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Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The LT are proinflammatory mediators that play an important role in disease states such as asthma (9, 10, 11). They are also produced in high levels in pneumonia (12) and acute lung injury (13). Recently, LT have been implicated as having a role in host defense against microbial infection (14). LT synthesis from AA is initiated by the Ca2+-dependent activation of 5-LO, acting in concert with the AA-binding protein, 5-LO-activating protein (FLAP) (15), to form LTA4. LTA4 is the precursor for formation of the two major groups of LT, LTB4 and the peptidoleukotrienes LTC4, D4, and E4 (16).
Among the other macrophage mediators stimulated by LPS is NO. This is formed from the amino acid L-arginine by NO synthase (NOS). An inducible form of NOS (iNOS) is well known to be transcriptionally up-regulated by LPS (17, 18). NO is an important endogenous regulator of pro- and anti-inflammatory processes (19, 20). It has been shown to modulate COX activity in a complex manner, either up-regulating (21, 22) it or down-regulating (23) it in various systems. Although NO activation is stimulated by 5-LO products (24, 25), little is known about the effect of NO on 5-LO metabolism.
In this study we evaluated the effects of prolonged exposure to LPS on LT synthesis by alveolar macrophages (AM). AM are the resident inflammatory cell of the pulmonary alveolar space and have a greater capacity for LT synthesis than do other mononuclear phagocytic populations (26). We now report that, in contrast to the reported effects of short term LPS exposure, prolonged LPS treatment of AM in vitro suppressed LT synthesis. The mechanism of LPS suppression involved reduced activity of both 5-LO and FLAP. Most of the down-regulatory effect of LPS on LT synthesis was accounted for by the generation of NO following induction of iNOS by LPS. By comparison, PBM failed to exhibit induction of iNOS following LPS treatment, and they showed substantially less suppression of LT synthesis than did AM. These effects of prolonged LPS exposure and the effect of NO on LT synthesis provide novel and clinically relevant insights into the regulation of LT synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rat AM was studied because it is a good model of human AM 5-LO metabolism and has been well characterized in our laboratory (27). AM were obtained from 150-g specific pathogen-free female Wistar rats as previously described (26). Lavaged cells were >90% AM; they were >98% AM following adherence, as determined by differential staining. Viability was >95%, as assessed by trypan blue exclusion. PBM were isolated from whole blood by Ficoll-Hypaque centrifugation and adherence as previously described (28). Cells were >90% PBM by differential staining, and viability was >95% by trypan blue exclusion. Isolated AM and PBM were resuspended in LPS-free DMEM at 0.5 x 106/ml and were plated as follows: 0.2 ml/well in 96-well plates for enzyme immunoassay (EIA), 1 ml/well in 24-well plates for [3H]AA release studies, and 5 ml/50-mm culture plate for immunoblot analysis. Cells adhered for 1 h at 37°C in a humidified atmosphere of 5% CO2/95% O2. Nonadherent cells were removed by washing twice with DMEM, and adherent cells were cultured in DMEM containing 10% FCS, with or without various concentrations of LPS (Escherichia coli serotype 0111:4B, Sigma, St. Louis, MO) for various time periods.
Quantitation of 5-LO metabolism in intact cells
The maximal capacity for 5-LO metabolism in intact cells was measured by EIA (Cayman Chemicals, Ann Arbor, MI) determination in cell-free supernatants of the predominant 5-LO product, LTB4. Following incubation with or without LPS, the cells were washed three times in DMEM and subsequently incubated with the Ca2+ ionophore A23187 (1 µM) to stimulate the release and metabolism of endogenous AA or exogenous AA (50 µM). This high concentration of AA has been shown to activate 5-LO enzyme activity as well as providing substrate for LT synthesis (28, 29). The EIA results were confirmed by reverse phase HPLC analysis. Briefly, cells were prelabeled overnight with [3H]AA in the presence or the absence of LPS. There was no effect of LPS on cellular uptake of radioactivity (data not shown). The eicosanoid profile was determined by HPLC analysis of 3H-radiolabeled eicosanoids (thromboxane B2, PGE2, LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE)) released from A23187-stimulated cells as previously described (30). To assess total AA release, cells were stimulated in the presence of 0.1% BSA, which binds AA and prevents both metabolism and reacylation.
