The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moghaddami, N.
Right arrow Articles by Ferrante, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moghaddami, N.
Right arrow Articles by Ferrante, A.
The Journal of Immunology, 2003, 171: 2616-2624.
Copyright © 2003 by The American Association of Immunologists

Unique Effect of Arachidonic Acid on Human Neutrophil TNF Receptor Expression: Up-Regulation Involving Protein Kinase C, Extracellular Signal-Regulated Kinase, and Phospholipase A2 1

Nahid Moghaddami*,{dagger}, Maurizio Costabile*,{ddagger}, Phulwinder K. Grover*, Hubertus P. A. Jersmann*,{dagger}, Zhi H. Huang2,*,{dagger}, Charles S. T. Hii* and Antonio Ferrante3,*,{dagger},{ddagger}

* Department of Immunopathology, Women’s and Children’s Hospital, North Adelaide, South Australia; {dagger} Department of Pediatrics, Adelaide University, Adelaide, South Australia; and {ddagger} School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Adelaide, South Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10–20 min) and dose-dependent (0.5–30 µM) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the {omega}3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A2. The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} is a highly pleiotropic cytokine that plays a key role in inflammation, defense against microbial pathogens, and cancer (1). Among the important properties of TNF is its ability to promote neutrophil-microbial killing (2, 3). This cytokine acts via two types of receptors, a 55-kDa TNFR1 (CD120a) and 75-kDa TNFR2 (CD120b) (4). Although both receptors show strong similarities in amino acid sequence in their extracellular domain (both exhibiting cysteine-rich repeats in this region), they exhibit little similarity in the sequence of their intracellular domain, consistent with their use of different signal transduction pathways (4).

Both TNFR are expressed on neutrophils (5). Numbering 500-6000 receptors (6), the TNFR on the surface of neutrophils are susceptible to cleavage following cell activation (7, 8). Neutrophils stimulated with LPS, fMLP, GM-CSF, and opsonized microbial pathogens show down-regulation of surface TNFR expression (7, 8, 9, 10). Thus, it is evident that many exogenous and endogenous inflammatory mediators generated in inflammatory exudates regulate neutrophil responses by down-regulating TNFR expression. This has at least two implications. First, neutrophil responses to TNF become down-regulated, and second, the released receptor fragments act as TNF inhibitors.

During inflammation, activation of the cytosolic phospholipase A2 (cPLA2) 4 releases arachidonic acid (AA) from membrane phospholipids (11, 12). At concentrations found in biological fluids, AA stimulates neutrophil superoxide production, degranulation (13, 14, 15), and the expression of complement receptors type 3 (CR3) (15). In this manner, AA can perpetuate the inflammatory reaction. Because regulation of TNFR expression is considered of importance in inflammation, it was of interest to determine whether or not AA modulated the expression of TNFR on neutrophils. The data presented demonstrate that AA plays a unique role in the regulation of TNFR expression on neutrophils during inflammation. Although all agonists studied to date have been shown to down-regulate the expression of TNFR, AA caused a marked up-regulation of these receptors in a protein kinase C (PKC)/extracellular signal-regulated kinase (ERK)/PLA2-dependent manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fatty acids and derivatives

AA, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and linolenic acid (LNA) were purchased from Sigma-Aldrich (St. Louis, MO); 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and AA methyl ester (ME) were prepared, as described previously (16). Stocks of the fatty acids and derivatives were mixed with dipalmitoyl phosphatidylcholine (DPC) in chloroform in a ratio of fatty acid:DPC of 1:4 (w/w), respectively. The solvent was evaporated under nitrogen, and the lipids were dispersed by sonication in HBSS (Ystrom System Ultra Sonicator, Westwood, NJ). These were then added to the cells at the indicated concentrations. Control cells received equivalent concentration of vehicle (DPC or ethanol).

Biochemicals

LPS from Escherichia coli, serotype 0127:B8, lucigenin, and fMLP were purchased from Sigma-Aldrich. These were dissolved in HBSS and DMSO, respectively. Arachidonyltrifluoromethyl ketone (AACOCF3), a cPLA2 inhibitor, and the PKC inhibitor, GF109203X, were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The mitogen-activated protein kinase/ERK kinase-1 (MEK-1) inhibitor, PD 098059, was obtained from New England Biolabs (Beverley, MA). Indomethacin (IM) and nordihydroguaiaretic acid (NDGA) were purchased from Sigma-Aldrich. GF109203X and PD098059 were prepared in DMSO. AACOCF3, NDGA, and IM stocks were prepared in ethanol. All of these inhibitors were diluted with HBSS immediately before use. Control cells received the appropriate dilutions of vehicle.

Neutrophils

Neutrophils were isolated from blood of healthy volunteers by the rapid single-step method involving centrifugation of blood on Hypaque-Ficoll (3). The neutrophils, harvested from the lower leukocyte band, were 96–99% pure and >99% viable, judged by their ability to exclude trypan blue.

Endothelial cells

HUVEC were isolated from umbilical cords by collagenase digestion (17). The identity of endothelial cells was confirmed by morphology (contact-inhibited cobblestone morphology) and by positive staining for factor VIII-related Ag using peroxidase-conjugated rabbit anti-human von Willebrand factor Ab (DAKO, Glostrup, Denmark A/S) and 3,3'-diaminobenzidine. The cells were plated in gelatin-coated 96-well microtiter plates (Linbro; Flow Laboratories, McLean, VA) and were used when cultures were confluent (36–48 h).

Neutrophil chemiluminescence response

Neutrophil chemiluminescence was measured by the reduction of lucigenin, which is a measure of superoxide production by the cells, as described previously (18). Neutrophils (1 x 106) were preincubated with AA (10 µM) or vehicle at 37°C for 30 min. The cells were transferred to an LB 953 Autolumat Plus tube luminometer (Berthold Technologies, Bad Wildbad, Germany) and TNF (1000 U/ml) or HBSS and 500 µl of 250 µg/ml lucigenin were added. The light emission was recorded over 12 min, and the data were imported into Microsoft Excel (Redmond, WA). Peak rate of chemiluminescence (cpm) was calculated.

Culture of HL-60 cells

Human promyelocytic leukemia HL-60 cells were maintained at a density of <1 x 106/ml in RPMI 1640 medium supplemented with 10% FCS and antibiotics in a humidified atmosphere of 5% CO2 in air at 37°C. The cells were matured along the granulocytic pathway with retinoic acid (100 nM) for 8–9 days (19). Maturation was verified by morphology after staining with Giemsa. The viability of the differentiated cells was 95%, as judged by the ability to exclude trypan blue.

