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Published online before print September 4, 2008
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From The Sansom Institute and School of Pharmacy and Medical Sciences,* University of South Australia, Adelaide; the Department of Immunopathology, Children, Youth, and Women’s Health Services, North Adelaide; and the School of Paediatrics and Reproductive Medicine, University of Adelaide, Adelaide, Australia
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Abstract |
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-6-polyunsaturated fatty acids and has been suggested to regulate inflammatory responses because it inhibits tumor necrosis factor (TNF) mRNA production in the human monocytic cell line THP-1. Here we demonstrate that 4-HNE inhibits TNF and interleukin-1β production in human monocytes in response to lipopolysaccharide. The main action of 4-HNE occurred at the pretranscriptional level; there was no effect on TNF mRNA production or stability when 4-HNE was added after stimulation. The mechanism of action of 4-HNE appears to be downstream of lipopolysaccharide-receptor binding. In the human monocytic MonoMac 6 cell line, 4-HNE caused selective inhibition of the activity of the mitogen-activated protein kinases p38 and ERK1/ERK2, but not JNK. However, in monocytes, the activities of all three kinases were inhibited, suggesting that the effects of 4-HNE were exerted at points upstream of ERK1/ERK2 and JNK as the levels of the phosphorylated kinases were reduced. In contrast, p38 phosphorylation was not inhibited, suggesting that 4-HNE affects kinase activity. 4-HNE also inhibited nuclear factor-
B activation in monocytes. In view of the roles of p38, ERK1/ERK2, JNK, and nuclear factor-B in inflammation, the data suggest that 4-HNE, at nontoxic concentrations, has anti-inflammatory properties, most likely through an effect on these signaling molecules, and could lead to the development of novel treatments for inflammatory diseases.
-6-polyunsaturated fatty acids such as linoleic acid and arachidonic acid.1,2 Previous reports have shown that a 15-lipoxygenase-derived metabolite of arachidonic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production in monocytes at both the pretranscriptional level and by accelerating TNF mRNA degradation.3 Since it has been shown that 15-HPETE breaks down to 4-HNE through enzymatic and nonenzymatic pathways4-6 and previous studies showed the inhibition of TNF production by 4-HNE,7 it is possible that 15-HPETE may exert some of its effects through the more stable breakdown product, 4-HNE.
At very high concentrations >100 µmol/L, 4-HNE is acutely toxic to mammalian cells and leads to cell death within 1 hour or less. However it is unlikely to reach such concentrations in cells and organs, although such concentrations could be built up locally in peroxidizing membranes for a short time.8
It has been suggested that 4-HNE may accumulate in membranes at concentrations of 5 to 20 µmol/L in response to oxidative insults.1,2
The effects of such concentrations include the stimulation of neutrophil chemotaxis, modulation of adenylate cyclase activity, and stimulation of phospholipase C.1
Besides its effects on neutrophils, 4-HNE has displayed various other pro-inflammatory effects. At 0.1 to 10 µmol/L it up-regulated TGF-β mRNA in J774 murine macrophages and human U937 promonocytes differentiated with phorbol 12-myristate 13-acetate.9
The aldehyde (25 to 50 µmol/L) also displayed pro-inflammatory activity in inducing COX-2 mRNA in RAW264.7 murine macrophages by activating the p38 MAPK kinase pathway10,11
and chemotactic activity toward monocytes/macrophages.12
More recently however, 4-HNE was found to inhibit nuclear factor (NF)-B activation in a dose-dependent manner by inhibiting phosphorylation of the inhibitory protein, IB
, in the monocytic cell line THP-1.7
4-HNE has also been found to inhibit the metabolism of arachidonic acid to form cyclooxygenase and lipoxygenase metabolites,13
as well as inhibiting the production of adhesion molecules ICAM-1, VCAM-1, and E-selectin.14
These results suggest that 4-HNE plays a role in regulating the inflammatory reaction.
