www.elsevier.com/locate/jpedsurg

Induction and modulation of apoptosis in neonatalmonocytes by polyunsaturated fatty acidsB

Brian Sweeneya,*, Prem Puria, Denis J. Reena,b

aChildren’s Research Centre, University College Dublin, Our Lady’s Hospital for Sick Children, Dublin K1H 8L1, IrelandbConway Institute of Biomolecular and Biomedical Research, University College Dublin,

Our Lady’s Hospital for Sick Children, Dublin K1H 8L1, Ireland

0022-3468/$ – see front matter D 2007

doi:10.1016/j.jpedsurg.2006.12.024

B Supported by a Health Research

ship Grant.

* Corresponding author. Children’

Ottawa, Ontario, Canada K1H 8L1. Tel

613 7384297.

E-mail address: bsweeney@cheo.on

Index words:Monocytes;

Apoptosis;

Human;

Lipid mediators;

Inflammation;

Fatty acids

Abstract Polyunsaturated fatty acids (PUFAs), known modulators of the immune response, are the

source of essential fatty acids in total parenteral nutrition–dependent patients. Critically ill infants on

TPN have an increased incidence of sepsis, and lipid emulsions depress various immune functions.

Recent studies have demonstrated that PUFAs induce apoptosis in various tissue cells in vitro and ex

vivo. The susceptibility of neonatal monocytes, as major early effector cells in the host response to

sepsis, to PUFA-mediated apoptosis and the mechanisms associated with PUFA-induced apoptosis were

investigated. Both n-3 and n-6 PUFAs induced rapid, dose-dependent cell death in purified monocytes.

Polyunsaturated fatty acids induced significant activation of upstream caspases 8 and 9 as well as

caspase 3. The PUFA treatment resulted in a 4-fold increase in oxidative stress and a loss of monocyte

mitochondrial potential compared with carrier controls (P b .05). The addition of cyclosporin, which

blocks the development of mitochondrial transition pores, completely abolished the proapoptotic effects

of PUFAs. Although Trolox (Sigma Aldrich) reduced PUFA-induced intracellular oxidative stress in

neonatal monocytes, apoptosis was not blocked by this potent antioxidant. The data identify PUFAs as

potent inducers of monocyte apoptosis, which can occur independently of the induction of oxidative

stress, by using a mitochondrial dependent pathway. The TPN-dependent infant may be particularly

sensitive to such PUFA effects, having a relatively poor capacity to both use and clear PUFAs.

D 2007 Elsevier Inc. All rights reserved.

The introduction of lipid emulsions for total parenteral

nutrition (TPN) has been a major breakthrough in medicine.

The most frequently used lipid formula in TPN is based on

Elsevier Inc. All rights reserved.

Board Research Training Fellow-

s Hospital of Eastern Ontario,

.: +1 613 7377600x2196; fax: +1

.ca (B. Sweeney).

soybean oil and contains predominantly long-chain fatty

acids of the n-6 series (linoleic acid) [1]. More recently, fish

oil–based lipid emulsions, which are rich in fatty acids of the

n-3 polyunsaturated fatty acids (PUFAs) (a-linolenic acid),

have been used [2]. Furthermore, it has been demonstrated

that the use of soybean oil–derived lipid formula results in

significant elevation of n-6 PUFAs such as arachidonic acid

(AA), whereas the use of fish oil–based lipid formula results

in an elevation of n-3 PUFAs such as docosahexaenoic acid

(DHA) and eicosapentaenoic acid (EPA) [3]. The n-3 and n-6

fatty acids have long been recognized to have potent effects

Journal of Pediatric Surgery (2007) 42, 620–628

1 Dr. Jaydeep Sarma, Welcome Trust Research Fellow, University

of Edinburgh Medical School, Edinburgh, Scotland.