Expression of CR3 ({alpha}-chain) and TNFR

Surface expression of the {alpha}-chain of CR3 (CD11b), TNFR1, and TNFR2 was measured by flow cytometry. Neutrophils at 106 cells/ml were treated with agonists or vehicle for the times indicated in the figure legends. The vehicle did not affect the expression of CD11b or TNFR. The cells were washed in ISOTON II supplemented with 0.1% BSA and then incubated for 30 min on ice with saturating concentrations of a PE-conjugated mAb to human CD11b or isotype IgG1 control (BD Biosciences, San Jose, CA), or anti-human TNFR1 and TNFR2 mAb (or {gamma}1/{gamma}2 isotype-matched mAb) (R&D Systems, Minneapolis, MN). The cells were then incubated with FITC-conjugated goat anti-mouse IgG Ab (AMRAD Operations, Melbourne, Australia) for 30 min (for TNFR), fixed with paraformaldehyde (1% w/v), and analyzed on a BD Biosciences FACS analyser. Mean fluorescence intensity (MFI) was obtained and corrected by subtraction of values for isotype-matched negative controls

Assay for soluble TNFR2

The amount of soluble TNFR2 was quantified by a commercially available ELISA kit (R&D Systems). In this assay, an immobilized anti-TNFR2 mAb is used to capture the receptor, which is then detected by a peroxidase-conjugated polyclonal Ab. According to the manufacturer, in this immunoassay, TNF does not show any significant cross-reactivity and exhibits only a low level interference (10% decrease in the observed value using TNF at 5 ng/ml).

Statistics

Results are expressed as the mean ± SEM of 3–10 experiments, using neutrophils from healthy donors. Statistical analyses were performed by the two-tailed Student’s t test for paired or unpaired data using Graphpad Software (Cricket Software, 1985, Philadelphia, PA) or by ANOVA. Where appropriate, multiple comparisons with one control were performed using Dunnett’s modification.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of TNFR expression by AA

Neutrophils were treated with 30 µM of AA or the equivalent amount of vehicle for 30 min at 37°C and then examined for TNFR expression by flow cytometry. The results showed that AA caused a marked increase, ~8-fold, in the expression of TNFR1 and TNFR2 (Fig. 1). No change in receptor expression was seen in response to the vehicle. The histograms demonstrate that a major proportion of the neutrophil population showed an increase in TNFR1 and TNFR2 expression when treated with AA (Fig. 1, B and C). There was a clearly dramatic shift in TNFR expression by the majority of cells. In seven experimental runs, the mean ± SEM of the population of cells with an increase in TNFR1 and TNFR2 was 78.7 ± 4.0 and 73.5 ± 5.0, respectively.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1. Effect of AA on TNFR expression on neutrophils. Neutrophils were treated with 30 µM AA or vehicle for 30 min at 37°C and then examined for the expression of TNFR1 and TNFR2 by flow cytometry. A, The results in MFI are expressed as mean ± SEM of 11 experiments. The MFI (mean ± SEM) for the vehicle was 31 ± 3 and 34 ± 2 for TNFR1 and TNFR2, respectively. Significant difference between AA and vehicle: *, p < 0.0001 for AA vs control. Representative histograms of AA-stimulated cell surface expression for TNFR1 (B) and TNFR2 (C) on neutrophils are shown.

 
AA increased the expression of TNFR1 over a concentration range of 0.5–30 µM (Fig. 2A). An increase was evident at 0.5 µM (p < 0.01), and this continued to increase dramatically as the concentration reached 30 µM. A similar trend in results was seen with respect to the expression of TNFR2 (Fig. 2A). In comparison, treatment of neutrophils with AA only caused a 1.7-fold increase in the expression of CD11b (Fig. 2A). This effect was AA concentration dependent, reaching significance at 10 µM (p < 0.05), and a maximum increase in CD11b occurred at 30 µM.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 2. A, Effect of varying concentrations of AA on neutrophil TNFR and CD11b expression. Neutrophils were incubated with 0.5–30 µM AA or vehicle and then examined for the expression of TNFRI ({blacksquare}), TNFR2 (•), and CD11b ({diamondsuit}) by flow cytometry. The results, percentage of increase in MFI, are presented as the mean ± SEM of four experiments. The MFI (mean ± SEM) for vehicle-treated cells was 16 ± 2, 19 + 4, and 599 ± 90 for TNFR1, TNFR2, and CD11b, respectively. Significance of differences between AA-treated and untreated cells: p < 0.001 for CD11b; p < 0.0001 for TNFR1 and TNFR2, ANOVA. B, Effect of treatment time on AA-stimulated increase in TNFR expression. Neutrophils were incubated with 30 µM AA or vehicle for the indicated times, and then examined for the expression of TNFR1 ({diamondsuit}) and TNFR2 ({blacksquare}) by flow cytometry. The results, percentage of increase in MFI, are presented as the mean ± SEM of three experiments. The MFI (mean ± SEM) for the vehicle for time 0 was 26 ± 4 and 29 ± 4 for TNFR1 and TNFR2, respectively. Significance of difference between control and treatment with AA for both receptors, p < 0.0001, ANOVA.

 
To gain further understanding on the mechanisms of the AA-induced increase of TNFR expression, the effect of varying the treatment time was examined. An increase in TNFR1 expression was evident after 10-min treatment with AA. This reached a maximum by 20–30 min (Fig. 2B). At 40 min, the degree of enhancement in expression began to decrease. The findings were similar in this relationship with respect to TNFR2 expression (Fig. 2B).

Alterations in the TNF-induced superoxide response in AA-activated neutrophils

In view of the increased expression of TNFR on neutrophils, it was of interest to determine whether or not this translated into a functional difference when such neutrophils were exposed to TNF. In these investigations, neutrophils were first incubated for 30 min with AA or vehicle and then stimulated with TNF or diluent. The chemiluminescence response was measured over time as a marker of superoxide produced by the neutrophils. Neutrophils that had been pretreated with AA showed increased superoxide production in response to TNF compared with those treated with vehicle (Fig. 3).



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. The chemiluminescence response of AA-treated neutrophils stimulated with TNF. Neutrophils (1 x 106/ml) were treated with AA (10 µM) or vehicle for 30 min, and then TNF (1000 U/ml) or HBSS was added. Superoxide production was determined by chemiluminescence, as described in Materials and Methods. Results shown are from the mean ± SEM of triplicates for each of two donors. Significance of difference between AA and AA + TNF: p < 0.001.