The signs and symptoms of a diverse range of such seemingly unrelated diseases as rheumatoid arthritis, multiple sclerosis, cancer, septic shock, and hematopoietic disorders such as aplastic anemia and myelodysplastic syndrome can be attributed to the overproduction of TNF. The overproduction of TNF can be acute and intense, as in the case of sepsis, or chronic and low grade, as in the case of chronic disorders such as rheumatoid arthritis. In the case of bacterial sepsis, endotoxins cause intense production of TNF from monocytes and macrophages resulting in the clinical manifestations of septic shock.15 The damaging effects of such high and intense TNF levels are attributable to its capacity as a master initiator of the cytokine cascade leading to the end-organ failure that often proves to be fatal.
The major source of secreted TNF is cells of the monocyte/macrophage lineage and one of the most potent stimuli for inducing TNF release is bacterial LPS. LPS forms complexes with the plasma protein LBP (LPS binding protein) and is transferred to the CD14 receptors a membrane protein found specifically on monocytes/macrophages. CD14 then transfers LPS to a complex of TLR4 (Toll-like receptor) and MD-2 (myeloid differentiation factor) and thereby initiates the LPS response.16,17
The main pathways involved in the activation of monocytes/macrophages by LPS include the p38, ERK, and JNK MAPKs and the IKKβ pathways.16-18
These in turn activate the various transcription factors involved in TNF expression and monocyte/macrophage activation including Elk-1, c-jun, c-Fos, ATF-1, ATF-2, SRF, CREB, and NF-B. The ability of 4-HNE to inhibit monocyte/macrophage activation and subsequent TNF production has implications in the regulation of chronic inflammatory responses in diseases such as rheumatoid arthritis in which anti-TNF antibody therapy is currently practiced.19
To gain better insights into the potential role of 4-HNE as a regulator of pathogenesis and its relationship to the anti-inflammatory properties of 15-HPETE, we have examined its mechanism of action on human monocyte/macrophage TNF production at the pre- and posttranscriptional level in particular its effects on the MAP kinase pathways, because these are known to play a role in production of TNF by monocytes.20 We present evidence that its prime action is at the pretranscriptional level involving inhibition of selective members of the MAPK family.
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Materials and Methods |
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Mononuclear leukocytes (MNLs) were prepared from the blood of healthy volunteers using an adaptation of the Hypaque/Ficoll method.21 Briefly, blood was carefully layered onto a solution of Angiograffin (Schering, Munich, Germany)/Ficoll400 (Pharmacia Biotech, Uppsala, Sweden)/sodium diatrizoate (Sigma Chemical Co., St. Louis, MO) of density 1.114. After centrifugation at 600 x g for 30 to 35 minutes, the leukocytes resolved into two bands. The lower density MNLs in the top band were harvested and washed twice with RPMI medium (JRH Biosciences, Lenexa, KS).
Monocytes/macrophages were enriched from the MNL fraction by density gradient centrifugation.22 Briefly, MNLs were layered onto a 46% iso-osmotic Percoll gradient and centrifuged at 600 x g for 30 minutes at room temperature. The monocytes formed a band at the interface of the gradient and the lymphocytes formed a pellet at the bottom of the tube. Once harvested, the monocytes were washed twice in ice-cold RPMI medium with 1 mmol/L ethylenediaminetetraacetic acid by centrifugation at 4°C.
The human monocytic cell line MonoMac 6 was maintained at 37°C with 5% CO2 air and high humidity at a density of 1 x 106cells/ml in 250-ml culture flasks. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (JRH Biosciences), 4 mmol/L L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (CSL, Victoria, Australia), 1x nonessential amino acids solution, and 1 mmol/L sodium pyruvate (Sigma Chemical Co.).
