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* Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, and
Laboratório Inflamação e Imunidade, Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and Departments of
Internal Medicine and
Pathology, and
¶ Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112
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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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(PPAR) (4). Other
oxidized phospholipids are generated by the oxidative attack on LDL
with less defined mechanisms of action (5, 6). Formation
of all of these types of biologically active phospholipid oxidation
products results from a series of radical catalyzed chemical reactions,
and so is unregulated. Oxidized LDL and phospholipids isolated from oxidized LDL particle induce neutrophil adhesion and smooth muscle cell proliferation in vitro through activation of the PAF receptor (1). These also stimulate the PAF receptor in vivo, and much of this effect results from C4 analogs of PAF derived from the oxidation of arachidonoyl-containing phosphatidylcholines (2). PAF is an inflammatory phospholipid in which the sn-1 ether bond, the short sn-2 acetyl residue, and the phosphocholine head group are recognized by a single G protein-coupled PAF receptor at picomolar levels (7). Oxidatively generated PAF analogs differ from PAF by the presence of longer and sometimes derivatized residues at the sn-2 position of the glycerol backbone and, although less potent than PAF, are considerably more abundant than PAF after oxidation of polyunsaturated phosphatidylcholines.
Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine whose role in the pathogenesis of inflammatory diseases is extensive (8). By example, MCP-1 has a key role in conditions ranging from acute respiratory distress syndrome to rheumatoid arthritis (9). MCP-1 is detected in the bronchoalveolar lavage fluid of allergic asthmatic patients (10) and in bronchoalveolar lavage fluid and plasma of patients with interstitial lung diseases (11), suggesting that it may also play a fundamental role in development of allergic responses as well (12). MCP-1 is essential for monocyte recruitment in in vivo models of inflammation, and impaired monocyte accumulation has been noted in MCP-1-deficient mice stimulated by thioglycolate (13).
Oxidized LDL particles stimulate MCP-1 synthesis by vascular cells (14, 15), and MCP-1 is expressed in inflamed macrophage-rich areas of atherosclerotic lesions (16). Extravasated macrophages in these lesions express MCP-1 receptors (17), and its overexpression increases atherogenesis in susceptible mice (18). Conversely, mice doubly deficient for the MCP-1 receptor (CCR2) and apolipoprotein E on a Western diet show decreased lesion formation as compared with atherosclerosis-susceptible apolipoprotein E-deficient mice (19). Activation of the PAF receptor stimulates MCP-1 synthesis by monocytes in vitro, but only when they are tethered to P-selectin and receive appropriate adhesion-dependent cosignals (20). Whether oxidized phospholipids act on PAF receptor-bearing cells and expand the acute inflammatory response through cytokine synthesis in in vivo oxidative, inflammatory conditions is not known.
We investigated the mechanisms of the inflammatory effects of oxidatively fragmented phospholipids extracted from oxidized LDL on leukocyte accumulation in an animal pleurisy model. We characterized the profile of chemokine expression and assessed the involvement of MCP-1 on leukocyte influx and lipid body formation in MCP-1-deficient mice. The results showed that MCP-1 is an early response that amplifies inflammation through the production of another lipid mediator, leukotriene B4 (LTB4). These results also show that neutrophils, which are not a primary target of MCP-1 in vitro, primarily are recruited into the inflammatory environment through the induction of 5-lipoxygenases and LTB4 by MCP-1. In vivo, MCP-1 is a pleiotrophic regulator with direct and secondary effects on the development of an inflammatory response even when cells possess receptors for PAF.
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Materials and Methods |
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[
-32P]UTP was purchased from NEN Life
Sciences (Boston, MA); mck-5 in vitro hybridization
kit and RNase protection kit were from BD PharMingen (San Diego,
CA); anti-5-lipoxygenase rabbit IgG was from Cayman Chemicals (Ann
Arbor, MI); anti-rabbit biotinylated IgG was from Vector
Laboratories (Burlingame, CA); aqua Polymount was from Polyscience
(Warrington, PA); BN 52021 was from Biomol (Plymouth Meeting, PA);
C4-PAF,
1-O-hexadecyl-2-butanoyl-sn-glycero-3-phosphocholine
was from Avanti Polar Lipids (Alabaster, AL); aminopropyl columns were
from J. T. Baker (Phillipsburg, NJ); PAF,
1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine
was from Sigma-Aldrich (St. Louis, MO); Pefabloc was from Pentapharm
Laboratories (Basel, Switzerland); LTB4 was
detected by immunoenzymatic assay with a kit from Cayman Chemicals,
while the kit for MCP-1 detection was from R&D Systems (Minneapolis,
MN); Ultraspec RNA isolation system was from Biotecx Laboratories
(Houston, TX); zileuton was obtained from Abbott Laboratories (Abbott
Park, IL). Swiss mice were maintained by the Oswaldo Cruz Foundation
breeding facility. MCP-1-/- mice and their
backcrossed have been previously described (13) and were
kindly provided by C. Gerard (Pelmutter Laboratory, Children’s
Hospital, Harvard Medical School, Boston, MA).