 
Effect of different types of long chain polyunsaturated fatty acids

Previous work has demonstrated that the anti-inflammatory effects of {omega}3 polyunsaturated fatty acids are related to their metabolism into poorly proinflammatory metabolites compared with AA metabolites and inhibition of cytokine production (20, 21, 22, 23). However, many neutrophil functions have been shown to be increased by both {omega}3 and {omega}6 fatty acids. These include activation of respiratory burst, degranulation, and CR3 receptor expression (15). It was therefore of interest to see whether the {omega}3 fatty acids also increased the expression of TNFR on neutrophils. We compared the effects of {omega}6 fatty acids, AA, with the {omega}3 fatty acids, LNA, EPA, and DHA.

To examine these effects, neutrophils (1 x 106) were treated with 20 µM of one of the following fatty acids: EPA, DHA, AA, and LNA, or the equivalent amount of vehicle (ethanol) for 30 min at 37°C, and then examined for expression of TNFR1 and TNFR2. The results showed that while neutrophils treated with AA displayed a 6-fold increase in TNFR1 expression, DHA and EPA significantly decreased the expression of TNFR1 (Fig. 4). LNA had no significant effect on the TNFR1 expression on neutrophil (Fig. 4). Similar results were obtained for TNFR2 expression in neutrophils treated with 20 µM fatty acids (Fig. 4).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4. The effects of different types of polyunsaturated fatty acids, DHA, EPA, LNA, and AA, on TNFR expression on neutrophils. The cells (1 x 106) were treated with 20 µM of fatty acid (DHA, EPA, LNA, and AA) or an equivalent amount of diluent (ethanol) for 30 min at 37°C. Then the cells were processed for flow cytometry analysis to determine the expression of TNFR. The results are presented as the mean ± SEM of three experiments. The mean ± SEM of fluorescence intensity for the diluent ethanol was 29 ± 3 for TNFR1 and 28 ± 2 for TNFR2 (*, p < 0.001; **, p < 0.0001, fatty acid vs control).

 
LPS and fMLP down-regulate TNFR expression

It has been previously reported that both LPS and fMLP reduce TNFR expression (7); thus, it was therefore relevant to examine the effects of these agonists under conditions in which AA increased both TNFR1 and TNFR2 expression. Neutrophils were incubated with LPS (0.1 µg/ml), fMLP (10-6 M), or vehicle alone at 37°C for 30 min, and expression of TNFR was determined. LPS caused a 30–35% reduction in TNFR1 and TNFR2 expression (Fig. 5). fMLP reduced the expression of both receptors by 55–60% (Fig. 5). In contrast, fMLP increased the expression of CD11b by ~13-fold (Fig. 5). These results are consistent with the findings of Berger et al. (24), who reported that expression of CD11b on the surface of neutrophils was increased several-fold by fMLP.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of LPS and fMLP on TNFR1, TNFR2, and CD11b expression. Neutrophils were incubated with 0.1 µg/ml LPS, 10-6 M fMLP, or vehicle at 37°C for 30 min, and then examined for the expression of TNFR1 (A), TNFR2 (B), and CD11b (C). The results, expressed as percentage of control responses, are presented as the mean ± SEM of four experiments. The MFI (mean ± SEM) for the vehicle was 28 ± 2, 32 ± 3, and 734 ± 110 for TNFR1, TNFR2, and CD11b, respectively. Significance of difference between treated and control cells: *, p < 0.0001.

 
Effect of fMLP on TNFR expression on AA-activated neutrophils

TNFR down-regulation is believed to be due to proteolytic cleavage. The unique effect of AA on TNFR on neutrophils could be related to interference with the proteolytic activity in the neutrophil. If this were the case, then it would be expected that pretreatment of neutrophils with AA should prevent fMLP-induced down-regulation of TNFR. Neutrophils were treated with 30 µM AA for 30 min and then with 10-6 M fMLP, and the effect on TNFR1 and TNFR2 expression was examined. The results showed that fMLP did not cause a down-regulation in expression of TNFR1 and TNFR2 in AA-stimulated neutrophils (Fig. 6). In fact, in the presence of AA, fMLP caused a further and significant increase in TNFR1 and TNFR2 expression (Fig. 6).



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 6. A, The ability of fMLP to alter TNFR expression on AA-activated neutrophils. The leukocytes were pretreated with AA for 30 min and then exposed to fMLP. The results are presented as mean ± SEM of three experiments. The base and AA MFI was 11 ± 4 and 365 ± 54 for TNFR1; 26 ± 4 and 648 ± 76 for TNFR2. Significant difference between fMLP-treated and nontreated cells: *, p < 0.05; **, p < 0.01. B, Effect of AA and fMLP on the release of TNFR2 from neutrophils. Neutrophils were treated with either AA or vehicle for 30 min and then stimulated with fMLP diluent. Then the soluble TNFR2 was assayed in the supernatants. Results are presented as mean ± SEM of four experiments. Significant difference between ethanol and AA :a, p < 0.05; between DMSO and fMLP treatment :b, p < 0.05; between AA and AA + fMLP treatment :c, not significant, paired t test.

 
Effect of AA on TNFR release from neutrophils

Neutrophil activation by various agonists has been shown to lead to the release of soluble TNFR in fluids as a consequence of proteolytic cleavage of the extracellular portion of the receptor. Neutrophils were treated with either AA or vehicle and then with or without fMLP. The supernatants were collected, and the amount of soluble TNFR2 was determined by ELISA. The data shown in Fig. 6 demonstrate that fMLP induced the release of soluble TNFR. In contrast, AA caused a decrease in basal receptor release. It was also evident that if neutrophils had been pretreated with AA, fMLP failed to cause a release of TNFR in the culture fluids (Fig. 6). In these experiments, we used a longer incubation period of 30 min than previous studies (7) because we wanted to maintain conditions consistent with the present experiments on the expression of TNFR. It is likely that the high basal release of receptors was due to the increased incubation time. Because of this increase, the release induced by fMLP is also likely to be underestimated.

