Elevation of oleate-activated phospholipase D activity during thymic atrophy

Abstract

Various phospholipases are thought to be associated with the in vitro apoptosis of thymocytes. In the present study, the in vivo phospholipase D (PLD) activity of rat thymus was studied after whole-body X-irradiation or injection of dexamethasone (DEX). Using exogenous [14C]dipalmitoyl phosphatidylcholine (PC) as the substrate, an elevation of oleate-activated PLD activity was observed during thymic atrophy. The activity increases were sevenfold at 48 hr after 5-Gy irradiation and fourfold at 72 hr after injection of 5 mg/kg DEX. The elevation of PLD activity appeared to parallel extensive thymus shrinkage. An increased level of thymic phosphatidic acid (PA), the presumed physiological product of PLD action on PC, was also detected. By comparing the acyl chains of PA with those of other phospholipids, PA appeared to originate from PC. To assess the role of PLD during thymic atrophy, thymocytes and stromal cells were isolated. Although thymocytes themselves exhibited significant PLD activation, the major elevation in PLD activity (greater than fourfold) was found in isolated stromal cells. PLD was also activated during in vitro phagocytosis of apoptotic thymocytes by the macrophage-like cell line P388D1. This in vitro phagocytosis was significantly inhibited by PLD action blockers, such as 2,3-diphosphoglycerate and 1-butanol. These observations strongly suggest that the alteration of oleate-activated PLD activity is part of an in vivo event in the progression of thymic atrophy, including phagocytic clearance of apoptotic thymocytes.

Figure 1

Verification of assay conditions for phospholipase D (PLD) activities in rat thymus (a). PLD activity assays were carried out with or without 5 mm oleate, 12·6 µm phosphatidylinositol 4,5-bisphosphate (PIP₂), 60 µm guanosine 5′-(3-O-thio)triphosphate (GTPγS), and 1 µm ADP-ribosylation factor (ARF). The reaction mixtures contained 200 µg of protein from crude membranes for oleate-activated PLD or solubilized membrane fraction for ARF-dependent PLD, as described in the Materials and methods. (b) Effects of fatty acids on PLD activity in thymus membranes. Data represent mean values ± standard error (SE) of at least two independent experiments performed in duplicate.

Figure 2

Effect of whole-body irradiation on the oleate-activated phospholipase D (PLD) activity in thymus. (a) Effect of X-irradiation dose. Wistar rats were irradiated with an X-ray generator CLINAC 600C (4 MV) and PLD activities were determined 24 hr after irradiation. The PLD assay was carried out with 200 µg of crude membrane protein in the presence of 5 mm oleate as an activator. Data represent mean values ± standard error (SE) of at least four independent experiments performed in duplicate. (b) Effect of postirradiation time. The oleate-activated PLD activities were determined during thymic atrophy induced by 5-Gy X-irradiation at the indicated time-points after irradiation. Data represent mean values ± standard error (SE) of two to seven experiments for PLD activities (**P < 0·01, *P < 0·03) and means of two experiments for thymus weight.

Figure 3

Effect of dexamethasone (DEX) injection on the oleate-activated phospholipase D (PLD) activity in thymus. (a) Effect of DEX dose. DEX was injected intraperitoneally and the oleate-activated PLD activities were determined 48 hr after injection. Data represent mean values ± standard error (SE) of two independent experiments performed in duplicate. (b) Effect of postinjection time. The oleate-activated PLD activities were determined during thymic atrophy induced by 5 mg/kg DEX injection at the indicated time-points after injection. Data represent mean values ± standard error (SE) of three to nine experiments for PLD activities (**P < 0·01, *P < 0·05) and means of two experiments for thymus weight.

Figure 4

In vivo degradation of thymocyte DNA during thymic atrophy. (a) DNA fragmentation 6 hr after whole-body X-irradiation or dexamethasone (Dex) injection. Thymocytes were isolated, and extracted DNAs were electrophoresed on a 1% agarose gel. Lane M, 1-kb ladder marker; C, phosphate-buffered saline (PBS)-injected control; 5 Gy, 5-Gy X-irradiation; Dex, 5 mg/kg dexamethasone injection. (b) Time-course of the DNA degradation induced by X-irradiation. Rats were subjected to whole-body irradiation with 5-Gy X-ray and DNA samples were extracted at the indicated time-points (hr).

Figure 5

Distribution of the oleate-activated phospholipase D (PLD) activity between thymocytes and stromal cells. Thymocytes and stromal cells were isolated 48 hr after 5-Gy X-irradiation and injection of 5 mg/kg dexamethasone (Dex). Each assay was carried out with 60 µg of tissue homogenate in the presence of 5 mm oleate. Data represent mean values ± standard error (SE) of three experiments.

Figure 6

Phagocytosis of apoptotic thymocytes by P388D1 macrophages. (a) Effect of apoptotic thymocytes on the phospholipase D (PLD) activity of P388D1 macrophages. P388D1 cells were labelled with [³H]palmitic acid and incubated with or without apoptotic thymocytes for 1 hr, as described in the Materials and methods. For control data, the PLD activity of P388D1 cells in the freshly prepared thymocytes was also determined. PLD activity was presented as percentage formation of [³H]phosphaytidylbutanol (PBt) from total labelled lipids. Data represent mean values ± standard deviation (SD) of triplicate determinations. (b) The effect of PLD action blockers, 2,3-diphosphoglycerate (2,3-DPG) and 1-butanol (1-BuOH), on the phagocytotic capability of P388D1 macrophages. P388D1 cells were incubated with apoptotic thymocytes for 1 hr in the presence or absence of 2,3-DPG (10 mm), 1- and 2-butanol (0·3%, vol/vol), or phospho-l-serine (PLS, 1 mm), as described in the Materials and methods. Data are presented as numbers of phagocytosed thymocytes per 100 P388D1 cells. Data represent mean values ± standard deviation (SD) of three determinations from random fields. (Inset) Effect of 2,3-DPG on the PLD activity of P388D1 macrophages. [³H]-Labelled P388D1 cells were incubated for 30 min in the presence or absence of 2,3-DPG (10 mm). The 2,3-DPG effect in the presence of apoptotic thymocytes (2,3-DPG + apoptotic thy) was also measured. PLD activity was presented as percentage formation of [³H]PBt from total labelled lipids. Data represent mean values ± standard deviation (SD) of triplicate determinations.

Figure 7

Alteration of phosphatidic acid (PA) during thymic atrophy. (a) The PA content of rat thymus at 24 hr after 5-Gy X-irradiation. Thymus total lipid extract was subjected to high-performance liquid chromatography (HPLC) chromatography and the level of PA was estimated as described in the Materials and methods. Results were expressed as weight percentage of the total lipid (means ± standard error of three experiments). (b) Analysis of fatty acid side-chains of PA. Major phospholipids (including PA) were separated from rat thymus at 48 hr after injection of 5 mg/kg dexamethasone, and their acyl chains were analysed as described in the Materials and methods. For comparison, the acyl chain composition of DAG was also analysed. Data are presented as percentage molar ratios of fatty acids in each lipid species.