Purification and oxidation of human LDL
Human LDL was isolated, as described elsewhere (1). Briefly, LDL was purified from plasma of healthy volunteers after adjusting the density to 1.3 with potassium bromide. A gradient was formed from this plasma and saline overlaying it by centrifugation for 200 min at 150,000 x g. After centrifugation, the band of lipoproteins with densities between 1.019 and 1.062 was collected and dialyzed against PBS. The isolated LDL (200 µg protein/ml) was treated with a PAF acetylhydrolase inhibitor (200 µM Pefabloc) before oxidation with CuSO4 (10 µM) for 18 h at 37°C. Control LDL particles were those not subjected to oxidation.
Separation of lipids
The lipids were extracted from LDL by the method of Bligh and Dyer (21). Neutral lipids, fatty acids, and phospholipids were separated by aminopropyl chromatography using chloroform-isopropanol (2:1 v/v); acetic acid (2%) in diethyl ether; and methanol, respectively (22). The phospholipid fraction was further separated on a reversed-phase HPLC with a mobile phase of methanol (84%), acetonitrile (15%), and deionized water containing 1 mM ammonium acetate (1%), as previously described (2). The fractions were dried under a stream of N2, reconstituted with chloroform:methanol (2:1) containing BHT (10 µM), and stored at -20°C. Bioactive fractions and control fractions were suspended in HBSS/BSA (HBSS containing 0.01% BSA) and sonicated before use.
Effect of oxidized phospholipids on calcium mobilization in human neutrophils
Blood from healthy volunteers was drawn into a syringe containing sodium citrate (to yield a final concentration of 3.2%), and then layered over Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and centrifuged (30 min at 500 x g) as in the neutrophil purification method described by Zimmerman et al. (23). Neutrophil pellets were suspended in 2 ml 0.2% NaCl for hemolysis, followed by the addition of 2 ml 1.6% NaCl before centrifugation (5 min at 400 x g). The neutrophils were washed with HBSS (calcium and magnesium free) and counted, and their number was adjusted to 5.5 x 106/ml before incubation with fura 2 (2 µM) for 45 min at 37°C in the dark. Ca2+ transients were determined in the fura 2-loaded neutrophils, as before (2).
Pleurisy model
Swiss mice (20–25 g) from Oswaldo Cruz Foundation breeding unit were kept at constant temperature (25°C) with free access to diet and water in a room with a 12-h light/dark cycle. The experiments in this study received prior approval from the Oswaldo Cruz Institute’s animal welfare committee. Mice received an intrathoracic (i.t.) injection of pooled HPLC fractions 6–8 containing material derived from oxidized LDL, which activated a Ca2+ flux in polymorphonuclear cells, or with the surrounding inactive fractions (fractions 2–4, 10, and 11) from the same chromatographic separation of oxidized LDL. We also used the corresponding fractions 6–8 from the chromatographic separation of polar lipids extracted from unoxidized LDL as a control. The stored HPLC fractions were dried under nitrogen and resuspended in sterile HBSS containing 0.01% BSA just before use. The HBSS/BSA solution alone was administered i.t. into naive animals as a further control. Synthetic C4-PAF when tested as an agonist was reconstituted in HBSS/BSA and injected i.t. at 1 µg per cavity. After 3, 6, 12, and 24 h, the injected animals were sacrificed in a CO2 gas chamber, and the thoracic cavity was opened and washed with 1 ml HBSS. These pleural washes were recovered, and their volume was measured. When MCP-1-deficient mice or their backcrossed controls (13) were used, this analysis was performed at the fixed time of 6 h after stimulation.