Effects of AA on TNFR expression in HL-60 cells

AA significantly reduced the surface expression of both TNFR1 and TNFR2 on HL-60 cells (Fig. 7A). However, when the HL-60 cells were matured by retinoic acid to neutrophil-like cells, AA caused an increase in the expression of both receptors in these cells (Fig. 7A).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 7. A, Effect of AA on TNFR expression by HL-60 and matured HL-60 cells. Cells were treated with 30 µM AA or vehicle for 30 min at 37°C, and then examined for the expression of TNFR1 (filled bar) and TNFR2 (open bar). The results (percentage of control) are presented as the mean ± SEM of three experiments. The MFI (mean ± SEM) for control HL-60 cells was 103 ± 43 and 332 ± 73 for TNFR1 and TNFR2, respectively, and for matured HL-60 cells was 70 ± 13 and 126 ± 19 for TNFR1 and TNFR2, respectively. B, Effect of varying AA treatment time on TNFR1({blacksquare}) and TNFR2 ({square}) expression on matured HL-60 cells. The results, percentage of increase in MFI, are presented as the mean ± SEM of three experiments, The MFI (mean ± SEM) for vehicle was 30 ± 4 and 29 ± 6 for TNFR1 and TNFR2, respectively. C, Effect of AA on TNFR expression on HUVEC. HUVEC were treated with 30 µM AA at 37°C for 30 min, and the expression of TNFR1 (filled bar) and TNFR2 (open bar) was determined. The results, MFI, are presented as the mean ± SEM of three experiments. Significance of difference between control and AA-treated cells: *, p < 0.01; **, p < 0.001; B, p < 0.001 ANOVA.

 
The effect of AA treatment time on TNFR expression on neutrophil-like cells was also examined. The results show that AA (30 µM) stimulated an increase in TNFR expression in a time-dependent manner (Fig. 7B). An increase in TNFR1 and TNFR2 expression was evident after 10 min and reached a maximum by 20–30 min (Fig. 7B).

AA induces down-regulation of TNFR on vascular endothelial cells (HUVEC)

The lining of the vascular endothelium controls the trafficking of cells and molecules into underlying tissues, and is therefore a potential primary target for the action of inflammatory mediators. Cytokines such as TNF and IL-1 dramatically alter many responses of cultured endothelial cells in vitro that are believed to play key roles in the development of atherosclerosis, inflammation, and septic shock (25). Both TNFR1 and TNFR2 have been detected on the surface of HUVEC (26). We therefore analyzed the effect of AA on TNFR on HUVEC. Treatment of HUVEC with 30 µM AA for 30 min resulted in a decrease in the expression of both the TNFR1 and TNFR2 on the surface of endothelial cells (Fig. 7C).

The effects of 15-HPETE and ME derivatives on the expression of TNFR

AA is a substrate for lipoxygenases and cyclooxygenases. The first step in the oxygenation of AA by the lipoxygenase pathway results in the formation of HPETE, which is the precursor of monohydroxyeicosatetraenoic acids as well as the leukotrienes. Some of these AA metabolites can regulate neutrophil responses. However, our previous studies in neutrophils have demonstrated that modification of AA by the addition of a hydroperoxy moiety or converting AA to its ME results in the loss of biological activity (15). To investigate whether the unmodified AA structure was required for the up-regulation of TNFR, neutrophils were incubated for 30 min with 15-HPETE (30 µM), and the expression of TNFR was determined. The data in Fig. 8 demonstrate that 15-HPETE did not alter the expression of either TNFR1 or TNFR2, in contrast to AA. These results demonstrate that the addition of a hydroperoxy group to AA results in a loss in the ability to stimulate TNFR1 and TNFR2 expression. Converting AA to its ME, which masks the carboxyl group, also resulted in loss of this property of AA (Fig. 8).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 8. Effects of 15-HPETE and the ME of AA on TNFR1 and TNFR2 expression. Neutrophils were incubated with 30 µM of AA, its ME derivative, 15-HPETE, or vehicle at 37°C for 30 min, and then examined for the expression of TNFR1 (A) and TNFR2 (B). The results, percentage of control, are presented as the mean ± SEM of four experiments. The MFI (mean ± SEM) for vehicle was 30 ± 4 for TNFR1 and 29 ± 6 for TNF2. Significance of difference between control and AA treatment: *, p < 0.0001. Effect of NDGA, a lipoxygenase inhibitor, on the expression of TNFR1 (C) and TNFR2 (D) on neutrophils. Cells were preincubated with NDGA (10 µM) or vehicle for 15 min at 37°C, followed by treatment of neutrophils with 30 µM AA for 30 min at 37°C. TNFR expression was measured. The results, percentage of AA response, are expressed as mean ± SEM of three experiments. The MFI (mean ± SEM) for TNFR1 was 30 ± 3 and 154 ± 41 for vehicle and AA, respectively. The MFI (mean ± SEM) for TNFR2 was 35 ± 9 and 173 ± 43 for vehicle and AA, respectively. Effect of IM, a cyclooxygenase inhibitor, on the expression of TNFR1 (E) and TNFR2 (F) on neutrophils. Cells were incubated with 100 µM IM or vehicle for 15 min at 37°C, followed by treatment of neutrophils with 30 µM AA for 30 min at 37°C. TNFR expression was measured. The results are expressed as mean ± SEM of three experiments. The MFI (mean ± SEM) for TNFR1 was 30 ± 3 and 154 ± 41 for vehicle and AA, respectively. The MFI (mean ± SEM) for TNFR2 was 35 ± 9 and 173 ± 43 for vehicle and AA, respectively. Significance of difference between the absence and presence of IM: *, p < 0.0001.

 
The role of lipoxygenases and cyclooxygenases

The effects of NDGA, a nonselective lipoxygenase inhibitor, and IM, a cyclooxygenase inhibitor, on AA-induced TNFR expression in neutrophils were also examined. Neutrophils were pretreated with either NDGA (10 µM) or IM (100 µM) or with vehicle for 15 min at 37°C, before being stimulated with 30 µM AA or with vehicle for 30 min at 37°C. NDGA did not affect the ability of AA to up-regulate the expression of either TNFR1 or TNFR2 (Fig. 8). Interestingly, IM caused a significant enhancement of the AA-induced increase in both TNFR1 and TNFR2 (Fig. 8).

The role of PKC, ERK1/ERK2, and cPLA2

We have previously demonstrated that AA stimulates the activities of PKC, ERK1/ERK2, and phospholipase A2 (27, 28). To understand the mechanisms by which AA caused the up-regulation of TNFR, the chemical inhibitors, GF109203X, PD098059, and AACOCF3, were used to target PKC, ERK1/ERK2, and the cPLA2, respectively. These inhibitors have been used previously in neutrophils with respect to other agonists such as fMLP. Thus, GF109203X has been reported to inhibit fMLP-stimulated p47phox phosphorylation, superoxide production, and ERK1/ERK2 activation (29, 30). PD98059 has been reported to inhibit fMLP-stimulated chemotaxis, p47phox phosphorylation, and superoxide production (29, 31, 32), and AACOCF3 has been shown to inhibit fMLP-stimulated H+ efflux (33).