Stimulation of Leukocytes
MNLs/monocytes were plated on a 96-well U-bottom plate (Nalge Nunc International, Rochester, NY) at 2 x 105 cells/well. Cells were pretreated as stated in the figure legends, by the addition of 50 µl of MNLs (4 x 106/ml) and 50 µl of the appropriate solution of 4-HNE (Cayman Chemical Co., Ann Arbor, MI) in RPMI 1640 medium and incubated at 37°C with 5% CO2 air and high humidity for the appropriate time. After pretreatment with 4-HNE, cells were stimulated by the addition of 100 µl of LPS (Sigma Chemical Co.) in RPMI 1640 medium supplemented with 5% heat-inactivated AB serum. Stimulation was allowed to proceed for 72 hours at 37°C with 5% CO2 air and high humidity. The cell-free supernatant was removed and stored at –70°C for cytokine analysis via enzyme-linked immunosorbent assay.
Quantitation of TNF and Interleukin (IL)-1β in Culture Supernatants
Cytokine levels in cell-free supernatants were measured using a four step enzyme-linked immunosorbent assay.23 Readings were made from TNF/IL-1β standards (NIBSC, Hertfordshire, UK) on a standard curve.
TNF mRNA Measurements and Determination of Rate of Decay
The transformation of TNF insert plasmid into competent cells and probe purification was done as previously described.24 MonoMac 6 cells (1 x 106) were induced as described in the figure legends.24 In all cases, cells were stimulated with 10 ng/ml of LPS in the presence of 10% (v/v) heat-inactivated FCS (final concentrations). Total cellular RNA for the slot blots was prepared by the Trizol (Invitrogen, Carlsbad, CA) method and analyzed as previously described.24 Rates of TNF mRNA were measured essentially as described previously using Actinomycin D to block further transcription.3
Measurement of MAP Kinase Activation
Measurement of p38 and ERK activity was performed as described previously.25 Briefly, precleared lysates were incubated with anti-p38 or anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After precipitation with protein A Sepharose, kinase activity was conducted in the presence of 32P-ATP. A solid phase assay for JNK activity was used as described previously.25 Briefly, lysates were incubated with GST-jun (1 to 79), which is coupled to glutathione Sepharose. After extensive washing, the beads were isolated for kinase assays in the presence of 32P-ATP. To determine MAP kinase phosphorylation, lysates were subjected to Western blotting as described.25 The blots were probed with anti-ACTIVE ERK antibody (Promega Corp., Madison, WI) and sequentially stripped and reprobed with anti-ACTIVE p38 (Promega Corp.), anti-phospho JNK (Santa Cruz Biotechnology), and finally anti-ERK2 antibody (Santa Cruz Biotechnology), to assess loading. The order in which the anti-active MAP kinase antibodies was used varied between experiments.
Measurement of NF-B Activation
The activation of NF-B was assessed by measuring the degradation of cytoplasmic IB- by Western blotting. Monocytes were pretreated with 4-HNE and stimulated with 10 ng/ml of LPS, after stimulation (15 minutes) cells were lysed and the level of IB- analyzed by Western blotting as previously described.26
LPS Binding Assay
The binding of LPS to monocytes was measured using flow cytometry. Monocytes were treated as described in the figure legends before the addition of fluorescein isothiocyanate-LPS (Sigma Chemical Co.) (1 µg/ml) for 30 minutes at 4°C. The cells were washed twice with 1 ml of Isoton II (Coulter, Brookvale, Australia) and resuspended in 500 µl of Isoton II. The fluorescence intensity of the samples was measured immediately using a flow cytometer (Becton Dickinson, Mountain View, CA).
Statistical Analysis
All data were analyzed for significance using the Tukey or Student-Newman-Keuls test for multiple comparisons. All graphical and statistical analyses were done using Graphpad Prism version 3.00 (Graphpad Software, San Diego, CA).
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Results |
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Previously it has been reported that 4-HNE inhibited LPS-induced TNF production in THP-1 cells.7
To ensure that this was not specific to a macrophage cell line we examined effects of 4-HNE on peripheral blood monocyte TNF and IL-1β production. The cells were pretreated with varying concentrations of 4-HNE (5 to 20 µmol/L) and then stimulated with 10 ng/ml of LPS. 4-HNE was found to inhibit the production of TNF induced by LPS in a concentration-dependent manner (Figure 1)
. 4-HNE attenuated both TNF and IL-1β production with IC50 of 5.1 and 6.8 µmol/L, respectively.
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MonoMac 6 cells pretreated with 4-HNE showed a similar concentration-related inhibitory effect on TNF mRNA production (Figure 2A)
to that seen with monocytes and MNLs, with an IC50 of 4.2 µmol/L. To ascertain whether 4-HNE also accelerated the decay of TNF mRNA, MonoMac 6 cells were treated with 10 µmol/L 4-HNE for different times relative to LPS stimulation (Figure 2B.)
The aldehyde inhibited TNF mRNA when added before or at the same time as LPS but not if added after LPS addition. This suggests that it does not affect posttranscriptional events such as TNF mRNA stability. Not surprisingly, unlike 15-HPETE our data showed that 4-HNE had no effect on the level of TNF mRNA if added 60 minutes after stimulation with LPS (Figure 2C)
. Even when the concentration of 4-HNE was increased to 50 µmol/L, there was no effect on TNF mRNA levels. This indicates that there is no effect of 4-HNE on posttranscriptional events, at least at this level. To ascertain that 4-HNE did not influence TNF mRNA stability we examined the effects of treating LPS-prestimulated cells with 4-HNE in the presence of Actinomycin D. Then the levels of TNF mRNA were followed throughout time. The results in Figure 2D
show that the rates of TNF mRNA decay are the same in the presence and absence of 4-HNE.
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A starting point in dissecting the pretranscriptional effect of 4-HNE is to examine effects at the LPS binding step. The effects of 4-HNE on LPS binding to the human monocytes was assessed. Cells were pretreated for 1 hour with 20 µmol/L 4-HNE before the addition of 1 µg/ml of fluorescein isothiocyanate-labeled LPS. 4-HNE did not affect LPS binding to the monocytes (Figure 3)
.
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LPS is known to use MAP kinases to promote the production of TNF.20
Thus, the effects of 4-HNE on LPS-induced activation of these intracellular signaling pathways was examined using MonoMac 6 cells. These were pretreated with 10 µmol/L 4-HNE for 1 hour and then stimulated with LPS (10 ng/ml) for 15 minutes before assessing kinase activity, measured by the radioactive kinase assay. 4-HNE was shown to significantly inhibit p38 and ERK activity induced by LPS (Figure 4, A and B
, respectively). In contrast to this, not only was LPS-induced JNK activity not inhibited, but at the same concentration, 4-HNE was able to stimulate JNK activity (Figure 4C)
.
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. The aldehyde did not inhibit the LPS-induced phosphorylation of p38 (Figure 4E)
. Surprisingly, unstimulated MonoMac 6 cells showed a high baseline phosphorylation of the 46-kDa form of JNK (Figure 4F)
. Phosphorylation of the 54-kDa form of JNK was not observed, even on prolonged exposure to the film. What is interesting and unexplained is that JNK activity was affected by the treatments without a significant change in the degree of phosphorylation of the p46 and p54 JNK forms (Figure 4F)
. Since Zhang and colleagues27
showed that 4-HNE increased the phosphorylation of ERK1/ERK2 and p38 at earlier time points, we examined the effect of the aldehyde at these earlier time points. The results showed that 4-HNE also caused an increase in the levels of phospho-ERK1/ERK2 and p38 in MonoMac 6 cells at 5, 10, and 30 minutes (data not presented). In contrast, no change in phospho-JNK was observed (data not shown).
Because our work extended to the human peripheral blood monocytes, it was relevant to see if these changes in intracellular signaling were correlated in normal cells. Interestingly with monocytes, the results showed that 4-HNE inhibited the LPS-induced increase in enzymatic activity of all three MAP kinases (Figure 5, A–C)
. Although both ERK and JNK phosphorylation induced by LPS were inhibited by 4-HNE, there was no effect on p38 phosphorylation (Figure 5, D–F)
. This suggests that 4-HNE may directly inhibit p38 kinase activity in primary monocytes. As observed with MonoMac 6 cells, there was a high baseline phosphorylation of the p46 kDa form of JNK but the treatments predominantly affected the phosphorylation status of the p54 kDa JNK.
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B Activation
Although it has been previously reported that 4-HNE inhibits NF-B activation in THP-1 cells, it was evident that these cells show constitutive nuclear NF-B.28
We found similar results with our studies on MonoMac 6 cells (data not presented). Because this raises the issue as to the validity of these results when relating these to normal cells, we examined the effects of 4-HNE on LPS-induced NF-B activation in monocytes, by quantitating the levels of cytoplasmic IB-. Treatment with 10 µmol/L 4-HNE significantly inhibited the LPS-induced IB- degradation (Figure 6)
.
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Discussion |
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5 µmol/L for both cell types. We also found that the inhibitory effect of 4-HNE was not restricted to TNF because similar effects were observed in the ability to produce IL-1β. At these concentrations 4-HNE was not toxic to either monocytes or MonoMac 6 cells.
These effective concentrations are attainable in vivo during an inflammatory reaction suggesting that 4-HNE may regulate monocyte/macrophage function. Under noninflammatory conditions, sub-µmol/L levels of 4-HNE in the plasma have been reported and are suggested to be the resting physiological concentrations of 4-HNE. However, it is relevant that this can reach up to 45 µmol/L during inflammation/pathophysiology,1
and 1 mmol/L in cellular membranes.2
Up to 100 µmol/L of 4-HNE has been reported in hepatocytes. Others have estimated that 4-HNE concentrations of 150 mmol/L are present in the lipid phase of strongly oxidized LDL.29
Human monocytes exposed to hemozoin, a malarial parasite pigment, generate 50 µmol/L of 4-HNE.30
Our findings demonstrate that 4-HNE has anti-inflammatory effects at levels generated in cells, tissues, and fluids (5 to 20 µmol/L).
To attempt to increase our understanding of the mechanism involved we examined whether the action of 4-HNE was at a pre- or posttranscriptional level. Unlike the previously reported effects of 15-HPETE,3
4-HNE acted only at a pretranscriptional level. No evidence was found to suggest that it acted at a posttranscriptional level, the aldehyde had no effects on TNF mRNA stability. Thus, although 15-HPETE can be converted to 4-HNE, it is evident that the effects of this hydroperoxide on TNF mRNA stability cannot be accounted for by the generation of 4-HNE. Previously Page and colleagues7
reported that 4-HNE inhibited NF-B activation in THP-1. Here we demonstrate in human monocytes that 4-HNE can indeed inhibit the NF-B pathway, a transcriptional factor involved in the production of TNF. Difficulties were encountered with measurement of NF-B using the MonoMac 6 cell line, in that high levels of baseline activation were detected (data not presented). Levels of cytoplasmic IB- were significantly lower in MonoMac 6 cells than in monocytes, suggesting a high level of baseline activation. The ability of 4-HNE to inhibit LPS-induced NF-B is likely to contribute to the inhibition of TNF production.
The research has identified other key mechanisms of the regulation of macrophage function by 4-HNE, summarized in Table 1
. Consistent with previous data showing that 4-HNE transiently increased p38 phosphorylation in the murine macrophage cell line, RAW264.7,10,11
our data show that the aldehyde caused a transient increase in the phosphorylation of p38 and ERK1/ERK2 in MonoMac 6 cells (data not presented). Although pretreatment with 4-HNE did not affect LPS-induced p38 phosphorylation, the aldehyde inhibited LPS-stimulated increase in p38 activity in the human monocytic cell line MonoMac 6. LPS-induced increase in p38 activity in monocytes was also inhibited without an accompanying reduction in p38 phosphorylation. This suggests a direct inhibitory effect of 4HNE on p38 kinase activity. Furthermore, we found that 4-HNE inhibits LPS-stimulated ERK activity in MonoMac 6 and primary monocytes. Although 4-HNE inhibited LPS-stimulated JNK activity in peripheral blood monocytes, JNK activity in MonoMac 6 cells was increased by the aldehyde. Previous reports have established that inhibition of ERK1/ERK2, p38, or JNK alone is sufficient to inhibit cytokine production.31-33
For example blocking p38 inhibits production of TNF in monocytes. Our data suggest that the combined effects of the inhibition of p38, ERK, and JNK is likely to be responsible for the profound inhibitory effect of 4-HNE on the production of TNF in human macrophages.