Pleural wash samples were diluted in Turk fluid (2% acetic acid) for total leukocyte counts using Neubauer chambers. Differential analysis was performed on cytosmears treated with May Grunwald-Giemsa stain. Lipid body formation in leukocytes was evaluated on cytosmears stained with osmium (24). Briefly, cells on cytosmears were fixed with 3% formalin and stained with cacodilic acid and 1.5% osmium. After 30 min, the cytosmears were washed with water and incubated with 1% thiocarbohydrazide for 3 min, and then with cacodilic acid and osmium for 5 min. Intracellular lipid body content in these cells was then evaluated by counting them in 50 cells magnified under an optical microscope. Pleural wash fluid samples were centrifuged to pellet the cells, and the supernatant from this procedure was stored at -70°C for ELISA determinations. The pelleted cells were resuspended in Ultraspec RNA isolation reagent (Biotecx Laboratories).
Immunocytochemistry
Cytosmear preparations from pleural washes were fixed in 3% formaldehyde, permeabilized with a solution of 0.05% saponin in HBSS, treated with a biotin blocking solution (Vector Laboratories), and then blocked with 10% normal goat serum. After washing, these cytospin preparations were incubated overnight at 4°C with the primary rabbit polyclonal anti-mouse 5-lipoxygenase Ab (diluted 1/150 in 0.05% saponin/HBSS). Preimmune rabbit serum was used as control. After three washes of 5 min in 0.05% saponin/HBSS, the preparations were incubated with biotin-conjugated goat anti-rabbit IgG. The immunoreactive 5-lipoxygenase in cells was then identified by ABC Vectastain glucose-oxidase kit (Vector Laboratories) following the manufacturer’s instructions. The resulting glucose-oxidase immunostaining was visualized under light microscopy (25).
In vivo treatment with receptor antagonists and inhibitors
Swiss mice were treated with an i.p. dose (20 mg/kg) of the PAF antagonist BN 52021 30 min in advance of the i.t. injection of oxidized phospholipids. When the animals were to be treated with zileuton (50 µg per cavity), this 5-lipoxygenase inhibitor was injected i.t. immediately before the injection of oxidized phospholipids or C4-PAF. BN 52021 was dissolved in saline, while zileuton was solubilized in a DMSO stock solution such that the final DMSO concentration in the injectate was 0.01%.
In vitro lipid body formation in murine peritoneal macrophages
The peritoneal cavities of untreated wild-type and MCP-1-deficient mice were washed with PBS to recover resident macrophages. The cell concentration in these washings was adjusted to 106 cells/ml before these suspended cells were stimulated with PAF (1 µM), oxidized phospholipids (HPLC fractions 6–8, or 2, 10, and 11), C4-PAF (1 µM), or vehicle (HBSS/BSA 0.1%) at 37°C. After 1 h, the cells were cytocentrifuged onto glass slides (105 per slide) and stained with osmium, and the number of lipid bodies in 50 consecutively scanned macrophages was determined as above.
RNase protection assay
RNA was purified according to the Ultraspec RNA protocol (Biotecx Laboratories), and the RNase protection assays were performed according to the Riboquant protocol (BD PharMingen). In brief, leukocytes derived from the pleural cavities from each experimental group were pooled, and their total RNA was isolated. RNA (10 µg) from each pool was hybridized with the mck-5 RNase protection probe (BD PharMingen) using a previously synthesized radioactive probe. Hybridized RNA was treated with RNase, isolated and loaded on a denaturing polyacrylamide gel, and developed at 50 W. The resulting gel was adsorbed onto a filter paper, dried, and exposed to film for quantification by autoradiography (26).
ELISA for chemokines and LTB4
LTB4 was detected by immunoenzymatic assay, according to the manufacturer’s instruction (Cayman Chemicals). The chemokine MCP-1 was quantitated in cellular supernatants by sandwich ELISA according to the MCP-1 detection kit’s (R&D Systems) accompanying protocol.
Statistical analysis
The data are represented as mean ± SEM and were statistically analyzed by means of ANOVA, followed by a Newman-Keuls-Student test with significance level set at p < 0.05.
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Results |
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We previously demonstrated that phospholipids extracted from
oxidized LDL could be fractioned by reversed-phase HPLC to obtain
bioactive fractions, eluting at min 6–8, with proinflammatory activity
both in vitro and in vivo (2). In the present study, we
confirmed that the current fractions of oxidized phospholipids induced
a transient rise in the concentration of free intracellular calcium in
neutrophils (data not shown). This biochemical change indicated that
the neutrophils had been activated, so we next performed an experiment
in vivo to characterize the nature of the inflammatory response to
these oxidatively generated agonists. We found that injection of these
fractions into the pleural cavity of mice induced the formation of an
exudate with a distinct increase in cellularity compared with animals
receiving nonactive fractions (Fig. 1
A). Analysis of the cellular
composition of the cells recovered from animals injected with
biologically inactive fractions, in this case the HPLC fractions
surrounding those that contained PAF-like activity, showed that the
recovered cells were mainly resident macrophages (Fig. 1B).