Neutrophils were pretreated with the PKC inhibitor,GF109203X, for 10 min at 37°C, and then stimulated with 30 µM AA or vehicle. After further 30-min incubation at 37°C, expression of TNFR was determined. The results presented in Fig. 9A demonstrate that GF109203X dose dependently inhibited the AA-induced increase in expression of TNFR1 on neutrophils (p < 0.001). The inhibition was 40 and 80% at concentrations of 1 and 100 nM of GF109203X, respectively. The expression of TNFR2 was also inhibited by GF109203X (p < 0.05), although the degree of inhibition was less than that observed for TNFR1 (Fig. 9A). Thus, PKC was required for the increased expression of TNFR induced by AA, in particular for TNFR1.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 9. Effect of inhibitors of intracellular signaling molecules on AA-induced increase in TNFR1 and TNFR2 expression on neutrophils. Cells were pretreated with either GF109203X (for 10 min), PD098059 (for 45 min), or AACOCF3 (for 5 min) at 37°C, followed by treatment of neutrophils with 30 µM AA for 30 min at 37°C. Then TNFR expression was measured by flow cytometry. The results are presented as percentage of the AA response expressed as mean ± SEM of three to four experiments. The mean MFI for TNFR1 was 17 and 232 for vehicle and AA, respectively, in the absence of inhibitor. The mean of MFI for TNFR2 was 31 and 402 for vehicle and AA, respectively, in the absence of inhibitor. There were no significant effects of the inhibitors on baseline expression of TNFR. Significance of difference between the absence and presence of inhibitor: *, p < 0.05; **, p < 0.001, Dunnett’s Modification.

 
To investigate the role of ERK1/ERK2 in AA-induced up-regulation of TNFR expression, the effect of PD098059, a MEK-1 inhibitor, was examined. This inhibitor has been shown to inhibit ERK activity in neutrophils (31). Neutrophils (1 x 106) were pretreated with PD098059 or vehicle for 45 min at 37°C, followed by 30 µM AA or vehicle for 30 min at 37°C. Treatment of neutrophils with PD098059 caused a dose-dependent inhibition of AA-induced increase in TNFR1 expression (Fig. 9B). Maximum inhibition (almost 85%) of TNFR1 expression occurred at a PD098059 concentration of 37.5 µM (p < 0.001). In comparison, 37.5 µM PD098059 only had minimal, but nonsignificant effect (30% inhibition) on AA-induced increase in expression of TNFR2 (Fig. 9B). These results demonstrate that the MEK-ERK pathway is involved in regulating AA-stimulated expression of TNFR1 to a greater extent than TNFR2.

To determine the role of cPLA2, the effects of the pharmacological inhibitor of cPLA2, AACOCF3, on the AA-induced increase in the expression of TNFR were examined. Neutrophils were pretreated with AACOCF3 or vehicle for 5 min at 37°C before being stimulated with 30 µM AA or vehicle for 30 min at 37°C.

Pretreatment of neutrophils with AACOCF3 significantly inhibited the AA-stimulated increase in TNFR1 expression (Fig. 9C). Maximum inhibition (80–90%) occurred at an AACOCF3 concentration of 0.5 and 5 µM (p < 0.001). In contrast, the inhibitory effect of AACOCF3 on TNFR2 expression was less prominent (30–50%) (p < 0.05) compared with its effects on TNFR1 expression. These effects were very similar to those observed with the PKC and MEK inhibitors (Fig. 9, A and B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Surprisingly, while all agonists studied to date cause the down-regulation of surface TNFR expression, our data demonstrate that AA induced a marked increase in the expression of both TNFR1 and TNFR2 on neutrophils. This increase was substantial, some 8-fold, compared with a 2-fold increase in CR3 expression. An AA concentration-related effect was seen over 0.5–30 µM. Significant increases were seen at 0.5 µM (p < 0.05), and these were marked at 30 µM. This is well within the concentrations of AA generated in biological fluids during inflammation. AA has been found in inflammatory fluids in the hundreds of micromolar to millimolar amounts (34, 35). Thus, these findings suggest a novel role for AA in inflammation. The AA-stimulated expression of TNFR has relevance to regulation of inflammation, in which it is likely to have a role in ensuring, maintaining, and indeed increasing the ability of TNF to stimulate/prime neutrophils coming into an inflammatory site, and therefore promoting their antimicrobial function. Consistent with this was the finding that neutrophils pretreated with AA demonstrated a significantly increased superoxide release in response to TNF. This is of particular importance under conditions in which exogenous and endogenous inflammatory mediators cause the down-regulation of TNFR (7, 8, 9). Thus, neutrophil agonists, such as fMLP, LPS, the complement fragment C5a, leukotriene B4, GM-CSF, TNF, IFN-{gamma}, PMA, calcium ionophore (A23187), and platelet-activating factor, cause the down-regulation of TNFR expression on neutrophil surfaces through proteolytic cleavage of the extracellular portion of these receptors (7, 8, 9). Recently, it has been reported that a membrane-bound, nonmatrix serine metalloproteinase, possibly a disintegrin and metalloprotease-17 (36), may be involved in the cleavage of TNFR. Elastase has also been suggested to contribute to the release of the soluble TNFR (37).

The up-regulation of TNFR expression by AA was unique to neutrophils because AA-treated HL-60 cells showed depressed expression of TNFR. Interestingly, when matured to neutrophil-like cells, AA caused a significant increase in TNFR expression on these cells. This up-regulation of TNFR by AA is most likely due to the ability of neutrophils to readily mobilize TNFR from granules, upon cell activation, which is consistent with the AA-induced effect being rapid, causing a significant increase in TNFR expression in the first 10 min. Also, AA-treated HUVEC showed a significant reduction in TNFR expression.

AA is a complete neutrophil secretagogue, causing the release of constituents from both specific and azurophilic granules (15). It is most likely that this results in the mobilization of TNFR from storage granules to the surface membrane. However, to date, evidence has only been presented for the storage of TNFR1 in specific granules (38). TNFR2 was found to be associated only with the plasma membrane fraction. Thus, our data showing that TNFR2 was increased in expression to a similar extent as TNFR1 suggest that it is likely to be mobilized from other intracellular locations. Although some of the above agonists that cause TNFR down-regulation are weak secretagogues, others such as A23187 and fMLP in the presence of cytochalasin B (38) induce marked degranulation. It is therefore surprising that these agents do not increase receptor expression. This is most likely related to their ability to also stimulate proteolytic cleavage of the receptors, and thus, any increase in receptor expression is masked by loss of surface protein. Because AA caused a down-regulation of TNFR in both HL-60 cells and endothelial cells, it is unlikely that AA inhibits TNFR cleavage in these cells, and indeed is likely to stimulate proteolysis for this receptor. However, this may not apply to neutrophils because neutrophils that had been pre-exposed to AA showed a significant increase in fMLP-induced receptor expression. These findings would be best explained by suggesting that AA, by a mechanism yet to be identified, inhibits cleavage of receptors specifically in neutrophils. This was supported by the finding that AA inhibited the fMLP-induced release of TNFR in neutrophil cultures.