5-LO cell-free assay
The 5-LO activity of cell lysates (100 µg of total protein) or
of purified recombinant 5-LO (gift from Denis Riendeau, Merck Frosst,
Montreal, Canada) was determined in reaction mixtures containing 20
µM AA (Cayman Chemicals; including 
100,000 dpm of
[3H]AA (DuPont-New England Nuclear, Boston,
MA)) and 10 µM
13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic
acid (Cayman Chemicals) as activator in 1 ml of 50 mM Tris, 0.3 mM
CaCl2, 0.6 mM EDTA, 0.1 mM ATP, and 12 µg/ml
phosphotidylcholine (Avanti Polarlipids, Alabaster, AL) as previously
described (31, 32). After a 30-min reaction at room
temperature the reaction was stopped by adding 1 ml of ether/MeOH/1 M
citric acid (30/4/1, v/v/v) and centrifuging at 3500 rpm for 5 min. The
upper phase was removed, evaporated under nitrogen, and stored at
-70°C. Lipid residues were dissolved in 250 µl of acetonitrile and
analyzed by HPLC on a 5-µm Bondapak C18 column
(30 x 0.4 cm; Waters Associates, Milford, MA) using a mobile
phase of acetonitrile/water/trifluoroacetic acid at a flow rate of 2
ml/min as previously described (27). Radioactivity in 1 ml
of eluate fractions was quantitated by on-line radioactivity detection.
5-LO specific activity was calculated based on conversion of AA to
5-HPETE/5-HETE plus
LTB4/LTB4 isomers, and was
expressed as nanomoles per milligrams of protein per 10 min.
Immunoblot analysis of 5-LO, FLAP, and iNOS
The relative quantities of cellular 5-LO, FLAP, and iNOS proteins were determined by Western blot analysis. Crude lysates were prepared as previously described (27) and subjected to SDS-PAGE by the method of Laemmli on 10% acrylamide gels. Proteins were transferred overnight to nitrocellulose membranes, and probed with rabbit polyclonal Abs against human leukocyte 5-LO (1/3000 dilution), amino acid residues 41–52 of the human FLAP sequence (1/5000 dilution; both provided by Dr. J. Evans, Merck Frosst) (28), or iNOS (1/2000 dilution; Biomol, Plymouth Meeting, PA). After washing, blots were incubated for 1 h with HRP-conjugated anti-rabbit IgG (Amersham) at a dilution of 1/5000. Membranes were then washed and incubated for 1 min with ECL chemiluminescence detection reagents (Amersham) and exposed to film for varying time periods to ensure that densitometric quantitation was performed under conditions in which band density and exposure time were linearly related. Video densitometry was performed using NIH Image software (Scion, Frederick, MD).
Modulation of NO
The levels of NO in cell cultures were modulated by a number of reagents. Exogenous NO was provided by addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (33). Inhibition of NO generation was accomplished by the addition of an analogue of L-arginine, N-monomethyl-L-arginine (L-NMMA) (34). Finally, specific inhibition of iNOS-dependent NO generation was accomplished using the specific iNOS inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL) (35).
Data analysis
Where indicated, data were expressed as the mean ± SEM. Intergroup differences were analyzed by ANOVA, with statistical significance assessed by Scheffe’s test; p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AM treated with LPS overnight consistently demonstrated a marked
decline in LTB4 synthesis from endogenous AA.
This suppression of 5-LO metabolism was dose dependent (Fig. 1
A). A dose of 1 µg/ml
suppressed LT synthesis by 81.3 ± 6.3% (n = 6;
p < 0.001) compared with that in untreated cells (Fig. 1B). A 1-h incubation with LPS at any dose failed to
suppress LTB4 synthesis. In fact, consistent with
literature reports, such short term LPS pretreatment increased AA
release (data not shown). A modest decrease in
LTB4 synthesis was also seen in PBM treated
overnight with LPS (69.4 ± 9.5% of untreated cells;
n = 4; p = 0.02), but this difference
reached statistical significance only when comparing relative (data
expressed as a percentage of the value in untreated cells), as shown
above, and not absolute levels of LTB4 (as shown
in Fig. 1B).