In contrast, the exudate from animals receiving the bioactive fractions
isolated from oxidized LDL revealed a significant enrichment in the
neutrophil and eosinophil populations, as illustrated in Fig. 1C.
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D). This influx was accompanied by
mononuclear cell infiltration 6 h after injection (0.83 ±
0.21 x 106 to 4.1 ± 1.1 x
106) and by eosinophil accumulation from 6 to
24 h postinjection (Fig. 1E). Vehicle-injected animals
did not show a significant influx of either of these types of
leukocytes, nor was there an inflammatory response when the animals
were injected with inactive fractions surrounding those that contained
this PAF-like activity. We analyzed the effect of the bioactive
fractions on the accumulation of T cell subtypes by flow cytometry to
find that the bioactive fractions did not increase the number of
CD3+, CD4+,
CD8+, or 
+ T
lymphocytes in pleural cavity of mice at 24 h poststimulus (data
not shown). PAF antagonists block the effects of oxidized phospholipids on leukocytes: potential involvement of C4-PAF
The polar phospholipids generated by LDL oxidation include
PAF-like lipids (1), PPAR agonists (4),
lysophosphatidic acid (27), as well as other products
(5, 6). We tested whether the inflammatory actions that we
had observed arose from oxidized phospholipids acting on the PAF
receptor on target leukocytes. Fig. 2
shows that pretreatment of the animals with the PAF receptor antagonist
BN 52021 inhibited the influx of both neutrophils (Fig. 2A)
and eosinophils (Fig. 2B) into the thoracic cavity at 6
h after injection of the biologically active fractions derived from
oxidized LDL. Previous studies using mass spectrometry identified
C4-PAF as a major component of the bioactive
fractions responsible for the PAF-like activity of oxidized LDL
(2). We found that synthetic C4-PAF
induced an increase in the number of leukocytes, both neutrophils (Fig. 2C) and eosinophils (Fig. 2D), recovered from the
pleural cavity of mice. The time relationship of this influx
recapitulated that induced by the lipids extracted from oxidized LDL
(compare Figs. 1 and 2).
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An enhanced burden of intracellular droplets, lipid bodies, is
characteristic of activated inflammatory cells (28) and
can mark an in vivo inflammatory environment. Cells isolated from the
pleural cavity of animals injected with HPLC fractions from unoxidized
LDL show few intracytoplasmic lipid inclusions (Fig. 3A), while cells isolated from
animals receiving PAF-like lipids isolated from oxidized LDL visibly
contained more lipid bodies (Fig. 3B). Quantitation of these
inclusions in animals injected with active fractions derived from
oxidized LDL showed a statistically significant increase in both the
number of lipid bodies per cell (Fig. 3C) and a marked
change in the distribution of these among cells (Fig. 3D)
when compared with the number and distribution of these structures in
animals injected with equivalent fractions recovered from the
chromatography of unoxidized LDL or those injected simply with
vehicle.
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Lipid bodies serve as sites for localization of esterified
arachidonate and eicosanoid-forming enzymes (25, 29), and
certain leukotrienes mediate some of the effects of PAF in vivo
(30, 31, 32). We asked whether the cellular response to
oxidized phospholipids required these eicosanoids. We first stained
cells for 5-lipoxygenase, which catalyzes the committed step in
leukotriene synthesis, to find that there was only a modest expression
of this enzyme in cells isolated from animals injected with the
inactive HPLC fractions that surround the elution position of PAF-like
lipids (Fig. 4, compare A with
B). In sharp contrast, cells isolated from animals injected
with either HPLC fractions containing the PAF-like activity derived
from oxidized LDL (Fig. 4C) or synthetic
C4-PAF (Fig. 4D) showed a distinct
increase in this rate-limiting enzyme. Interestingly, a large portion
of this immunostaining was punctate, potentially indicating that this
5-lipoxygenase was localized in lipid bodies, as has been observed
under other conditions (25).
