Effect of aging on Kupffer cell membrane phospholipid function: Modulation by vitamin E

Abstract

AIM: To investigate the mechanisms leading to the disordered response to aging liver Kupffer cells (KCs).

METHODS: The effects of aging on KC membrane signal transduction and eicosanoid production and their management with vitamin E (VE) were assessed by measuring inositol phospholipid (PI) metabolism, intracellular calcium responses, and prostaglandin Ea-2 (PGEa-2) production in response to the inflammatory signals endotoxin (LPS) and platelet activating factor (PAF).

RESULTS: Aging resulted in a significant alteration in signal transduction of PAF as both PI turnover and calcium response were significantly reduced in the 18- and 24-mo old groups, compared with the 6 mo-old group. Aging significantly reduced PGEa-2 production in response to LPS. VE pretreatment resulted in an increased PI turnover, calcium response and PGEa-2 production.

CONCLUSION: The aging KCs have a disordered membrane phospohlipid function and VE is an effective modulator on it.

Key Words: Liver/cytology, Phospholipids, Calcium, Prostaglandin, Vitamin E, Aging

INTRODUCTION

Liver Kupffer cells (KCs) play an important role in the inflammatory process through the production of eicosanoids and monokines. Aging has an important effect on KC, resulting in a disordered function, which is related with the susceptibility of the defense system of the aged host[1]. Membrane phospholipids provide structural integrity to the cell, and have many additional functions. These include: (1) serving as substrate for inflammatory mediators, i.e., eicosanoid production, and (2) transducing extracellular signals into appropriate intracellular responses. We examined the effect of aging on membrane phospholipid function of KCs and its management with vitamin E (VE) to elucidate the nature of their relationship.

MATERIALS AND METHODS

Animals and groups

Forty Wistar rats (Chinese Herb Research Institute of Sichuan Province) were divided into four groups (6, 12, 18 and 24-mo age groups). Each group was further randomly subdivided into two groups: VE pretreated group (VEG) and non-VE pretreated group (NVEG). Each group has 5 animals. Animals in the VEG were given an introperitoneal injection of 5% VE solution for 3 months (500 mg/kg/wk, 2 injections/wk) before experiments. The NVEG was given normal saline.

KC isolation

Liver non-parenchymal cells were isolated by a collagenase-perfusion method[2]. After anaesthesia (30 mg of barbital/kg body weight, intraperitoneally), the liver was perfused in situ with Ca²⁺-free Hanks balanced salt solution at 37 °C for 3 min. Then 0.05% collagenase (Sigma, type IV) was added and the liver was perfused for 4 min with Hanks balanced salt solution. After gentle shaking, the suspension was filtered and hepatocytes were sedimented at 50 g for 3 min. Non-parenchymal cells were collected and sedimented at 300 g for 10 min. The pellets of non-parenchymal cells were resuspended and cultured with RPMI 1640 medium containing 15 mmol/L HEPES, 0.05 U/mL insulin, 15 mmol/L L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin supplemented with 10% newborn calf serum. After 30 min, the non-adherent cells were deleted. The viability of KCs was greater than 90% as determined by trypan blue exclusion.

Measurement of prostaglandin E₂ (PGE₂) production

KCs (1 × 10⁶/mL) were plated in a 2-mL volume of medium in six-well plastic tissue culture trays (New York). The plated cells were allowed 6 h to adhere and recover from the isolation procedure, and then non-adherent cells were washed away. The adherent monolayers were then stimulated with lipopolysaccharide (LPS) from Escherichia coli O₁₁₁B₄ (Sigma). In 90 min, the conditioned supernatants were removed, filtered, and stored at -70 °C. The PGE₂ level of each conditioned supernatant was determined by radioimmunoassay according to the manufacturer’s protocol (Scientific Institute of Military Medicine).

Measurement of intracellular calcium

Intracellular calcium was measured by a method previously described[3]. KC suspensions were loaded with the fluorescent calcium probe Fura-2 for 40 min in culture medium. Uptake of the dye was confirmed by fluorescent microscopy. The cells were washed twice and resuspended in HEPES-Krebs-Rins buffer before measurement. Intracellular calcium was measured on a MPF-4 spectrofluorimeter at an excitation wavelength of 380 nm and an emission wavelength of 510 nm. Macrophages were added to the cuvette (2 million/mL) and kept in suspension by constant stirring. Fluorescence was recorded. Changes in intracellular calcium in response to the added signals were calculated from the changes in fluorescence after calibration. Triton X-100 (final concentration 0.09%) was used in calibration for Fmax and Fmin is determined by addition of 9 mmol/L EGTA.