Induction and modulation of apoptosis in neonatal monocytes 621

as modulators of the immune response and have been shown

to have differential effects on monocyte, lymphocyte, and

neutrophil function [4,5]. In addition to extensive in vitro

and ex vivo laboratory animal work, some human studies

have been conducted assessing various immune parameters

after dietary supplementation with n-3 PUFAs. This work

has been extensively reviewed [4], and some benefits of fish

oils have been shown in rheumatoid arthritis, psoriasis,

inflammatory bowel disease, systemic lupus erythematosus,

and post–renal transplantation patients [6]. Strikingly,

critically ill neonates on TPN have an increased incidence

of sepsis [7,8]; and additional studies have shown that lipid

emulsions depress monocyte chemotaxis [9], phagocytosis

[10], as well as complement [11] and cytokine production

[12]. Monocytes are recognized as major effector cells of the

immune system, playing a central role in the initiation,

development, and outcome of the innate immune response.

Monocyte survival is exquisitely regulated in vivo by various

pro- and antiapoptotic signals, survival being promoted

during the inflammatory response and diminished when

surplus to requirements or in the presence of recognized

immunosuppressive agents such as glucocorticoids [13].

Monocyte apoptosis has emerged as a central regulatory

event in hematopoiesis and inflammation [14], and some of

the elements of the complex system regulating the tight

control of monocyte survival are gradually emerging.

Regulation of monocyte apoptosis may be an important

homeostatic mechanism for controlling the number of

monocytes available to respond to infection, wound healing,

and tumor growth.

Recent studies have demonstrated that PUFAs are

capable of inducing apoptosis in vitro and ex vivo [15]. In

a rodent feeding model, Garrido et al have shown that

dietary supplementation with PUFAs results in depletion of

cellular antioxidants such as glutathione, thus rendering

them more susceptible to oxidative stress [16].

How PUFAs mediate cell death has yet to be fully

elucidated. Several potential mechanisms have been pro-

posed to explain their immunomodulatory effects, including

their effects on eicosanoid formation, signal transduction,

gene expression, and lipid peroxidation [17]. It is known

that lipid peroxidation and the metabolites of this process

are associated with increased oxidative stress [18]. Oxida-

tive stress, in turn, has been implicated as a significant

mediator of apoptosis [19]. Reactive oxygen species (ROS)

resulting from oxidative stress have been implicated in the

regulation of Fas-mediated monocyte apoptosis. This form

of monocyte apoptosis is associated with increased intra-

cellular levels of ROS and can be blocked with the

antioxidant n-acetylcysteine [14]. Synthesizing these data,

there is considerable evidence to support the concept that

agents that have a proapoptotic effect on monocytes may

have a potentially anti-inflammatory effect.

The aim of this study was to gain mechanistic insights

into PUFA-regulated apoptosis in monocytes in a human

neonatal model by determining upstream intracytoplasmic

events in apoptosis, specifically, the development of

oxidative stress, the loss of mitochondrial inner membrane

potential, and the activation of initiator and effector

caspases. If PUFAs have apoptogenic effects, identifying

the mechanisms whereby they may modulate the immune

response could have important implications for management

of infants on TPN. The TPN-dependent infant may be

particularly sensitive to such PUFA effects, having a

relatively poor capacity to both use and clear PUFAs [8].

1. Materials and methods

1.1. Monocyte isolation by negative selection

Umbilical cord blood monocytes were isolated by an

immunomagnetic bead negative selection method (Miltenyi

Biotech, Germany), using a modification of a protocol

provided by Dr Jaydeep Sarma1. Density gradient isolated

cord blood mononuclear cells (peripheral blood mononu-

clear cells; PBMCs), derived from acid citrate dextrose

anticoagulated whole blood, were washed twice in cation-

free Hanks balanced salt solution and counted. The PBMCs

were resuspended in an isolation buffer composed of cation-

free Hanks balanced salt solution, 4% autologous platelet

free plasma (APFP), and EDTA (250 lmol/L) at a volume

of 40 lL of isolation buffer per 107 PBMCs. Added to this

were 40 lL of APFP and 20 lL of hapten antibody cocktail

per 107 PBMCs. This mixture was incubated for 5 minutes

at 48C. The cells were then washed twice in the isolation

buffer before resuspension in 10 lL of isolation buffer,

40 lL of APFP, and 20 lL of antihapten microbeads per 107

cells. In addition, 10 lL per 107 cells of anti–glycophorin A,

anti-CD15, and anti-CD61 microbeads was added to remove

contaminating erythroblasts, neutrophils, and platelets,

respectively. The mixture was then incubated at 48C for

15 minutes. The cells were washed before resuspension in

500 lL of isolation buffer. The mixture was then passed

through a primed LS+/VS+ isolation column in a magnetic

field. The resulting negatively selected monocytes were

washed once in RPMI and 10% APFP. Their purity was

routinely greater than 85% as determined flow cytometri-

cally using CD45, CD14, and CD41 monoclonal antibodies.