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). We
investigated the role of 5-lipoxygenase metabolites on the pleurisy
induced by oxidized phospholipids or C4-PAF by
treating the mice with the 5-lipoxygenase inhibitor zileuton
immediately before stimulation. Injection of zileuton by itself did not
induce any change in the leukocyte count in the pleural cavity of
vehicle-injected animals (data not shown). However, treatment with
zileuton did significantly inhibit the accumulation of neutrophils in
response to the oxidized phospholipids recovered from oxidized LDL
(Fig. 5). This effect was specific for neutrophils because there was no
difference in the number of recruited macrophages or eosinophils
between control and zileuton-treated animals (data not shown). These
studies show that a product of the 5-lipoxygenase, potentially
LTB4, is required for the leukocyte response to
the newly generated polar phospholipids of oxidized LDL.
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Our finding that leukotriene synthesis was stimulated by oxidized
phospholipids suggested that the production and secretion of other
inflammatory mediators might also be increased, and so we assessed the
level of expression of mRNAs for chemokines by RNase protection. We
analyzed mRNA from the total leukocyte population present in the
pleural cavity 6 h after injection of the PAF-like lipids purified
from oxidized LDL and observed that there was increased expression of
mRNA for RANTES, macrophage-inflammatory protein (MIP)-1
,
MIP-1, MIP-2, IFN--inducible protein-10, and MCP-1 when compared
with the levels of expression of these cytokines by cells isolated from
animal receiving adjacent inactive HPLC fractions (Fig. 6). This pattern of enhanced cytokine
message expression was recapitulated by cells extracted from animals
receiving C4-PAF (Fig. 6).
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). Oxidized phospholipids induced inflammatory response is reduced in MCP-1-deficient mice
MCP-1 is implicated in inflammatory diseases (9, 16),
and, as described above, MCP-1 mRNA expression and protein production
were increased in mice injected with the inflammatory phospholipids
isolated from oxidized LDL. We investigated the involvement of MCP-1 in
the inflammation triggered by oxidized phospholipids using mice that
had been genetically engineered to be deficient in MCP-1
(MCP-1-/-). PAF-like lipids purified from
oxidized LDL, but not the surrounding inactive fractions (data not
shown), induced mononuclear cell (data not shown), neutrophil, and
eosinophil accumulation in thoracic cavity 6 h after stimulation
of wild-type animals (Fig. 7). In
contrast, in strain-matched MCP-1-/- mice there
was a marked deficit in the number of neutrophils (Fig. 7A)
and eosinophils (Fig. 7B) recruited into the pleural cavity
in these mice in response to the inflammatory phospholipids of
oxidized LDL.
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A). MCP-1 acted on the
exudate cells to promote lipid body accumulation as shown in
experiments with cultured peritoneal macrophages from wild-type and
MCP-1-/- animals. In vitro exposure to active
oxidized phospholipids failed to induce lipid body formation in cells
from MCP-1-/- mice under conditions in which
significant stimulation was observed in cells from wild-type mice (Fig. 8B). Thus, MCP-1 is required for a complete inflammatory
response to oxidized phospholipids.
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C). The lack of cellular response to oxidized phospholipids
in MCP-1-/- mice also was reflected in the
profile of mRNA expression. Although cells from wild-type mice had
similar profiles of mRNA expression compared with Swiss mice
leukocytes, cytokine mRNA expression in leukocytes from
MCP-1-/- mice differed dramatically. As shown
in Fig. 9, mRNA expression was
significantly decreased for all the chemokines probed, especially
RANTES, in which the signal practically disappeared in
MCP-1-/- mice. Taken together, these data
demonstrate that MCP-1 has a central, early role in the pleural
inflammation induced by oxidized, PAF-like phospholipids.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oxidation of LDL particles generates phospholipids with PAF-like biologic activity (1, 2) that can induce inflammation in a rat model of pleurisy (2). We sought to define the molecular mechanisms responsible for this response and so established a similar model in mice. The features of the murine model were similar to that of rats, in which there was a rapid influx of neutrophils that was maximal by 3 h and had resolved by 24 h. The influx of eosinophils was slower, but then this increased cellularity remained elevated for up to 24 h after exposure to the inciting oxidized phospholipids. This response was expected because PAF, as a biosynthetic product of activated inflammatory cells, is an important component of the inflammatory response in mice (33) that elicits an inflammatory exudate and edema (7).