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LPS has no effect on AA release in macrophages
We next wished to specifically examine the effects of LPS
pretreatment on AA release. AM phospholipids were prelabeled overnight
with [3H]AA and then stimulated with A23187.
Although all the 5-LO products were diminished following LPS treatment
(Fig. 2), the synthesis of
[3H]COX products (predominantly thromboxane)
and of 12-HETE was unchanged. This argues against a reduction in AA
release being responsible for the decrease in LT synthesis. This was
confirmed in experiments conducted to conclusively measure deacylation
of AA by stimulating cells in the presence of 0.1% albumin. Release of
AA from macrophages was not affected by LPS treatment (untreated cells,
1.64 ± 0.27% of incorporated radioactivity; LPS-treated cells,
1.93 ± 0.61%; n = 3), indicating that this was
not a mechanism for limiting LT synthesis.
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The reduction in LT synthesis from exogenous AA, albeit less pronounced than that observed with endogenous AA, suggested an inhibitory effect of LPS on the activity of the 5-LO enzyme itself. We further tested this possibility by performing cell-free 5-LO assays on crude cellular lysates of AM following treatment with or without LPS. Treatment of AM with LPS indeed resulted in reduced 5-LO activity in crude cell lysates (33.5 ± 11.5% of cells untreated with LPS; n = 2). The extent of this suppression of 5-LO activity by LPS treatment was greater than that of LT synthesis by intact AM incubated with exogenous AA, and not as great as that seen with A23187 stimulation of intact AM. These observations indicate that in addition to possible effects on FLAP function, LPS probably has a direct inhibitory effect on the 5-LO enzyme itself.
No effect of LPS on 5-LO and FLAP expression
A reduction in 5-LO metabolism that is slow in onset could
theoretically be explained by reduced 5-LO and/or FLAP protein
expression. The data presented to date suggested impairment of both
these components of 5-LO metabolism in LPS-treated cells. However,
Western blot analysis did not demonstrate any reduction in 5-LO or FLAP
expression in AM upon overnight treatment with LPS (Fig. 3). Therefore, the mechanism of the LPS
effect is likely to involve impaired 5-LO and FLAP actions.
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We next investigated whether new protein synthesis was necessary for the suppression of LT synthesis in AM treated with LPS overnight. Treatment with the protein synthesis inhibitor cycloheximide overnight blocked the inhibitory effect of LPS on 5-LO metabolism (169.5 ± 10.31% of LPS-treated AM; n = 3; p = 0.003). These observations suggested that LPS treatment induced de novo synthesis of a protein that, in turn, was responsible for reduced 5-LO metabolism in AM.
COX metabolism plays only a modest role in inhibition of 5-LO metabolism by LPS
One plausible candidate for such a newly synthesized protein is COX-2, because LPS is known to induce COX-2 in AM (5, 6), and PGE2 is known to inhibit LT synthesis (36, 37). Although A23187-stimulated PGE2 synthesis was indeed increased after LPS treatment (696.7 ± 196.6 vs 259.3 ± 59.5 pg/ml; n = 3; p = 0.01), relatively less of an increase was observed in cells incubated with exogenous AA (1629 ± 59.3 vs 1296.0 ± 138.2 pg/ml; n = 3; p = 0.09). Thus, there was dissociation between LPS-induced enhancement of PGE2 synthesis and inhibition of 5-LO metabolism under the two experimental conditions. Furthermore, the COX inhibitor indomethacin failed to abrogate the LPS-induced inhibition of LT synthesis (LPS-treated cells, 24% of untreated cells; LPS plus indomethacin-treated cells, 26% of untreated cells), conclusively demonstrating that enhanced PG synthesis did not play a significant role in the suppression of 5-LO metabolism.