The viability of freshly isolated negatively selected mono-

cytes was greater than 90% on ethidium bromide/acridine

orange staining. Purified monocytes were then incubated at

a concentration of 106/mL in RPMI 1640 and either 1% or

10% APFP in a 5% CO2 incubator at 378C in 24- or 96-well

ultralow attachment polystyrene flat-bottomed plates.

1.2. Preparation of PUFAs

Stock solutions of the n-3 PUFAs DHA and EPA and the

n-6 PUFA AAwere made up in a nitrogen atmosphere using

an Atmosbag (Sigma Aldrich) in 100% ethanol and were

Fig. 1 Cell death in monocytes treated with PUFAs. Negatively

selected blood monocytes were cultured for 2 hours in RPMI

and 1% APFP with carrier control or various concentrations of

the PUFAs AA, DHA, and EPA. The treated cells were then

harvested and triple stained with annexin V, CD14, and PI to

quantify the degree of cell death by flow cytometry as determined

by measuring annexin V positivity. Data are mean F 1 SD for

4 separate experiments (*P b .05 indicates a significant difference

from control).

B. Sweeney et al.622

stored in the dark at �208C to prevent peroxidation The

stocks were diluted at least 1000-fold in complete medium

to the test culture concentrations. Carrier controls consisted

of the addition of 0.1% ethanol in the absence of fatty acids.

They had no influence on cell viability at the concentration

used for all experiments. The doses of PUFAs used ranged

from 0 to 100 lmol/L and were based on previously

published cell line data [15,20].

1.3. Detection and quantification of monocytecell death

Cell death of monocytes was measured essentially

according to the protocol of Schmidt et al [13]. After the

designated culture period, carrier control– or fatty acid–

treated monocytes were harvested with ice-cold phosphate-

buffered saline (PBS), washed and resuspended in annexin

V binding buffer, and triple labeled with annexin V

fluorescein isothiocyanate (FITC), CD14 phycoerythrin

(PE), and propidium iodide (PI). Ten thousand events were

acquired on 3 channels of a FACscan flow cytometer

(Becton Dickinson, Oxford, UK) and data were analyzed

with CellQUEST software (Becton Dickinson, Oxford, UK).

Cell death was quantified by measuring the number of

apoptotic annexin V–positive and primarily or secondarily

necrotic PI-positive events.

1.4. Detection of active caspases 8 and 9in monocytes

CaspaTag caspase activity kits (Intergen, UK) were used

to detect active caspases 8 and 9 through the use of specific

carboxyfluorescein-labeled caspase substrates as per the

manufacturer’s protocol. Briefly, 300-lL aliquots of carrier

control– and PUFA-treated cells were incubated for 1 hour

in the dark at 378C with 10 lL of 30� working dilution of

the peptide substrates FAM-LETD-FMK or FAM-LEHD-

FMK to measure active caspase 8 or 9, respectively. The

cells were then washed twice in the wash buffer provided by

the manufacturer before flow cytometric analysis. The

resulting frequency histogram of number of events (y-axis)

versus FL1 fluorescein intensity (x-axis) was used to

determine the level of caspase activation.

1.5. Detection of caspase 3

An FITC-conjugated rabbit antiactive caspase 3 antibody

was used to detect active caspase 3 in monocytes after 2 hours

of culture in RPMI 1640 and 1% APFP as per the

manufacturer’s protocol (Pharmingen, CA). Briefly, carrier

control– or PUFA-treated cord monocytes were washed in

PBS and then fixed and permeabilized for 20 minutes at room

temperature, washed, and resuspended in Perm/Wash Buffer

(Pharmingen). Cells were then stainedwith antiactive caspase

3 antibodies for 1 hour at room temperature in the dark. The

permeabilized cells were then washed and resuspended in

Perm/Wash Buffer and analyzed by flow cytometry.