In addition to neutrophil accumulation, we find that the PAF-like lipids from oxidized LDL as well as C4-PAF induced monocyte and eosinophil accumulation in the thoracic cavity of mice. PAF is a potent and direct chemoattractant for eosinophils in vitro (34), and PAF induces eosinophil accumulation in the pleural cavity of rats, an effect secondary to the generation of a protein with specific eosinophil chemotactic activity (35). These observations support the conclusion that bioactive fractions of oxidized LDL phospholipids, C4-PAF, and PAF all trigger inflammatory response through the PAF receptor.
The oxidation of LDL generates a number of phospholipid oxidation
products that are still phospholipids. These include the PAF-like lipid
oxidation products in which the sn-2 residue of the minor
class of alkyl phosphatidylcholines is fragmented to a point in which
the highly selective PAF receptor recognizes and responds to these
products of a radical chemical reaction. Oxidation of this class of
phospholipids also generates products with slightly longer residues
with an 
-carboxy function that actives gene transcription through
PPAR (4). Oxidatively fragmented phospholipids
themselves are precursors for the vasoactive lipid
lysophosphatidylcholine because they are substrates for the
LDL-associated enzyme PAF acetylhydrolase (7). Other
biologically active phospholipids generated during the oxidation of LDL
include lysophosphatidic acid (3), isoprostane-containing
phosphatidylcholine (6), and fragmented diacyl
phosphatidylcholines (5). In this study, we find that a
synthetic PAF-like lipid induced all of the in vivo effects of the
inflammatory phospholipids isolated from LDL, leading us to conclude
that these PAF-like lipids underlie much of the inflammatory activity
of oxidatively modified LDL particles.
We analyzed the pattern of chemokine mRNA expression in the pleurisy
triggered by oxidized phospholipids and C4-PAF to
better understand the mechanisms implicated in the inflammatory
reaction to oxidized phospholipids. Both stimuli induced an increase in
mRNA for RANTES, MIP-1, MIP-1, MIP-2, IFN--inducible
protein-10, and MCP-1 in cells obtained from pleural cavity 6 h
poststimulation. Among those chemokines, RANTES, MIP-1, and MIP-1
have been implicated in the formation of atherosclerotic lesions
(36). The role of the C-C chemokine MCP-1 in the
pathophysiology of atherosclerosis, now recognized as a chronic
inflammatory condition, has been demonstrated in humans
(16) and animal models of this disease (18, 37, 38, 39, 40). MCP-1 participates in the development of allergic
inflammation (12), and is positioned for a similar role in
pleurisy.
In the latter model, the anti-MCP-1 Ab reduced peritoneal LTB4 levels and an LTB4 antagonist reduced both monocyte and neutrophil influx.
We find a marked dependence of monocytes, eosinophils, and neutrophils trafficking on the ability to increase MCP-1 levels in a closed compartment in response to a relevant inflammatory insult. The CCR2 receptor for MCP-1 normally is not expressed by neutrophils (41), and disruption of its gene does not block neutrophil influx into the peritoneum (42), so it is not readily apparent that loss of MCP-1 would impact neutrophil migration into the pleural cavity. However, anti-MCP-1 Abs reduced the neutrophilic influx into the lungs of mice infected with Cryptococcus neoformans (43) and briefly attenuated neutrophil influx in the more complex model of cecal ligation and puncture (44). We do not believe that this is a primary reaction to the disruption of the MCP-1 gene, but rather that the neutrophil accumulation induced by inflammatory oxidized phospholipids is secondary to the generation of 5-lipoxygenase products, most likely LTB4. We found enhanced levels of LTB4 in the pleural exudate, similar to the increased levels of LTB4 in a model of peritonitis in which MCP-1 induction of this mediator has a major role in leukocyte recruitment (44). LTB4 is a potent chemoattractant for neutrophils (45), and disruption of the gene for 5-lipoxygenase interferes with PAF-induced neutrophil recruitment (46). Inhibition of 5-lipoxygenase activity with zileuton shows that its products are essential for the recruitment of neutrophils, and not eosinophils or monocytes (data not shown), into the pleural cavity after introduction of PAF-like oxidized phospholipids.
We detected increased levels of LTB4 in the pleural fluid of animals injected with the active fractions isolated from oxidized LDL, and increased 5-lipoxygenase expression in the accompanying cells. MCP-1 stimulates monocyte LTB4 synthesis (44), suggesting that these cells were the source of this agonist. However, the influx of neutrophils markedly preceded the increase in infiltrating monocytes, a result that indicates that the resident macrophage population, which does not require MCP-1 for its establishment (13), is a more probable source of this eicosanoid.
PAF-like lipids isolated from oxidized LDL or pure C4-PAF induced lipid body accumulation in leukocytes recovered from the pleural cavity. Lipid body formation in response to PAF activation of the PAF receptor and a downstream G protein has been previously established (25). Lipid bodies are membraneless cytoplasmic inclusions whose numbers are increased in endothelial cells, eosinophils, neutrophils, and macrophages upon appropriate activation. In this study, we use this property to mark the development of an inflammatory milieu.
Lipid bodies may have a functional role in this model, as they localize esterified arachidonate and eicosanoid-forming enzymes to facilitate the production of eicosanoids by activated cells (24). The presence of increased numbers of lipid bodies in leukocytes stimulated by the oxidized phospholipids correlated with the increase in LTB4 levels found in the pleural exudate of stimulated animals, and we find that incubation of pleural leukocytes with oxidized phospholipids in vitro primes those cells for enhanced calcium ionophore-induced LTB4 production (E. F. de Assis et al., in preparation). We note that most of the 5-lipoxygenase detected by immunohistochemistry displays a granular cytoplasmic localization in cells isolated from animals injected with phospholipids isolated from oxidized LDL, as previously detected by Bozza et al. (24), in response to PAF, and so may be positioned to act in this way. Recently, a direct proof of the involvement of lipid bodies as sites of leukotriene production was provided by the demonstration of intracellular immunofluorescent localization of newly formed LTC4 within lipid bodies in chemokine-stimulated human eosinophils (47).
Pleurisy offers a quantitative way to examine the early events of acute inflammation. This model provides an easy way to detect cell influx and the release of chemical mediators in the inflammatory milieu. In this study, we demonstrated that PAF-like lipid oxidation products induce a rapid influx of neutrophils that progresses to a late phase marked by monocyte and eosinophil influx. Accumulation of each of these cells is characteristic of pleurisy evoked by distinct types of insults (48). Carrageenan-induced pleurisy, for example, creates an oxidative stress in which the neutrophilic infiltration and damage are attenuated by antioxidant treatment (49). PAF biologic activity is recoverable from pleural washes from patients with select etiologies that correlate with neutrophil and eosinophil numbers (50). There may be an oxidative component to pleurisy in which the events we define in this work come into play, but it is the quantitative aspects of the model that are particularly relevant. Oxidized LDL (51), oxidatively fragmented phospholipids (5), MCP-1 (16), and monocytic cells with MCP-1 receptors (17) are all found in atherosclerotic lesions, but individually assessing the role of each early in this prolonged inflammatory event is difficult. A quantifiable model of acute inflammation has connected oxidatively modified phospholipids with PAF-like activity derived from oxidized LDL to the accumulation of several classes of white blood cells through enhanced synthesis of MCP-1. The sequence of events following the stimulus by oxidized phospholipids and the generation of MCP-1 and other chemokines may be of fundamental importance in the early stages of atherosclerosis and other inflammatory disorders.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hugo C. Castro-Faria-Neto, Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, CEP 21045-900, Brazil. E-mail address: hcastro{at}gene.dbbm.fiocruz.br
3 Abbreviations used in this paper: LDL, low-density lipoprotein; i.t., intrathoracic; LTB4, leukotriene B4; MCP-1, monocyte chemoattractant protein 1; MIP, macrophage-inflammatory protein; PAF, platelet-activating factor; PPAR, peroxisome proliferator-activated receptor-.
Received for publication November 5, 2001. Accepted for publication February 11, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 C. M. Maya-Monteiro, P. E. Almeida, H. D'Avila, A. S. Martins, A. P. Rezende, H. Castro-Faria-Neto, and P. T. Bozza Leptin Induces Macrophage Lipid Body Formation by a Phosphatidylinositol 3-Kinase- and Mammalian Target of Rapamycin-dependent Mechanism J. Biol. Chem., January 25, 2008; 283(4): 2203 - 2210. [Abstract] [Full Text] [PDF]
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P. Pacheco, A. Vieira-de-Abreu, R. N. Gomes, G. Barbosa-Lima, L. B. Wermelinger, C. M. Maya-Monteiro, A. R. Silva, M. T. Bozza, H. C. Castro-Faria-Neto, C. Bandeira-Melo, et al. Monocyte Chemoattractant Protein-1/CC Chemokine Ligand 2 Controls Microtubule-Driven Biogenesis and Leukotriene B4-Synthesizing Function of Macrophage Lipid Bodies Elicited by Innate Immune Response J. Immunol., December 15, 2007; 179(12): 8500 - 8508. [Abstract] [Full Text] [PDF]
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 A. M. Meyer, L. D. Dwyer-Nield, G. Hurteau, R. L. Keith, Y. Ouyang, B. M. Freed, L. R. Kisley, M. W. Geraci, J. V. Bonventre, R. A. Nemenoff, et al. Attenuation of the pulmonary inflammatory response following butylated hydroxytoluene treatment of cytosolic phospholipase A2 null mice Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1260 - L1266. [Abstract] [Full Text] [PDF]
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H. D'Avila, R. C. N. Melo, G. G. Parreira, E. Werneck-Barroso, H. C. Castro-Faria-Neto, and P. T. Bozza Mycobacterium bovis Bacillus Calmette-Guerin Induces TLR2-Mediated Formation of Lipid Bodies: Intracellular Domains for Eicosanoid Synthesis In Vivo. J. Immunol., March 1, 2006; 176(5): 3087 - 3097. [Abstract] [Full Text] [PDF]
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 I. Sethy-Coraci, L. W. Crock, and S. C. Silverstein PAF-receptor antagonists, lovastatin, and the PTK inhibitor genistein inhibit H2O2 secretion by macrophages cultured on oxidized-LDL matrices J. Leukoc. Biol., November 1, 2005; 78(5): 1166 - 1174. [Abstract] [Full Text] [PDF]
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 A. Vieira-de-Abreu, E. F. Assis, G. S. Gomes, H. C. Castro-Faria-Neto, P. F. Weller, C. Bandeira-Melo, and P. T. Bozza Allergic Challenge-Elicited Lipid Bodies Compartmentalize In Vivo Leukotriene C4 Synthesis within Eosinophils Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 254 - 261. [Abstract] [Full Text] [PDF]
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C. Penido, A. Vieira-de-Abreu, M. T. Bozza, H. C. Castro-Faria-Neto, and P. T. Bozza Role of Monocyte Chemotactic Protein-1/CC Chemokine Ligand 2 on {gamma}{delta} T Lymphocyte Trafficking during Inflammation Induced by Lipopolysaccharide or Mycobacterium bovis Bacille Calmette-Guerin J. Immunol., December 15, 2003; 171(12): 6788 - 6794. [Abstract] [Full Text] [PDF]
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E. F. de Assis, A. R. Silva, L. F. C. Caiado, G. K. Marathe, G. A. Zimmerman, S. M. Prescott, T. M. McIntyre, P. T. Bozza, and H. C. de Castro-Faria-Neto Synergism Between Platelet-Activating Factor-Like Phospholipids and Peroxisome Proliferator-Activated Receptor {gamma} Agonists Generated During Low Density Lipoprotein Oxidation That Induces Lipid Body Formation in Leukocytes J. Immunol., August 15, 2003; 171(4): 2090 - 2098. [Abstract] [Full Text] [PDF]
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G. K. Marathe, G. A. Zimmerman, and T. M. McIntyre Platelet-activating Factor Acetylhydrolase, and Not Paraoxonase-1, Is the Oxidized Phospholipid Hydrolase of High Density Lipoprotein Particles J. Biol. Chem., January 31, 2003; 278(6): 3937 - 3947. [Abstract] [Full Text] [PDF]
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P. Pacheco, F. A. Bozza, R. N. Gomes, M. Bozza, P. F. Weller, H. C. Castro-Faria-Neto, and P. T. Bozza Lipopolysaccharide-Induced Leukocyte Lipid Body Formation In Vivo: Innate Immunity Elicited Intracellular Loci Involved in Eicosanoid Metabolism J. Immunol., December 1, 2002; 169(11): 6498 - 6506. [Abstract] [Full Text] [PDF]
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), bioactive (fractions 6–8) (
), or inactive
(fractions 2, 3, and 10) (
) HPLC fractions of oxidized
phospholipids. The cell analysis of the exudate revealed a significant
cell accumulation (A) with enrichment in the neutrophil
and eosinophil populations, as illustrated in C, whereas
B shows only mononuclear cells obtained from inactive
oxidized phospholipids-injected group. Neutrophil (D)
and eosinophil (E) counts were performed in samples
obtained from a cytospin of the pleural wash. Each point is the mean,
with SEM represented by vertical lines, from at least eight animals.
*, Statistically significant differences as compared with animals
injected with vehicle.