Because of the existence of multiple cellular targets for AA, it is difficult to readily decipher the mechanisms involved in TNFR up-regulation. Badwey et al. (39) pointed out that AA readily partitions into membranes that result in changes to the physical properties of the membranes (40). However, Corey and Rosoff (41) excluded a detergent-like action of AA on neutrophil oxidase activity, and our recent studies (42) and those of others (43) have demonstrated that cell activation by AA can occur via the ErbB receptor family. AA can be readily esterified into membrane phospholipids, and so can 15-HPETE (44), which did not alter the expression of TNFR. This suggests that membrane incorporation is insufficient for the observed action. The ME derivative is likely to cause membrane perturbation, but is unable to promote TNFR expression. Furthermore, we found no evidence for a need to convert AA to lipoxygenase and cyclooxygenase metabolites because inhibition of these enzymes did not prevent the actions of AA. Indomethacin actually enhanced the effect, and others have reported that leukotriene B4 decreases TNFR expression (7). Whether or not the stimulating effects of indomethacin are due to an effect through the cyclooxygenase pathway remains to be established.

AA induces the activation of several intracellular signaling molecules that have been shown to be responsible for its biological effects (15). Using chemical inhibitors of these pathways, we have identified some of the mechanisms by which AA stimulates TNFR expression on neutrophils. The effects on TNFR1 were very sensitive to GF109203X, PD098059, and AACOCF3, showing a role for PKC, ERK, and cPLA2, respectively. These inhibitors caused greater than 80% inhibition of the effect of AA. Although similar results were obtained with respect to TNFR2, the inhibitors caused only a 30% inhibition of this response. The reason for this difference in requirement of the signaling between the two receptors remains to be identified, but is consistent with the findings of Porteu and Nathan (38), who found the TNFR1 and TNFR2 to have different cellular localization.

The above findings are not surprising given that we have demonstrated in neutrophils that AA caused an increase in the intracellular concentration of Ca2+ (18) and activation of ERK1/ERK2 (45), which are essential for the activation of the cPLA2. Indeed, we have demonstrated that exogenous AA caused the activation of the cPLA2 and secretory PLA2 (28). This was accompanied by the release of [3H]AA from prelabeled cells, and this could be inhibited by AACOCF3. As with AA-induced up-regulation of TNFR, AACOCF3 also inhibited AA-stimulated superoxide production (28). Our results therefore support the existence of a signaling loop involving AA, which stimulates PKC and the mitogen-activated protein kinases, which together with an increase in intracellular Ca2+, result in the activation of the cPLA2.

The proinflammatory properties of AA are associated with several of its effects on neutrophil functions (15). These include the stimulation of oxygen radical production (13, 14, 39, 40, 41), degranulation and cell adhesion (15), and migration inhibition (46). This study has revealed a unique biological effect of AA on neutrophil TNFR expression that is likely to have a significant impact on our present concepts on mechanisms of regulation of the inflammatory reaction, and thus it is not surprising that AA and TNF cause synergistic responses in neutrophils (47). The belief that neutrophils dramatically decrease their expression of TNFR during inflammation as a result of exposure to a range of agonists is therefore questioned by our findings that AA caused a marked increase in the expression of these receptors. Thus, as the levels of AA increase during inflammation, there is a bias toward increased expression of TNFR and increased responsiveness to TNF. Furthermore, it has been argued that the release of TNFR from neutrophils provides a source of soluble TNFR to regulate inflammation and prevent TNF-promoted tissue damage (7, 8, 9). This may not only be of relevance in acute inflammation, but also in chronic inflammatory diseases that experience infiltration of large numbers of neutrophils during exacerbations of the diseases in which soluble TNFR can act as TNF inhibitors (48). However, once AA has been generated, neutrophil TNFR expression will be substantially increased, promoting inflammation and tissue damage. This is consistent with the value of anti-TNF therapy in diseases such as rheumatoid arthritis (49), and the high levels of the secretory PLA2 found in rheumatoid arthritis patients (50).

Particularly interesting was the finding that the {omega}3 polyunsaturated fatty acids, EPA, DHA, and LNA, not only failed to increase TNFR expression on neutrophils, but also that EPA and DHA significantly depressed the TNFR expression. Thus, in this study, we see another aspect of how {omega}3 fatty acids may function to inhibit the inflammation response. Incorporation of increased amounts of {omega}3 fatty acids in membrane phospholipids not only is likely to compromise the generation of highly inflammatory eicosanoids and cytokines, but also to prevent the increased expression of TNFR on neutrophils.


   Footnotes
 
1 This work received financial support from the National Health and Medical Research Council of Australia. Back

2 Current address: Department of Medicine, Rush-Presbyterian, St. Luke’s Medical Center, Chicago, IL 60612. Back

3 Address correspondence and reprint requests to Dr. Antonio Ferrante, Department of Immunopathology, The Women’s and Children’s Hospital, 72 King William Road, North Adelaide South Australia 5006. E-mail address: antonio.ferrante{at}adelaide.edu.au Back

4 Abbreviations used in this paper: cPLA2, cytosolic PLA2; AA, arachidonic acid; CR3, complement receptor 3; DHA, docosahexaenoic acid; DPC, dipalmitoyl phosphatidylcholine; EPA, eicosapentaenoic acid; ERK, extracellular signal-regulated kinase; HPETE, hydroperoxyeicosatetraenoic acid; IM, indomethacin; LNA, linolenic acid; ME, methyl ester; MEK, mitogen-activated protein kinase/ERK kinase; MFI, mean fluorescence intensity; NDGA, nordihydroguariaretic acid; PKC, protein kinase C; PLA2, phospholipase A2. Back