1.6. Determination of oxidative stress inmonocytes

Umbilical cord blood PBMCs were labeled with dichlor-

ofluorescin diacetate (DCFH-DA) (5 lmol/L) for 30 minutes

in an incubator, and intracellular oxidative stress of

monocytes was measured as described by Bass et al [21].

Briefly, DCFH-DA diffuses into the cell and is deacetylated

by intracellular esterases to form 2V, 7V-DCFH, which is

trapped within the cell. In the presence of hydrogen

peroxide, it is oxidized to become highly fluorescent.

Cumene hydroperoxide (CUHP) (50 lmol/L) was used as

a positive control of oxidative stress as described by others

[22]. Cells were also labeled with aCD14 PE antibody for

15 minutes before acquisition of 50,000 events on the flow

cytometer. Live monocytes were gated based on their CD14

and forward/side scatter characteristics.

1.7. Measurement of mitochondrial membranepotential by flow cytometry

To evaluate mitochondrial membrane potential (DCm),

the cationic lipophilic fluorochrome 3,3V-dihexyloxacarbo-cyanine iodide (DiOC6) was used as previously described

[23]. Briefly, umbilical cord blood PBMCs, at a concentra-

tion of 106/mL, were incubated for 20 minutes at 378C with

Induction and modulation of apoptosis in neonatal monocytes 623

DiOC6 (50 nmol/L) before 60 minutes with carrier control

or PUFA treatment. Cells were then washed in PBS before

flow cytometric analysis of gated live monocytes.

1.8. Statistics

The Mann-Whitney nonparametric U test was used to

compare the results of carrier control treatment with the

individual PUFA treatments using statistical analysis soft-

ware package (Instat, GraphPad Software, CA). P values

less than .05 were considered significant.

2. Results

2.1. PUFAs induce rapid cell death in monocytes

Purified umbilical cord blood monocytes were incubated

for 2 hours with carrier control or individual fatty acids. A

concentration-dependent significant increase (P b .05) in

PUFA-induced cell death was observed for each of the

Fig. 2 Polyunsaturated fatty acids induce caspase 8, 9, and 3 activation

C, D), caspase 9 (E, F, G, H), and caspase 3 (I, J, K, L) activation in

(100 lmol/L). These results are representative of 3 separate experiment

respective treatment is shown in the top right of each panel.

3 PUFAs under study over the concentration range 25 to

100 lmol/L (Fig. 1). At the highest PUFA concentration

(100 lmol/L), a significant proportion of monocytes were

PI positive (data not shown), suggesting that either primary

or secondary necrosis had taken place. Apoptosis and

necrosis have been reported to occur side by side in

response to an identical stimulus [24]. However, PI uptake

in the nucleus, traditionally considered an archetypal marker

of necrosis, has also been reported to be taken up at an early

stage in the apoptotic process, thus limiting its ability to

distinguish between apoptosis and necrosis [25]. Therefore,

to confirm that monocyte apoptosis was associated with

exposure to PUFAs, alternative markers of apoptotic cell

death were investigated. Upstream caspases 8 and 9 and the

effector caspase (caspase 3) activities were measured by

flow cytometry. Incubation of monocytes with PUFAs for

90 minutes resulted in activation of both caspase 8 and

caspase 9 (Fig. 2). These cells were also significantly PI

positive, suggesting that they were secondarily necrotic

(data not shown). This conclusion is supported by the recent

in monocytes. Flow cytometric histogram data of caspase 8 (A, B,

cord blood monocytes treated with control, AA, DHA, and EPA

s. The percentage of monocytes positive for each caspase after the

B. Sweeney et al.624

work by Denecker et al [26] who have demonstrated that

apoptotic cells in culture release activated caspases into

culture supernatants, whereas necrotic cells release unpro-

cessed caspases. Our data clearly showed massive caspase

activation, indicating that apoptosis was the dominant mode

of cell death in this model.

2.2. PUFAs activate caspase 3 in monocytes

Having determined that PUFA treatment resulted in

upstream caspase activation, it was decided to determine if

the downstream effector caspase (caspase 3) was also

activated. The level of active caspase 3 in cord monocytes

was 76 F 15%, 85 F 15%, 73 F 7% and 8 F 3% with AA,

DHA, EPA, and carrier control treatments, respectively

(Fig. 2), indicating that the total caspase cascade system

was activated.

Fig. 3 Effect of PUFAs on monocyte mitochondrial membrane pote

potentiometric probe DiOC6 before 1-hour treatment with carrier control (

figures show histograms of DiOC6 fluorescence of gated monocytes ba

fluorescence intensity is shown in the top right of each panel. Polyunsat

DiOC6 mean fluorescence intensity. Results are representative of 3 sepa

2.3. PUFAs induce a dissipation of mitochondrialmembrane potential in monocytes

The absence of known fatty acid membrane receptors on

monocytes that could trigger apoptosis suggested that

PUFA-induced apoptosis was more likely to be a conse-

quence of their action on intracellular targets, in particular

upon mitochondria. To test this possibility, mitochondrial

membrane potential (DCm) was measured in the presence of

PUFAs. Incubation of monocytes with PUFAs for 1 hour

resulted in loss of monocyte mitochondrial membrane

potential as determined by the potentiometric probe DiOC6

using flow cytometry (Fig. 3). The individual PUFAs tested

resulted in excess of a log order decrease in DiOC6

fluorescence. These data suggest that PUFA-induced apo-

ptosis is associated with significant mitochondrial mem-

brane depolarization as reflected by dissipation of DCm.

ntial. Peripheral blood mononuclear cells were labeled with the

A) or 50 lmol/L of the PUFAs AA (B), DHA (C), or EPA (D). The

sed on forward/scatter and CD14 expression characteristics. Mean

urated fatty acid treatment was associated with marked decrease in

rate experiments.

Fig. 4 Cyclosporin A inhibition of PUFA-induced apoptosis in

monocytes. Negatively selected cord blood monocytes were

incubated in 1% APFP in the presence or absence of CsA

(50lmol/L) for 30 minutes before the addition of carrier control

or one of the PUFAs AA, DHA, or EPA (100 lmol/L). After

2 hours of further incubation, the cells were harvested and triple

stained with annexin V, CD14, and PI to quantify the degree of cell

death by flow cytometry. Cyclosporin A significantly inhibited

PUFA-mediated apoptosis in all cases (*P b .05). Data are mean F1 SD for 3 separate experiments.

Fig. 5 Fatty acids induce oxidative stress in monocytes.

Peripheral blood mononuclear cells were labeled with DCFH-DA

(5 lmol/L) before the addition of carrier control or the fatty acids

AA, DHA, or EPA (50 lmol/L) for 30 minutes. Cumene

hydroperoxide (50 lmol/L) was used as a positive control for

induction of oxidative stress [19]. Cells were also labeled with CD14

to enable flow cytometric identification of monocytes by flow

cytometry. The fluorescence of the deacetylated probe is expressed

relative to carrier control fluorescence for each of the test fatty acids

and cumene hydroperoxide (CUHP). Data are mean F 1 SD for

4 separate experiments. All the fatty acids as well as CUHP induced

significant oxidative stress relative to carrier control (*P b .05).

Induction and modulation of apoptosis in neonatal monocytes 625

2.4. Cyclosporin A protects against PUFA-mediatedapoptosis in monocytes

One of the many biological activities of cyclosporin A

(CsA) is its function as a potent inhibitor of loss of DCm

[27]. The effect of CsA on PUFA-induced monocyte

apoptosis was therefore measured. Negatively selected

monocytes, in RPMI 1640 and 1% APFP, were pretreated

with CsA (50 lmol/L) for 15 minutes before the addition of

carrier control or PUFA (100 lmol/L) treatments. This dose

of CsA has been reported to prevent apoptosis in a model of

ceramide-mediated apoptosis in the human monocytic U937

cell line [28]. After a further 2 hours of incubation, the cells

were harvested; and cell death was measured as already

described (Fig. 4). Carrier control treatment was associated

with 16 F 1.4% cell death compared with 66 F 5.7%, 76 F8.5%, and 47 F 4% cell death with AA, DHA, and EPA

treatment, respectively. However, CsA pretreatment com-

pletely blocked PUFA-mediated cell death and reduced the

level of death down to that of carrier control treatment.

These results provided further evidence for an effect of

PUFAs on mitochondrial integrity.

2.5. PUFAs induce oxidative stress in monocytes

The apoptogenic effects of PUFAs via mitochondrial

membrane perturbation and caspase activation are likely

to be a consequence of their potent oxidizing effects on

cellular substrates. Oxidation of DCFH was used to measure

PUFA-induced intracellular oxidative changes in human

monocytes. Polyunsaturated fatty acids induced significant

substrate oxidation in neonatal monocytes (P b .05)

compared with carrier control–treated cells (Fig. 5). The

fatty acid treatment was associated with approximately

a 4-fold increase in oxidative stress compared with

carrier control.

B. Sweeney et al.626

2.6. Antioxidants inhibit oxidative stress inmonocytes but do not protect againstPUFA-mediated apoptosis

The effect of antioxidants on PUFA-induced apoptosis in

monocytes was therefore investigated. Trolox, a potent

water-soluble antioxidant analogue of a-tocopherol [22],

has been shown to be a potent antioxidant in a CUHP model

of oxidation-induced apoptosis. Trolox (200 lmol/L)

significantly inhibited DCFH oxidation (P b .05) resulting

from CUHP (50 lmol/L) treatment (data not shown).

However, Trolox completely failed to abolish the proapop-

totic effects of PUFAs on neonatal monocytes (Fig. 6).

These findings suggest that although PUFA-induced apo-

ptosis is associated with oxidative stress, the resultant

Fig. 6 Trolox does not inhibit PUFA-mediated apoptosis in

monocytes. Negatively selected monocytes were incubated for

1 hour in the presence or absence of Trolox (200 lmol/L) before

the addition of carrier control or one of the PUFAs (100 lmol/L)

AA, DHA, or EPA. After a further 2 hours of culture, cells were

harvested, washed, and triple stained with annexin V FITC, CD14

PE, and PI before undergoing flow cytometric analysis to quantify

the level of cell death. Data are mean F 1 SD for 3 separate

experiments.

intracellular oxidative changes are not central to the

apoptotic process induced by PUFAs.

3. Discussion

Polyunsaturated fatty acids are key nutrients for human

cells. They may be oxidized as an energy source or stored as

triglycerides or glycogen. Alternatively, they may be taken

up by cells and used for the synthesis of phospholipids,

cholesteryl esters, prostaglandins, and other potent signaling

agents [29]. The concentration of nonesterified or free fatty

acids (FFAs) in serum varies widely (0.25-3.0 mmol/L)

[29]. The liberation of FFAs from lipolysis is likely to

produce a very high transient local concentration in the

region of the microenvironment of cells including the

capillary epithelium [29,30]. It has also been reported

that administration of the lipid component of TPN

results in a significant elevation of FFAs in very low birth

weight infants [31]. Neonates on TPN have a higher

incidence of sepsis, and sepsis is in turn associated with a

marked elevation in FFA levels resulting from insulin

resistance [32].

Apoptosis has emerged as a process of fundamental

biological importance, playing a vital role in the develop-

ment and homeostatic regulation of the immune system and

other tissues [33]. It has recently been reported that elevated

levels of PUFAs trigger apoptosis of human endothelial cells

[34]. Within the immune system, monocytes and macro-

phages act as the sentinels of the innate immune response to

pathogens. There is a paucity of data with respect to the

apoptogenic effect of PUFAs on cells of the immune system.

This study is the first to describe their effect as potent

inducers of apoptosis in human monocytes.

Using annexin V and PI staining of monocytes, the data

clearly show that PUFAs induce apoptotic cell death; and

this was further confirmed at the level of caspase activation.