Determination of inositol phospholipid turnover

KCs (2 × 10⁶/mL) were cultured overnight in PRMI 1640 with 5% calf serum containing 3.0 μCi/mL of [³H]inositol. After washing, the cells were incubated for 15 min in culture medium containing 10 mmol/LiCl. The signal to be tested for stimulation of phospholipid (PI) turnover was added and the cells were incubated for 1-5 min. The cell monolayers were placed on ice, the medium was removed, and the cells were extracted with chloroform:methanol:water (1:1:0.9) into aqueous and lipid phases. The counts in the aqueous phase corresponded to total inositol phosphates[4]. The increases in total counts compared to controls indicated a signal-induced turnover of inositol phospholipid.

RESULTS

PGE₂ productions in various groups

The production of PGE₂ in response to LPS was significantly reduced in KCs from the 18- and 24-mo age groups in comparison with the 6-mo age group. In addition, with the increase of LPS concentration, the production of PGE₂ of the 6-mo group increased with the peak at 100 ng/mL and that of the 24-mo group peaked at 10 ng/mL. VE pretreatment had no significant effect on the production of PGE₂ by KCs from the 6-mo age group, but had an apparent effect on that by KCs from the 24-mo age group (Table 1).

Table 1 Prostaglandin E₂ production capacities of Kupffer cells from various age groups after lipopolysaccharide stimulation.

Group	0 ng/mL	10 ng/mL	100 ng/mL	10 μg/mL
6 mo
NVEG	921.78 ± 102.46	836.47 ± 90.18	3224.42 ± 396.53d	2926.02 ± 323.47d
VEG	985.23 ± 89.34	1011.47 ± 104.15	3553.64 ± 304.81	3018.74 ± 297.57
24 mo
NVEG	315.94 ± 26.54b	1013.19 ± 97.47d	634.73 ± 56.07bd	194.91 ± 21.78bd
VEG	713.48 ± 65.50bf	1278.78 ± 113.21bdf	1455.67 ± 121.34bdf	1337.46 ± 122.59bdf

^bP < 0.01 compared with 6 mo;

^dP < 0.01 compared with 0 ng/mL;

^fP < 0.01 compared with NVEG. NVEG: non-VE pretreated group; VEG: VE pretreated group.

Intracellular calcium responses of each age group

Table 2 shows that the basal level of [Ca⁺⁺]i increased with age. After the stimulation of platelet activating factor (PAF), the [Ca⁺⁺]i levels of four age groups were comparable with significant decreases of the net increase in the 18- and 24-mo age groups. KCs of 18 or 24 months old from vitamin E-fed animals showed a significant attenuation of basal level of [Ca⁺⁺]i and a better calcium response in comparison with NVEG.

Table 2 Intracellular calcium concentrations in various age groups before and after platelet activating factor stimulation (mean ± SD, pg/mL, n = 5).

Group (mo)	PAF	[Ca++]i		Δ[Ca++]		%
		NVEG	VEG	NVEG	VEG	NVEG	VEG
6	-	105.7 ± 16.1	111.5 ± 12.1
	+	266.1 ± 31.3d	285.3 ± 27.5	160.4 ± 22.4	173.8 ± 19.7	151.8	155.8
12	-	111.4 ± 19.3	109.8 ± 9.7
	+	262.8 ± 27.6d	276.3 ± 30.3	151.4 ± 21.9	166.5 ± 16.0	135.9	151.6
18	-	151.4 ± 21.4	118.4 ± 10.8f
	+	254.5 ± 36.7db	255.9 ± 27.0	103.1 ± 27.9a	137.5 ± 14.8a	68.1b	116.1bf
24	-	164.3 ± 20.8	130.7 ± 12.1f
	+	249.3 ± 28.4db	255.7 ± 24.1	85.0 ± 24.3b	125.0 ± 14.0bf	51.7b	95.6bf

^aP < 0.05 compared with 6 mo;

^bP < 0.01 compared with 6 mo;

^dP < 0.01 compared with PAF (-);

^fP < 0.01 compared with NVEG. NVEG: non-VE pretreated group; VEG: VE pretreated group; PAF: Platelet activating factor

Inositol phospholipid metabolism of each age group

It was found that the total inositol phospholipid decreased with age and that of the 18- or -24-mo old group was significantly decreased compared with that of the 6 mo-old group. When stimulated with PAF, KCs of the four groups had significantly increased levels with a fact that the total inositol phospholipid of the 18- or -24-mo old group was still significantly lower. VE pretreatment resulted in significant elevations in the 18- and/or 24-mo old groups in the control and PAF groups compared with NVEG. In addition, LPS stimulation resulted in no change of inositol phospholipid in each group (Table 3).