Received for publication December 23, 2002. Accepted for publication June 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Aggarwal, B. B., J. Vilcek. 1992. In Tumor Necrosis Factor: Structure Functions and Mechanism of Action: Immunology Services 56:1.-600. Marcel Dekker, New York.
  2. Klebanoff, S. J., M. A. Vadas, J. M. Harlan, L. H. Sparks, J. R. Gamble, J. M. Agosti, A. M. Waltersdorph. 1986. Stimulation of neutrophils by tumor necrosis factor. J. Immunol. 136:4220.[Abstract]
  3. Ferrante, A., A. J. Martin, E. J. Bates, D. H. Goh, D. P. Harvey, D. Parsons, D. A. Rathjen, G. Russ, J. M. Dayer. 1993. Killing of Staphylococcus aureus by tumor necrosis factor {alpha}-activated neutrophils: the role of serum opsonins integrin receptors, respiratory burst and degranulation. J. Immunol. 151:4821.[Abstract]
  4. MackEwan, D. J.. 2001. TNF receptor subtype signalling: differences and cellular consequences. Cell. Signal. 14:477.
  5. Brockhams, M., H. J. Schoenfold, E. J. Schlaeger, W. Hunziker, W. Lesslauer, H. Loetscher. 1990. Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 87:3127.[Abstract/Free Full Text]
  6. Larrick, J. W., D. Graham, K. Toy, L. S. Lin, G. Senyk, B. M. Fendly. 1987. Recombinant tumor necrosis factor causes activation of human granulocytes. Blood 69:640.[Abstract/Free Full Text]
  7. Porteu, F., C. Nathan. 1990. Shedding of tumor necrosis factor receptors by activated human neutrophils. J. Exp. Med. 172:599.[Abstract/Free Full Text]
  8. Schleiffenbaum, B., J. Fehr. 1990. The tumor necrosis factor receptor and human neutrophil function: deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation. J. Clin. Invest. 86:184.
  9. Ohman, H. B., M. Campos, L. McDougall, M. J. Lawman, L. A. Babiuk. 1990. Expression of tumor necrosis factor-{alpha} receptors on bovine macrophages, lymphocytes and polymorphonuclear leukocytes, internalization of receptor-bound ligand, and some functional effects. Lymphokine Res. 9:43.[Medline]
  10. Ferrante, A., A. J. Martin, E. J. Bates, I. C. Kowanko, D. P. Harvey, D. Parsons, D. A. Rathjen G. Russ, J. M. Dayer. 1994. Interaction of Staphylococcus aureus with human neutrophils and the down-regulation of TNF receptors. J. Immunol. 152:3998.[Abstract]
  11. Dennis, E. A.. 1997. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22:1.[Medline]
  12. Haeggstrom, J. Z., C. N. Serhan. 1999. Update on arachidonic acid cascade: leukotrienes and lipoxins in disease models. C. N. Serhan, and P. A. Ward, eds. Molecular and Cellular Basis of Inflammation 51. Human Press, Totowa.
  13. Badwey., J. A., J. T. Curnutte, M. L. Karnovsky. 1981. Cis-polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils. J. Biol. Chem. 256:12640.[Abstract/Free Full Text]
  14. Hardy, S. J., B. S. Robinson, A. Poulos, D. P. Harvey, A. Ferrante, A. W. Murray. 1991. The neutrophil respiratory burst: responses to fatty acids, FMLP and phorbol ester suggest divergent signalling mechanisms. Eur. J. Biochem. 198:801.[Medline]
  15. Ferrante, A., C. S. Hii, Z. H. Huang, D. A. Rathjen. 1999. Regulation of neutrophil function by fatty acids. D. Gabrilovich, ed. In The Neutrophils: New Outlook for the Old Cells Vol. 4:79. Imperial College Press, London.
  16. Ferrante, J. V., Z. H. Huang, M. Nandoskar, C. S. Hii, B. S. Robinson, D. A. Rathjen, A. Poulos, C. P. Morris, A. Ferrante. 1997. Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid and arachidonic acid: inhibition of TNF production. J. Clin. Invest. 99:1445.[Medline]
  17. Huang, Z. H., E. J. Bates, J. V. Ferrante, A. Poulos, B. S. Robinson, A. Ferrante. 1997. Inhibition of stimuli-induced endothelial cell intercellular adhesion molecule -1, E-selectin and vascular cellular adhesion molecule-1 expression by arachidonic acid and its hydroxy- and hydroperoxy-derivatives. Circ. Res. 80:149.[Abstract/Free Full Text]
  18. Hardy, S. J., B. S. Robinson, A. Ferrante, C. S. Hii, D. W. Johnson, A. Poulos, A. W. Murray. 1995. Polyenoic very-long-chain fatty acids mobilize intracellular calcium from a thapsigargin-insensitive pool in human neutrophils: the relationship between Ca2+ mobilization and superoxide production induced by long- and very-long-chain fatty acids. Biochem. J. 311:689.
  19. Breitman, T. R., S. E. Selonick, S. J. Collins. 1980. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Natl. Acad. Sci. USA 77:2936.[Abstract/Free Full Text]
  20. Sperling, R. I., R. A. Lewis, K. F. Austen. 1986. Regulation of 5-lipoxygenase pathway product generation in human neutrophils by n-3 fatty acids. Prog. Lipid Res. 25:101.[Medline]
  21. Boudrean, M. D., P. S. Chanmugam, S. B. Hart, S. H. Lee, D. H. Hwang. 1991. Lack of dose response by dietary n-3 to n-6 fatty acids at a constant ratio of n-3 to n-6 fatty acids in suppressing eicosanoids biosynthesis from arachidonic acid. Am. J. Clin. Nutr. 54:111.[Abstract/Free Full Text]
  22. Endres, S., R. Ghorbani, V. E. Kelly, K. I. Georgilis, G. Lonnemann, J. W. Van der Meer, J. G. Cannon, T. S. Rogers, M. S. Klempner, P. C. Weber, et al 1989. The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N. Engl. J. Med. 320:265.[Abstract]
  23. Calder, P. C.. 1997. n-3 Polyunsaturated fatty acids and cytokine production in health and disease. Ann. Nutr. Metab. 41:203.[Medline]
  24. Berger, M., J. O’Shea, A. S. Cross, T. M. Folks, T. M. Chused, E. J. Brown, M. M. Frank. 1984. Human neutrophils increase expression of C3bi as well as C3b receptors upon activation. J. Clin. Invest. 74:1566.
  25. Dinarello, C. A., B. J. Pomerantz. 2001. Proinflammatory cytokines in heart disease. Blood Purif. 19:314.[Medline]
  26. Mackay, F., H. Loetscher, D. Stueber, G. Gehr, W. Lesslauer. 1993. Tumor necrosis factor {alpha} (TNF-{alpha})-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J. Exp. Med. 177:1277.[Abstract/Free Full Text]