Suppression of 5-LO metabolism in LPS-treated AM involves NO generation
Because LPS also enhances NO production, we examined the effect of
this reactive nitrogen intermediate on 5-LO metabolism. When
L-NMMA, an analogue of L-arginine that
suppresses cellular NO synthesis, was coincubated overnight with
LPS-treated AM, it dose-dependently increased LT synthesis (Fig. 4). At L-NMMA doses
>10 µg/ml, LPS-induced suppression was completely overcome, and at
doses >100 µg/ml, LTB4 synthesis far exceeded
that in the cells untreated with LPS. The effect of treatment of
LPS-untreated AM with L-NMMA showed a trend
toward an increase in 5-LO metabolism, but this did not reach
statistical significance (Fig. 4).
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A). Furthermore, overnight
treatment of rat AM with SNAP alone in the absence of LPS was able to
significantly suppress 5-LO metabolism (Fig. 5A). Finally,
when purified 5-LO enzyme was incubated for 5 min with the NO donor,
SNAP, there was a decrease in cell-free enzymatic activity (Fig. 5B). These observations confirm a role for NO in the
suppression of 5-LO metabolism by LPS, and indicate that this
suppression involves at least in part a direct effect on 5-LO.
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A). Consistent with the fact
that suppression of 5-LO metabolism was much more modest in PBM than in
AM, PBM failed to demonstrate iNOS induction by LPS (Fig. 6A). Furthermore, in view of the controversy on the
elaboration of NO by human AM, we examined the effect of LPS overnight
on 5-LO metabolism and NO generation. LPS treatment of human AM for
16 h did not suppress LT synthesis and was not associated with
iNOS induction (data not shown). However, exogenous NO, in the form of
SNAP, suppressed human AM 5-LO product formation.
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B). Selective iNOS inhibitors had no effect on LT synthesis
in LPS-treated PBM (data not shown). These data suggest that iNOS was
an important source of NO, which resulted in reduced 5-LO metabolism in
LPS-treated AM. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS is present on the outer membrane of Gram-negative bacteria and plays an important role in inflammatory reactions that occur in response to these infections (1). It is well known that LPS activates macrophages to release proinflammatory mediators, including superoxide (38), cytokines (39), NO (17), and AA metabolites (40). Short term (2-h) treatment with LPS has been reported to prime PBM for increased AA release and enhanced LT synthesis following stimulation with FMLP (41). However, this priming effect waned after 2 h. Other investigators have demonstrated that i.v. LPS increased ex vivo synthesis of LTB4 by rat AM on day 1, but this returned to control levels on day 3 (42). LPS for 1–2 h in vitro primed human AM for A23187-stimulated LTB4 synthesis (8, 43).
Our data suggest that multiple mechanisms underlie the reduced 5-LO metabolism by LPS. As discussed above, the effect is not at the phospholipase A2 level, because there was no reduction in AA release itself or in the synthesis of eicosanoids derived from AA via the COX and 12-LO pathways with LPS treatment. LPS globally suppressed the formation of all 5-LO metabolites, but did not affect the expression of 5-LO or FLAP. Separate effects on the function of these two proteins appear to be involved. First, cell-free 5-LO activity was reduced. NO has been proposed to inactivate lipoxygenases by reducing the ferric enzyme to the ferrous form, which is inactive (33, 44). This also explains why LT synthesis is reduced in intact cells incubated with exogenous AA. An additional inhibition by LPS of FLAP activity was suggested by the observation that a reduction in LT synthesis was less marked when the cells were incubated under conditions that are relatively FLAP independent (exogenous AA) than when they were stimulated under conditions that are highly FLAP dependent (endogenous AA mobilized by A23187).
Induction of COX-2-dependent synthesis of PGE2 by LPS treatment represented one possible mechanism by which LT synthesis was suppressed in AM. However, a number of lines of evidence argue against such a mechanism. First, the degree of PGE2 augmentation by LPS was quite modest, consistent with previous observations in AM from the rat (45). Second, PG production was greater in LPS-treated AM incubated with exogenous AA, whereas the suppressive effect of LPS was more pronounced in A23187-treated cells. Finally, inhibition of PG production with indomethacin did not restore LT synthetic capacity in LPS-treated AM. The nature of the interaction of NO with COX has proven controversial. NO has been reported to both augment (21, 22) and suppress (23) COX activity in various systems. In our studies inhibition of NO induction tended to augment COX product synthesis to a minor degree.