Table 3 Total inositol phospholipid in various age groups (mean ± SD, cpm × 1000, n = 5).

	6 mo	12 mo	18 mo	24 mo
NVEG
Control	48.5 ± 4.3	46.3 ± 4.5	39.5 ± 4.3a	30.8 ± 4.4b
LPS	52.4 ± 3.8	49.7 ± 5.6	42.8 ± 4.6a	34.8 ± 3.9b
PAF	78.7 ± 6.1f	78.1 ± 8.4f	62.9 ± 6.5bf	49.5 ± 5.4bf
VEG
Control	53.1 ± 4.0	56.2 ± 6.3	49.9 ± 5.0c	39.3 ± 2.9bc
LPS	51.8 ± 3.4	53.2 ± 4.9	48.8 ± 4.6	38.1 ± 4.0b
PAF	80.5 ± 7.3f	77.2 ± 8.1f	72.4 ± 6.8f	63.8 ± 6.4bdf

^aP < 0.05 compared with 6 mo;

^bP < 0.01 compared with 6 mo;

^cP < 0.05 compared with NVEG;

^dP < 0.01 compared with NVEG;

^fP < 0.001 compared with control. NVEG: non-VE pretreated group; VEG: VE pretreated group; LPS: Lipopolysaccharide; Platelet activating factor.

DISCUSSION

Recent interest has focused on the effect of aging on the defense system of the host, in which liver macrophages (KCs) play an important role. It has been found that the aged KCs have disordered responsiveness and phagocytosis function whose mechanisms are thought to arise from altered composition and function of cell membrane phospholipids[1,5]. In addition to providing structural integrity to the cell, membrane phospholipids and the fatty acids provide the precursors to a number of inflammatory mediators and are involved in signal transduction pathways that initiate inflammatory responses. The aged KCs have altered phospholipid functions as demonstrated by the reduced production of PGE₂ and disordered signal transduction response to PAF.

The responsiveness of an inflammatory cell to an extracellular signal depends on its ability to transduce the signal into appropriate intracellular messengers that propagate the signal into the desired response. Membrane inositol phospholipids are intimately involved in the signal transduction process. When the appropriate signal is encountered with membrane receptor, inositol phospholipids are first phosphorylated and hydrolyzed to produce intracellular second messengers in a receptor-mediated and G-protein-coupled event. The second messengers include inositol phosphates, of which the triphosphate has been demonstrated to result in an increased intracellular calcium concentration; and diacylglycerol, which activates protein phosphorylation via protein kinase C (PKC). Eventually, through intermediary steps, the changes in calcium concentration and activation of PKC produce the desired cellular response. Platelet activating factor activates macrophages through this signal transduction pathway. Studies have demonstrated that PAF signals an increase in intracellular calcium and inositol phospholipid metabolism in macrophages[4]. This study showed that ageing altered the intracellular calcium and inositol phospholipid metabolism of KCs exposed to PAF.

The ability to modulate intracellular calcium responses to extracellular signals has sweeping implications on the functional responsiveness of macrophages since intracellular calcium plays an important role in several functions. These include superoxide production for killing, chemotaxis, phagocytosis, activation of phospholipases for arachidonate metabolism, and interleukin-1 and tumor necrosis factor production.

Our investigation suggests that the disordered functions of aged KCs may contribute to the suppression of eicosanoid production by KCs as well as the attenuation of KC responsiveness due to a reduced ability to transduce the inflammatory response.

It has been widely accepted that the age-associated damage to membrane phospholipids is mediated by free radicals which, once generated, are capable of initiating random chain reactions and destroying the integrity of the substance[6]. VE is thought to act as a biological antioxidant by protecting polyunsaturated lipids against peroxidative attack[7]. Our results showed that the pretreatment of VE protects the KC membrane phospholipids from age-related damage, implying that vitamin E is an effective modulator for KC membrane phospholipids.

KCs contribute to hepatic inflammation and cytotoxicity through the production of several proinflammatory cytokines, including eicosanoids. It has been postulated that KCs are responsible for hepatic dysfunction and liver failure in progressive sepsis and inflammation. Our results showed that VE offers the potential ability to regulate the inflammatory response by increasing the PGE₂ production, which would be of great significance in clinical practice.