The accompanying activation of caspases 8, 9, and 3 may

provide some insights into the possible mechanisms of

PUFA-induced apoptotic cell death in monocytes. Caspase

8 plays a critical role in the Fas/tumor necrosis factor

receptor death-inducing signaling pathway, whereas caspase

9 is activated by mitochondrial cytochrome c, apoptotic

protease activating factor 1, and deoxyadenosine triphos-

phate. It might therefore be appropriate to speculate that

caspase 8 activation in our model reflects the interaction of

PUFAs with a death receptor. However, as recently shown

by Kurita-Ochiai et al [35], in a butyric acid model of T-cell

apoptosis, caspase 8 activation can occur independently of

the Fas pathway. To date, there is no fatty acid receptor

that is recognized as a death receptor; but recent data

have identified membrane fatty acid transporters, which

are implicated in the active uptake of long-chain fatty

acids [36].

Fatty acid treatment resulted in a combination of caspase

9 activation and loss of mitochondrial membrane potential

Induction and modulation of apoptosis in neonatal monocytes 627

in our model. In addition, the use of CsA completely

blocked fatty acid–induced apoptosis. These data suggest

that the PUFAs mediate apoptosis, at least in part, through

the mitochondrion. This conjecture is consistent with other

work that has recently demonstrated CsA-sensitive apopto-

sis induced by fatty acids in various cells [27,37,38].

Our finding that PUFAs induce significant oxidative

stress in monocytes is not surprising in light of published

data that have long recognized PUFAs as a potential source

of oxidative stress [39]. By definition, they possess

multiple double bonds, which are readily susceptible to

peroxidation; and the lipoperoxides resulting from this

process are potential mediators of apoptosis [18]. Interest-

ingly, defective mitochondria have also been identified as a

major and primary source of free radicals, which can lead

cells into a death cascade [40]. In addition, modulation of

the oxidation state of the cell has been shown to have

dramatic effects on the level of apoptosis in various

models. For example, apoptosis induced by tumor necrosis

factor a results in a rapid rise in intracellular ROS; and

inhibition of this stress using n-acetylcysteine as an

antioxidant has been shown to inhibit apoptosis in this

model [41]. Recently, Arita et al showed that PUFAs

induce oxidative stress that was associated with significant

apoptosis after 6 hours of treatment in HL-60 cells and

found that dimethyl sulphoxide, which has antioxidant

properties, could be protective against the proapoptotic

effects of fatty acid treatment [42]. Therefore, it was

surprising that Trolox, a potent antioxidant, was not

protective in our model. However, Finstad et al were

unable to inhibit EPA-induced apoptosis in the HL-60

monocytic cell line using either a-tocopherol or ascorbic

acid (50-100 lmol/L) [43]. They argue that their findings

support the concept that PUFAs may regulate cellular

processes such as apoptosis independently of lipid perox-

ide/oxidative stress pathways, which is consistent with our

findings in primary human monocytes.

As previously stated, neonates receiving TPN are prone

to developing an elevation of FFA levels [31], especially in

the presence of sepsis [32]. Whether this in turn can result in

monocyte apoptosis, despite the incorporation of antiox-

idants into lipid emulsions, is a critical question that needs

to be addressed. Oxidative stress–independent induction of

apoptosis by PUFAs as demonstrated in this study is

potentially yet another mechanism whereby these nutrients

may modulate the immune response.

Further in vivo studies are needed to explore the

relevance of our findings in the context of TPN-dependent

neonates. In vivo, there is evidence that patients on TPN

have levels of the PUFAs AA, DHA, and EPA in the range

of approximately 100 to 200 lmol/L [3]. However, in

blood, the solubilization and transport of PUFAs are made

possible by their binding to albumin. This results, in the

physiological setting, in FFA levels of 5 to 50 nmol/L [44].

We did not determine the FFA level in our model system;

but it is likely to have been considerably higher than this

because we cultured using 1% plasma, whereas in vivo, the

neonatal plasma concentration is approximately 50%. As

yet, there are no in vivo data that indicate that there is

increased monocyte apoptosis occurring in neonates on

TPN. Our laboratory did consider conducting such experi-

ments, but current monocyte isolation techniques require a

significant volume of blood to isolate sufficient mono-

cytes to conduct these experiments, which we currently

cannot justify either ethically or clinically. We are planning,

however, to perform in vitro coincubation experi-

ments of purified umbilical cord monocytes with both

physiological and pharmacological levels of commercially

available lipid emulsions to determine if they induce

monocyte apoptosis.

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