There is less information available about the interactions of NO and 5-LO, and the information that is available is derived from complex experimental models. In platelet-neutrophil cocultures, exogenous nitroprusside had opposing effects on 5-LO-derived products, reducing LT synthesis and increasing lipoxin synthesis (46). Others have shown in a rabbit leukocyte-perfused heart model that treatment with L-arginine reduced cysteinyl-LT levels, which was associated with reduced coronary perfusion pressure (34). Our work extends the observation that endogenous NO inhibits lipoxygenase metabolism (47). Although the above studies suggest that NO may inhibit lipoxygenase metabolism, other investigators have noted that NO up-regulates the activity of lipoxygenases (48, 49), including 5-LO (50), in different models.
What are the clinical implications of our findings? Although human AM may not elaborate NO under normal conditions, there is evidence that NO is produced in AM in inflammatory states, e.g., acute lung injury (51), emphysema (52), and idiopathic pulmonary fibrosis (53). In addition, we have demonstrated that exogenous NO suppresses 5-LO metabolism in human AM. Furthermore, in the human lung other cell types, e.g., epithelial and endothelial cells, elaborate large amounts of NO, which, in turn, may suppress AM LT synthesis. The reduction in the synthesis of proinflammatory, smooth muscle constrictive, and edemagenic LTs may help to explain the salutary effects of inhaled NO in various models. These effects include relaxation of airway tone in asthmatics (54, 55) and of pulmonary vascular tone in pulmonary hypertension (56, 57), and inhibition of neutrophil migration (58) and endothelial permeability in models of acute lung injury (59). Therefore, exogenous treatment with NO may be a method of suppressing excessive 5-LO product formation that contributes to the pathogenesis of asthma (60), idiopathic pulmonary fibrosis (61), and acute lung injury (62). In contrast, prolonged exposure of macrophages to LPS is relevant in patients who survive the acute phase of sepsis secondary to Gram-negative bacteria (63). Our data predict that the macrophage’s capacity to elaborate LT would be compromised under these circumstances. This, in turn, might be expected to impair host defense mechanisms, because LT enhance the phagocytosis and killing of micro-organisms (14, 64, 65).
In summary, we have demonstrated for the first time that exposure of AM to LPS for a prolonged period suppresses LT synthesis in vitro via an NO-dependent mechanism. The induction of iNOS with associated NO generation results in reduced 5-LO and FLAP activity. This reduction in 5-LO metabolism in the setting of prolonged LPS exposure may represent an endogenous means to limit the inflammatory response, but may at the same time increase susceptibility to infection. iNOS inhibitors may have utility in restoring LT synthetic capacity and thereby enhancing host defense capabilities in subjects surviving sepsis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael J. Coffey, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: AA, arachidonic acid; AM, alveolar macrophage; COX, cyclo-oxygenase; EIA, enzyme-linked immunoassay; 5-HETE, 5-hydroxyeicosatetraenoic acid; iNOS, inducible NO synthase; 5-LO, 5-lipoxygenase; FLAP, 5-LO-activating protein; LT, leukotriene; L-NMMA, N-monomethyl-L-arginine; L-NIL, L-N6-(1-iminoethyl)-lysine; PBM, peripheral blood monocyte; SNAP, S-nitroso-N-acetylpenicillamine.
Received for publication January 21, 2000. Accepted for publication July 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 21:311.
, and lipopolysaccharide. J. Clin. Invest. 95:1669.
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, p = 0.05 vs treated cells with LPS;
, p = 0.05 vs no LPS. B,
Cell-free 5-LO assay was performed on recombinant 5-LO enzyme incubated
with various concentrations of SNAP. A representative experiment of a
total of two is displayed.