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Our data highlight the need to exercise reservations on results from cell lines, as already previously reported.38 In comparison to MonoMac 6 cells, we found 4-HNE to cause the inhibition of all three kinases in response to LPS in monocytes. The monocytes did show an increase in levels of phospho-ERK and phospho-p54 kDa JNK in response to LPS. For ERK1/ERK2 and JNK the decrease in activity caused by 4-HNE correlated with the levels of phosphokinases, suggesting that 4-HNE acts on an upstream kinase. However with p38, no change in levels of LPS-induced phospho-p38 was induced by 4-HNE. This suggests that 4-HNE may act directly on p38 to inhibit its activity.
Although the effects of 4-HNE could be mediated through a depletion in the intracellular GSH pool, being metabolized to 4-HNE-GSH conjugate,39 this is unlikely. In human monocytes, the activation of ERK1/ERK2 by LPS is not influenced by the intracellular GSH status, unlike the phosphorylation of p38, which varies with GSH levels.37,40 In fact it has been reported that subjecting cells to H2O2, which depletes GSH41 actually stimulates MAP kinases.42
4-HNE at concentrations used in this study can inhibit the activation of certain PKC isozymes by inhibiting the ability of these isozymes to translocate to the membrane fraction, most probably by interfering with the interaction between the PKC isozymes and membrane phospholipids.43,44 An interaction between 4-HNE and phospholipids has also been reported44 and 4-HNE forms Michael adducts with alkenylphospholipids, especially the O-alk-1-enyl form of phosphatidylethanolamine.45 This could interfere with the functioning of phospholipid-dependent signaling molecules because of a perturbation in the microenvironment in which these signaling molecules are activated by or interact with their respective phospholipid regulators. Thus, it is also likely that there are upstream signaling molecules that are dependent on membrane phospholipids for activation involved in the effects of 4-HNE. We found no evidence that 4-HNE prevented the binding of LPS to its receptor, suggesting that signaling molecules proximal to TLR4 may be affected by these changes in the lipid environment. Although there is also the possibility that 4-HNE directly modifies the MAP kinases through a Michael addition reaction, this is unlikely to contribute to the observed changes in MAP kinase activity in the MonoMac 6 given that the effects of 4-HNE on the three MAP kinases are not only incongruent but there are also dissimilarities between MonoMac 6 and peripheral blood monocytes for the same MAP kinases, eg, JNK kinase activity.
Microbial invasion is likely to lead to the release of 4-HNE that could thus modify innate immunity and in this manner even influence the adaptive immune response. It is also widely appreciated that excessive monocyte/macrophage activation and overexpression of TNF and its relative IL-1β lies at the heart of many inflammatory diseases such as rheumatoid arthritis and septic shock. Therefore endogenous inhibitors of TNF/IL-1β production and monocyte/macrophage activation, such as 4-HNE, could provide a novel pathway for the development of novel pharmaceuticals. Furthermore, the dual nature of 4-HNE in possessing both protective and destructive properties may need to be given careful consideration when anti-oxidant supplementation is being practiced.
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Acknowledgements |
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Footnotes |
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Supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia and WCH Foundation.
Accepted for publication July 7, 2008.
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References |
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B in cells of the monocyte lineage. Biochem J 1994, 304:87-94[Medline]This article has been cited by other articles:
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  F. Crea and F. Andreotti The unstable plaque: a broken balance Eur. Heart J., June 27, 2009; (2009) ehp266v1. [Full Text] [PDF]
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