resting and proliferating CD4+ and CD8+ T cells is examined in this study.

Results: Splenic T cells were cultured in the presence or absence of anti-CD3 mAb for 48 h then labeled with [3H]AA for 20 min. In unstimulated cells, labeled AA was preferentially incorporated into the phosphoglycerides, phosphatidylcholine (PC) followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). During a subsequent chase in unlabeled medium unstimulated CD4+ and CD8+ T cells demonstrated a significant and highly selective transfer of free, labeled AA into the PC pool. In contrast, proliferating CD4+ and CD8+ T cells distributed labeled [3H]AA predominantly into PI followed by PC and PE. Following a chase in AA-free medium, a decline in the content of [3H]AA-PC was observed in association with a comparable increase in [3H]AA-PE. Subsequent studies revealed that the cold AA content of all PE species was increased in proliferating T cells compared with that in non-cycling cells, but that enrichment in AA was observed only in the ether lipid fractions. Finally, proliferating T cells preincubated with [3H]AA exhibited a significant loss of labeled arachidonate in the PC fraction and an equivalent gain in labeled AA in 1-alk-1'-enyl-2-arachidonoyl-PE during a chase in unlabeled medium.

Conclusion: This apparent unidirectional transfer of AA from PC to ether-containing PE suggests the existence of a CoA-independent transacylase system in T cells and supports the hypothesis that arachidonoyl phospholipid remodeling may play a role in the regulation of cellular proliferation.

Figure 1

[³H]AA incorporation in T cells. Splenic T cells were cultured for 48 hr with (open circles) or without (closed circles) anti-CD3 mAb, then incubated with [³H] AA (1 μCi/ml) for up to 60 min. At varying times, cells were harvested and labeled AA incorporation was quantified. Inset, Tritiated thymidine incorporation, assessed daily for 3 days, in T cells cultured with (open circles) or without (closed circles) anti-CD3 mAb. The results are the mean and standard error of 3 separate experiments.

Figure 2

The distribution and turnover of [³H]AA in proliferating and unstimulated T cells. Splenic T cells, previously cultured as above for 48 hours with (right panel, open circles) or without (left panel, closed circles) anti-CD3 mAb, were labeled with [³H]AA (1 μCi/ml) for 20 min at 37°C. The cells were then washed and cultured at 37°C for up to 60 min in BME with 1% BSA. At the initiation of culture (zero time) and at various times thereafter, cells were harvested, membrane lipids were extracted and free AA and phosphoglycerides were resolved by TLC. The concentration of free [³H]AA and of [³H]AA within different phospholipid molecular species was measured by liquid scintillation counting. Data are the mean and standard error of 4 separate experiments.

Figure 3

Incorporation and turnover of [³H] AA within membrane phospholipids of proliferating and resting CD4⁺ and CD8⁺ T cells. Splenic T cells were culture as above for 48 hr with (open symbols) or without (closed symbols) anti-CD3 mAb, then enriched for CD4⁺ T cells (dashed triangles) or CD8⁺ T cells (circles) by negative selection. Following labeling with [³H]AA (1 μCi/ml) for 20 min, the cells were then washed and cultured at 37°C for 60 min in BME with 1% BSA. At the initiation (zero time) and conclusion of culture (60 min), cells were harvested, membrane lipids were extracted and free AA and phosphoglycerides were resolved by TLC. The concentration of free [³H]AA and of [³H]AA within different phospholipid molecular species was measured by liquid scintillation counting. Data are the mean of 2 experiments.

Figure 4

AA content within phospholipids of proliferating and unstimulated splenic T cells. Splenic T cells, previously cultured as above for 48 hours with (open bars) or without (closed bars) anti-CD3 mAb were harvested, phospholipids were isolated by TLC, fatty acids were derivatized to methyl esters and arachidonoyl-methyl ester content was determined by gas chromatography. Data are the mean and standard error of 4 experiments.

Figure 5

AA content within PC and PE molecular species of proliferating and quiescent T cells. Splenic T cells, previously cultured as above for 48 hours with (open bars) or without (closed bars) anti-CD3 mAb, were harvested and PC and PE species were resolved by TLC. Phospholipids were further separated into diacyl-, 1-alkyl-2-acyl- and 1-alk-1'enyl-2-acyl-diaglycerides. Fatty acids were derivatized to methyl esters as above and arachidonoyl-methyl ester content was determined by gas chromatography. Data are the mean and standard error of 3 experiments.

Figure 6

A comparison of the content of diacyl- and ether-lipids within PC and PE species of proliferating and unstimulated T cells. Splenic T cells pooled from 4 mice, previously cultured as above for 48 hours with (open bars) or without (closed bars) anti-CD3 mAb, were harvested and PC and PE species were resolved by TLC. Phospholipids were further separated into diacyl-, 1-alkyl-2-acyl- and 1-alk-1'enyl-2-acyl-diaglycerides and the content of diacyl- and ether-lipids was quantified by elemental phosphorous measurement. Data are the mean and standard error of three experiments.

Figure 7

Movement of [³H]AA within PC and PE species of proliferating and unstimulated T cells. Splenic T cells pooled from 6 mice, cultured as above for 48 hr with (open bars) or without (closed bars) anti-CD3 mAb, were labeled with [³H]AA (1 μCi/ml) for 20 min. The cells were then washed and cultured at 37°C for 60 min in BME with 1% BSA. At the initiation (zero time) and conclusion of culture, cells were harvested and PC and PE species were resolved by TLC. Phospholipids were further separated into diacyl-, 1-alkyl-2-acyl- and 1-alk-1'enyl-2-acyl-diaglycerides and the content of diacyl- and ether-lipids was quantified by liquid scintillation counting. Data are the mean and standard error of 3 experiments. *p < .05 compared to arachidonoyl phospholipid content at the initiation of culture. **p < .01 compared to arachidonoyl phospholipid content at the initiation of culture.