  27. Hii, C. S., A. Ferrante, Y. S. Edwards, Z. H. Huang, P. J. Hartfield, D. B. Rathjen, A. Poulos, A. W. Murray. 1995. Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism. J. Biol. Chem. 270:4201.[Abstract/Free Full Text]
  28. Robinson, B. S., C. S. Hii, A. Ferrante. 1998. Activation of phospholipase A2 in human neutrophils by polyunsaturated fatty acids and its role in stimulation of superoxide production. Biochem. J. 336:611.
  29. Dewas, C., M. Fay, M. A. Gougerot-Pocidalo, J. El-Benna. 2000. The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils. J. Immunol. 165:5238.[Abstract/Free Full Text]
  30. Chang, L. C., J. P. Wang. 1999. Examination of the signal transduction pathways leading to activation of extracellular signal-regulated kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils. FEBS Lett. 454:165.[Medline]
  31. Hii, C. S., K. L. Stacey, N. Moghaddami, A. Murray, A. Ferrante. 1999. Role of extracellular signal-regulated protein kinase cascade in neutrophil killing of S. aureus and C. albicans and in migration. Infect. Immun. 67:31297.
  32. Nagata, T., M. Kansha, K. Irita, S. Takahashi. 2001. Propofol inhibits FMLP-stimulated phosphorylation of p42 mitogen-activated protein kinase and chemotaxis in human neutrophils. Br. J. Anaesth. 86:853.[Abstract/Free Full Text]
  33. Susztak, K., A. Mocsai, E. Ligeti, A. Kapus. 1997. Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2. Biochem. J. 325:501.
  34. Eissen, E. U.. 1993. Significance of plasma free fatty acid levels in human malaria with parasitaemia. Med. Sci. Res. 21:405.
  35. Yasuda, H. K., K. Kishivo, N. Izumi, M. Nakanishi. 1985. Biphasic literation of arachidonic acid and stearic acids during cerebral ischemia. J. Neurochem. 45:168.[Medline]
  36. Dri, P., C. Gasparini, R. Menegazzi, R. Cramer, L. Alberi, G. Presani, S. Garbisa, P. Patriarca. 2000. TNF-induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase. J. Immunol. 165:2165.[Abstract/Free Full Text]
  37. Porteu, F., M. Brockhaus, D. Wallach, H. Engelmann, C. F. Nathan. 1991. Human neutrophil elastase releases a ligand-binding fragment from the 75-KDa tumor necrosis factor (TNF) receptor: comparison proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils. J. Biol. Chem. 266:18846.[Abstract/Free Full Text]
  38. Porteu, F., C. F. Nathan. 1992. Mobilizable intracellular pool of p55 (type 1) tumor necrosis factor receptors in human neutrophils. J. Leukocyte Biol. 52:122.[Abstract]
  39. Badwey, J. A., J. T. Curnutte, J. M. Robinson, C. B. Berde, M. J. Karnovsky, M. L. Karnovsky. 1984. Effects of free fatty acids on release of superoxide and on change of shape by human neutrophils reversibility by albumin. J. Biol. Chem. 259:7870.[Abstract/Free Full Text]
  40. Steinbeck, M. J., J. M. Robinson, M. J. Karnovsky. 1991. Activation of the neutrophil NADPH oxidase by free fatty acids requires the ionized carboxyl groups and partitioning into membrane lipids. J. Leukocyte Biol. 49:360.[Abstract]
  41. Corey, S. J., P. M. Rosoff. 1991. Unsaturated fatty acids and lipoxygenase products regulate phagocyte NADPH oxidase activity by non-detergent mechanism. J. Lab. Clin. Med. 118:343.[Medline]
  42. Hii, C. S., N. Moghadammi, A. Ferrante. 2001. Stimulation of the PI3-kinase-Akt/PKB signalling pathway by arachidonic acid in human myeloid and endothelial cells: involvement of the ErbB receptors. J. Biol. Chem. 276:27246.[Abstract/Free Full Text]
  43. Dulin, N. O., A. Sorokin, J. G. Douglas. 1998. Arachidonate-induced tyrosine phosphorylation of epidermal growth factor receptor and Shc-Grb2-Sos association. Hypertension 32:1089.[Abstract/Free Full Text]
  44. Ochi, H., I. Morita, S. Murota. 1992. Mechanism for endothelial cell injury induced by 15-hydroxyeicosatetraenoic acid, an arachidonate lipoxygenase product. Biochim. Biophys. Acta 1136:247.[Medline]
  45. Hii, C. S., Z. H. Huang, A. Bilney, M. Costabile, A. W. Murray, R. A Rathjen, C. J. Der, A. Ferrante. 1998. Stimulation of p38 phosphorylation/activation by arachidonic acid in HeLa cells, HL60 promyelocytic leukaemic cells and human neutrophils: evidence for cell-type specific activation of MAP kinases. J. Biol. Chem. 273:19277.[Abstract/Free Full Text]
  46. Ferrante, A., D. H. B. Goh, D. P. Harvey, B. S. Robinson, C. S. Hii, E. J. Bates, S. J. Hardy, D. W. Johnson, A. Poulos. 1994. Neutrophil migration inhibitory properties of polyunsaturated fatty acids: the role of fatty acid structure, metabolism and possible second messenger systems. J. Clin. Invest. 93:1063.
  47. Li, Y., A. Ferrante, A. Poulos, D. P. Harvey. 1996. Neutrophil oxygen radical generation: synergistic responses to tumor necrosis factor and mono/polyunsaturated fatty acids. J. Clin. Invest. 97:1605.[Medline]
  48. Arend, W. P., J.-M. Dayer. 1990. Cytokine and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum. 33:305.[Medline]
  49. Feldmann, M.. 2002. Development of anti-TNF therapy for rheumatoid arthritis. Nat. Rev. Immunol. 2:364.[Medline]
  50. Jamal, O. S., P. G. Conaghan, A. M. Cunningham, P. M. Brooks, V. F. Munro, K. F. Scott. 1998. Increased expression of human type IIa secretory phospholipase A2 antigen in arthritic synovium. Ann. Rheum. Dis. 57:550.[Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2003 171: 2181-2182. [Full Text]  



This article has been cited by other articles:


Home page
 J. Nutr.Home page
J. Mane, E. Pedrosa, V. Loren, I. Ojanguren, L. Fluvia, E. Cabre, G. Rogler, and M. A. Gassull
Partial Replacement of Dietary (n-6) Fatty Acids with Medium-Chain Triglycerides Decreases the Incidence of Spontaneous Colitis in Interleukin-10-Deficient Mice
J. Nutr., March 1, 2009; 139(3): 603 - 610.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moghaddami, N.
Right arrow Articles by Ferrante, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moghaddami, N.
Right arrow Articles by Ferrante, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS