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* Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil;
Centro de Terapia Intensiva, Hospital Universitário Clementino Fraga Filho, and
Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and
Harvard Thorndike Laboratories and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, was also shown to colocalize within
lipid bodies. Prior stimulation of leukocytes to form lipid bodies
enhanced the capacity of leukocytes to produce leukotriene
B4 and PGE2. In conclusion, our studies
indicate that lipid bodies formed after LPS stimulation and sepsis are
sites for eicosanoid-forming enzymes and cytokine localization and may
develop and function as structurally distinct, intracellular sites for
paracrine eicosanoid synthesis during inflammatory
conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LTs and PGs are enzymatically formed oxidative derivatives of arachidonic acid (AA) that have a wide range of biological activities, including roles as paracrine mediators of inflammation as well as intracellular signals (6). Arachidonate is released from arachidonyl phospholipids, which may reside in various membranes within cells, by the actions of phospholipases (7). Although the enzymatic pathways for eicosanoid formation are well understood, the intracellular sites of action of these enzymes and the cellular sources of AA during inflammation remain less clear. Different studies have focused on the intracellular localization of eicosanoid-forming enzymes. Cyclooxygenases (COXs) are associated with cellular membranes, including the endoplasmic reticulum and nuclear membrane (8, 9). In contrast, 5- lipoxygenase (5-LO) localization is cell type specific and also varies according to the activation state of the cell. 5-LO was shown to localize within the nuclear environment of alveolar macrophages but was predominantly cytosolic in human neutrophils and resting peritoneal macrophages (10, 11, 12). Although translocation from cytosol to membrane may facilitate interactions of cytosol enzymes with membrane-bound arachidonate, there is increasing evidence that specific compartmentalization of eicosanoid formation within cells may relate to the different paracrine and intracrine functions of eicosanoids (9, 13). In addition to membranes, another lipid-bearing domain in cells contains lipid bodies.
Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that are candidates to play a major role in the formation of eicosanoid mediators during inflammation. Lipid bodies have been recognized to be abundant in cells engaged in inflammatory and degenerative processes (14, 15). For instance, increased lipid body numbers have been noted in eosinophils from patients with hypereosinophilic syndrome (16, 17), in leukocytes from inflammatory arthritis (18, 19), and from bronchoalveolar lavage of patients with acute respiratory distress syndrome (20). In addition to being sites of storage of neutral lipid and phospholipids, it has been recently shown that lipid bodies are also sites of localization of several enzymes related to eicosanoid metabolism including COX, LO, phospholipase (PL) A2, mitogen-activated protein kinase and phosphatidylinositide 3-kinase (17, 21, 22, 23, 24). That lipid bodies in leukocytes contribute to 5-LO and COX pathway eicosanoid formation is supported by findings that increase in lipid body numbers correlated with increased LTB4, LTC4 and PGE2 release by these cells when activated with submaximal concentrations of A23187 (19, 21, 25, 26, 27). Conversely, agents that inhibit lipid body formation in vitro inhibited the priming response for enhanced eicosanoid release (14, 19, 21, 28). Recently, 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 (26, 29).
Despite the recent advances on the understanding of the roles of leukocyte lipid bodies in arachidonate metabolism, little is known about the origins, composition, or functions of leukocyte lipid bodies in vivo. In this study, we have evaluated the occurrence and composition of lipid bodies from naturally occurring or experimentally induced sepsis and the functions that these structures play in innate immunity as inducible intracellular compartments involved in the heightened production of inflammatory mediators.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS from Escherichia coli (serotype 0127:b8) was obtained from Sigma (St. Louis, MO). Calcium ionophore A23187 was obtained from Calbiochem Novabiochem (La Jolla, CA). 1-acyl-2-(7-octyl BODIPY-1-pentanoyl)-sn-glycerol was obtained from Molecular Probes (Eugene, OR). Osmium tetroxide (OsO4) was obtained from Electron Microscopy Science (Fort Washington, PA), WEB2086 was from Boehringer-Ingelheim (Ingelheim, Germany), and SR27417 and BN52021 was kindly provided by Dr. P. Braquet (Institut Henri Beaufour, Paris, France).
Patients with severe sepsis and septic shock
Leukocyte lipid bodies were analyzed from 10 septic patients (median age, 53 years old) hospitalized in the critical care unit, University Hospital, Federal University of Rio de Janeiro or in Spanish Hospital, Rio de Janeiro, Brazil. Eight patients had septic shock, and two had severe sepsis, as defined by the Consensus Conference of the American College of Chest Physician and Society of Critical Care Medicine (30). Among septic patients, there were four women and six men. The median acute physiology assessment and chronic health evaluation were 17 (range, 6–25) at the time of hospital admission. Blood was collected within 72 h of the sepsis diagnostic. Six healthy volunteers (median age, 43 years old) were used as controls. Patients and volunteers were recruited after ethical committee approval and informed consent was signed.
Animals
CBA/J, C3H/HeN, and C3H/HeJ mice of both sexes weighing 20–25 g were used. CBA/J and C3H/HeN mice were obtained from the Oswaldo Cruz Foundation breeding unit. C3H/HeJ mice were obtained from Fluminense Federal University (Niterói, Brazil). The C3H/HeJ and C3H/HeN mice used as controls were age and sex matched and were raised and maintained under similar housing conditions. The animals were maintained in a room with constant temperature (25°C) and alternating light/dark cycle of 12 h and had free access to pelleted diet and water. Animals were maintained and treated according to the animal care guidelines of the Council for International Organizations of Medical Sciences and the protocols were approved by the Oswaldo Cruz Animal Welfare Committee.
Pleurisy
Pleurisy was induced in anesthetized mice through the intrathoracic (i.t) injection of LPS (125–1000 ng/cavity) or vehicle (sterile saline) in a final volume of 0.1 ml. The animals were killed in a CO2 chamber at different time points after LPS administration (1–96 h). The thoracic cavity was opened and rinsed with 1 ml of saline containing heparin (10 IU/ml). Leukocyte counts were performed in Neubauer chambers after diluting the samples in Turk solution (2% acetic acid). Differential leukocyte counts were performed in cytospin smears stained by the May-Grünwald-Giemsa method.
Polymorphonuclear neutrophil and mononuclear cell purification
The pleural fluid from LPS-stimulated or control animals were centrifuged for 10 min at 400 x g. The pellet was resuspended in 5 ml of Ca2+/Mg2+-free HBSS and placed on the top of a discontinuous Percoll gradient with densities of 57–72%. Cells were centrifuged for 30 min at 600 x g. Mononuclear cells were recovered from the interface, whereas polymorphonuclear neutrophils were recovered from the pellet (purity, >95%). The cells were washed twice in 10 ml of Ca2+-Mg2+-free HBSS.
Mouse peritoneal macrophages
Peritoneal macrophages (>90% macrophages) were collected by rinsing the peritoneal cavity from naive mice with 5 ml of PBS containing heparin (20 IU/ml).
Lipid body induction and treatments
LPS (125–1000 ng/cavity) was injected into the pleural cavity of mice, and its effect was analyzed at different time points (1–96 h). To study the effect of inhibitors, animals were pretreated with PAF receptor antagonists SR27417 (1 mg/kg), BN52021 (20 mg/kg), and WEB2086 (20 mg/kg i.p) 1 h before the injection of LPS (i.t., 500 ng/cavity). WEB 2086 and SR27417 were diluted in 0.1 N NaOH, neutralized with 0.1 N HCl, and volume completed with saline. BN52021 was diluted in saline. Vehicle alone were used as a control of experiment. Mouse peritoneal macrophages (106 cells/ml) were pretreated with anti-CD14 and anti-CD11b/CD18 mAb (BD PharMingen, San Diego, CA; 1 and 10 µg/ml), 5 min before the LPS administration (500 ng/ml) for 1 h at 37°C.
Lipid body staining and enumeration
While still moist, leukocytes on cytospin slides were fixed in 3.7% formaldehyde in Ca2+-Mg2+-free HBSS (pH 7.4), rinsed in 0.1 M cacodylate buffer, 1.5% OsO4 (30 min), rinsed in dH2O, immersed in 1.0% thiocarbohydrazide (5 min), rinsed in 0.1 M cacodylate buffer, restained in 1.5% OsO4 (3 min), rinsed in dH2O, and then dried and mounted. The morphology of fixed cells was observed, and lipid bodies were enumerated by light microscopy with a x100 objective lens in 50 consecutively scanned leukocytes.
LTB4 and PGE2 assays
Animals were stimulated i.t. with LPS (125–1000 ng/cavity) or vehicle for 6 h for lipid body formation. After that, pleural leukocytes were obtained for lipid body enumeration, and leukocytes were washed in Ca2+-Mg2+-free HBSS. Leukocytes (1 x 106 cells/ml) were resuspended in HBSS containing Ca2+-Mg2+ and then stimulated with A23187 (0.5 µM) for 15 min. Reactions were stopped on ice, and the samples were centrifuged at 500 x g for 10 min at 4°C. LTB4 and PGE2 in the supernatants were assayed by ELISA according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI).
Immunocytochemistry
Mouse leukocytes obtained 6 h after i.t. injection of LPS (500 ng/cavity) or human leukocytes obtained from septic patients were incubated or not with 1 µM of the fluorescent fatty acid-containing diglyceride, 1-acyl-2-(7-octyl-BODIPY-1-pentanoyl)-sn-glycerol, to fluorescently label lipid bodies. After the incubation, cells were washed twice in Ca2+-Mg2+- free HBSS, cytospin onto slides, and fixed in 3% formaldehyde at room temperature for 10 min. Fixed cells were permeabilized with 0.05% saponin/HBSS solution (5 min) and then blocked with 10% normal serum from animals where secondary Ab was raised. After a washing, cytospin preparations were incubated for 1 h at room temperature with the following primary Abs which were diluted in 0.05% saponin/HBSS solution: rabbit polyclonal serum anti-5-LO (1/150 dilution; Cayman Chemical, Ann Arbor, MI), polyclonal Ab anti-murine COX 2 (C terminus; Oxford Biomedical Research, Oxford, MI) (1:150 dilution) and purified goat IgG anti-murine TNF (1/50 dilution) and purified mouse IgG1 anti-hCOX-2 (1/50 dilution) (Transduction Laboratories, San Diego, CA). Nonimmune rabbit or goat IgG, at the same concentration as the primary Abs, were 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 (Vector Laboratories, Burlingame, CA) or biotin-conjugated rabbit anti-goat IgG (Sigma, St. Louis, MO). The immunoreactive in cells were then identified by the ABC Vectastain glucose-oxidase kit following the manufacturer’s instructions (Vector Laboratories). Glucose oxidase immunostaining was visualized under light microscopy, and fluorescent lipid bodies were identified under FITC filter.
Statistical analysis
Results were expressed as mean ± SEM and were analyzed statistically by ANOVA followed by the Newman-Keuls Student test with the level of significance set at p < 0.05. Correlation coefficients were determined by linear regression followed by analysis by Spearman correlation rank.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The i.t. injection of LPS (500 ng/cavity) induced an intense cell
accumulation within 6 h; this augmentation was due to a marked
increase in neutrophil numbers followed by significant increases in
mononuclear cell and eosinophil numbers at later time points (data not
shown) (31). The effect of LPS on lipid body formation in
leukocytes was investigated. We observed that LPS was capable of
inducing a dose-dependent increase on lipid body formation, with
maximum lipid body induction observed at dose of 500 ng/cavity (Fig. 1
a). This lipid body formation
was apparent within 1 h, maximum within 6 h and decreased
thereafter (Fig. 1b). Interestingly, blood leukocyte lipid
body number were not modified after i.t. LPS stimulation, thus
indicating that lipid body formation depends on leukocyte activation at
the inflammatory site (not shown).
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c), which precludes their recognition in
LPS-stimulated leukocytes. However, with appropriate lipid fixation and
staining with osmium tetroxide, increased lipid body numbers have been
observed in leukocytes after LPS administration (Fig. 1d),
compared with the control (Fig. 1e).
To analyze the effect of LPS on lipid body formation in different
pleural cell population, a discontinuous Percoll gradient was performed
for cellular separation. As shown in Fig. 2, LPS-induced a significant increase in
lipid body formation in neutrophils and mononuclear cells alike.
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in
leukocyte lipid bodies
We evaluated whether key eicosanoid-forming enzymes and
TNF- were localized at leukocyte lipid bodies after LPS stimulation
in vivo. The compartmentalization of 5-LO and COX-2, as well as the
proinflammatory cytokine, TNF-, to lipid bodies was analyzed by
immunocytochemistry using conditions of cell fixation and
permeabilization that prevent dissolution of lipid bodies. Lipid bodies
were visualized by endogenous labeling with the fluorescent fatty
acid-containing diglyceride, 1-acyl-2-(7-octyl
BODIPY-1-pentanoyl)-sn-glycerol (1 µM) (21).
Leukocytes stained with rabbit polyclonal serum anti-5-LO (Fig. 3a), anti-COX-2 (Fig. 3c) polyclonal Abs, in addition to present perinuclear
membrane and cytosolic staining, show punctate cytoplasmic staining
that matched with fluorescent fatty acid-labeled lipid bodies (Fig. 3, b and d, respectively). There was no
immunoreactivity when control nonimmune rabbit were used as control for
5-LO and COX-2 Abs (Fig. 3g), although fluorescent fatty
acid-labeled lipid bodies were strongly visualized (Fig. 3h). As shown in Fig. 3e, there was also a
significant intracellular staining of TNF- in LPS-stimulated
leukocytes that coincidentally matched with fluorescent fatty
acid-labeled lipid bodies (Fig. 3f). There was no
immunoreactivity when control nonimmune goat serum was used (data not
shown).
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Most LPS effects occur via interaction with membrane receptors.
The existence of membrane-bound receptors for LPS has been postulated
and an increasing number of membrane-bound LPS-binding proteins have
been identified (1, 3). As shown in Fig. 4a, pretreatment with
anti-CD14 (1 and 10 µg/ml) or anti-CD11b/CD18 (1 and 10
µg/ml) mAb significantly inhibited lipid body formation in vitro. The
specificity of the treatment was confirmed by the lack of effect of the
irrelevant Abs in inhibiting LPS-induced lipid body formation (Fig. 4a). Furthermore, anti-CD14 and anti-CD11b/CD18 mAb
were not able to inhibit PAF-induced lipid body formation (Fig. 4b).
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a) or in vitro (not shown),
demonstrating a requisite role for LPS receptor on lipid body
induction. Lipid body formation induced by PAF was equally effective
when cells from C3H/HeN and C3H/HeJ were compared after in vivo (Fig. 5a) or in vitro stimulation (not shown).
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b). Endogenous PAF signals for LPS-induced lipid body formation
PAF and PAF-like lipids are potent inductors of lipid bodies in
human and mouse leukocytes (21, 25, 32). Moreover, lipid
mediators, especially PAF, have been implicated as mediators of
LPS-induced inflammation (5). The role of PAF on
LPS-induced lipid body formation was investigated through the use of
three structurally unrelated PAF receptor antagonists. The animals were
pretreated (i.p.) with PAF receptor antagonists BN52021 (20 mg/kg),
SR27417A (1 mg/kg), and WEB2086 (20 mg/kg) 1 h before LPS
stimulation. Confirming previous results (33), the
pretreatment with PAF-receptor antagonists significantly inhibited the
pleural neutrophil infiltration induced by i.t. injection of LPS within
6 h, suggesting the involvement of PAF in LPS-induced pleurisy
(Ref. 33 and data not shown). As shown in Fig. 6, PAF antagonists inhibited LPS-induced
lipid body formation, suggesting an important role for endogenous PAF
in lipid body formation.
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Because leukocyte lipid bodies are sites of intracellular
localization of eicosanoid-forming enzymes (Refs. 17, 21 ,
and 24 and Fig. 3) and also stores of the eicosanoid
precursor AA (16), we analyzed whether LPS-induced
increases in lipid body numbers on leukocytes isolated from the mice
pleural cavity would correlate with LTB4 and
PGE2 production. After leukocytes were stimulated
in vivo with increasing concentrations of LPS for 6 h, lipid
bodies were enumerated, and replicated leukocytes were stimulated with
A23187 (0.5 µM). As shown in Fig. 7, LPS dose-dependently induced significant concordant increases in lipid
body numbers and priming for enhanced LTB4
(r = 0.917, p < 0.001) and
PGE2 (r = 0.986,
p < 0.001) generation. Similar results were obtained
when peritoneal macrophages were stimulated in vitro with LPS (data not
shown).
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As shown in Fig. 8a, lipid body numbers in
leukocytes from septic patients were significantly increased compared
with control healthy subjects. We also evaluated whether key
eicosanoid-forming enzymes were localized at leukocyte lipid bodies
from septic patients. The compartmentalization of 5-LO (Fig. 8b) and COX-2 (Fig. 8c) to lipid bodies was
demonstrable in leukocytes from septic patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mechanisms involved in LPS-induced lipid body formation were analyzed.
First, we investigated the need of LPS to interact with surface
receptors to induce lipid bodies in leukocytes. The current
understanding of the LPS receptor system consists of multiple LPS
receptors on the surface of phagocytes which, after interacting with
LPS, mediate remarkably diverse events. CD14 is likely the LPS receptor
primarily responsible for initiating LPS-induced cellular activation.
This is supported in part by the observation that pretreatment with
anti-CD14 drastically inhibit LPS-induced inflammatory mediator
production, cell activation, and death and by the findings that
CD14-deficient mice are highly resistant to LPS and Gram-negative
bacteria (42, 43, 44). The 
2 integrin
CD11/CD18 is important for phagocytosis of invading Gram-negative
bacteria and has been shown to bind LPS (45). Moreover,
CD11/CD18 is involved in LPS-induced activation of gene expression,
including induction of COX-2 and IL-12 (46, 47, 48).
LPS-induced macrophage lipid body formation occurs in a
dose-dependent manner, consistent with a receptor-dependent phenomenon.
In agreement, LPS-induced lipid body formation was inhibited by
anti-CD14 and anti-CD11b/CD18 mAb (Fig. 4), suggesting a role
for both CD14 and CD11b/CD18 receptors in lipid body formation.
Recent data have demonstrated that mammalian TLR proteins participate
in intracellular signaling initiated by Gram-negative bacterial LPS.
The Toll protein is a transmembrane receptor that displays resemblance
in its intracellular portion to the signaling domains of members of the
IL-1R family that mediate activation of NF-
B (1, 3). In
mice, Tlr4 appears to be the main LPS response pathway
involved in cell activation (49, 50, 51, 52). The involvement of
Tlr4 in LPS-induced lipid body formation was investigated by
using C3H/HeJ mice.
It has been recognized for several years that mice of the C3H/HeJ strain have a defective response to bacterial endotoxin (53, 54). Recently, the inability of C3H/HeJ mice to respond to LPS was characterized to be linked to a truncated and inactive Tlr4 (49, 50, 51, 52). Indeed, we observed that LPS administration into C3H/HeJ failed to induce pleural inflammation as measured by neutrophil influx (data not shown). Moreover, leukocytes from these animals did not present lipid body formation or punctate eicosanoid-forming enzyme localization after LPS administration indicating a requisite role for Tlr4 in LPS-induced lipid body formation. Our findings suggest that for lipid body induction CD14, CD11b/CD18, and Tlr4 must be coordinately engaged to deliver optimal signaling to leukocytes.
Several LPS-induced effects occur indirectly through the generation of cytokines and lipid mediators. One such mediator is the glycerophospholipid PAF. Indeed, pretreatment with PAF receptor antagonists are capable of significantly inhibit the inflammatory process and cardiovascular collapse induced by endotoxin in different experimental models (5). LPS have also been shown to induce PAF production both in vivo and in vitro (55, 56). Moreover, many groups have shown that patients undergoing septic shock and systemic inflammatory response syndrome have elevated levels of PAF or PAF-like substances in their plasma (57). We demonstrated that the pretreatment with three structurally unrelated PAF receptor antagonists inhibited the lipid body formation induced by LPS in vivo, suggesting that endogenous PAF plays an important role in this phenomenon. In agreement, we and others have previously demonstrated the ability of PAF and PAF-like lipids to induce lipid body formation in a receptor-mediated manner in human and murine leukocytes (21, 25, 27, 32). Although PAF may induce cell activation at intracellular binding sites (58), our results suggest that PAF is acting in a paracrine/autocrine way to induce lipid body formation, because the PAF receptor antagonist used in this study would act preferentially at membrane receptors.
The two major enzymes, 5-LO and COX-2, involved in the enzymatic conversion of AA into eicosanoids during inflammatory conditions were shown to localize within in vivo formed lipid bodies from septic patients or experimentally elicited by LPS administration in mice. In agreement, by using immunocytochemistry at the light level and ultrastructural postembedding immunogold, COX has been reported to localize at eosinophil lipid bodies, both in naturally formed lipid bodies in eosinophils from hypereosinophilic syndrome patients and in PAF-induced lipid bodies (17, 21, 24). Previous reports have used a nonselective anti-COX Ab, and our results are the first description of COX-2 localization in lipid bodies. Previous studies on the intracellular localization of 5-LO have shown that 5-LO localization is cell type specific and also varies according to the activation state of the cell. 5-LO was shown to localize within the nuclear environment of alveolar macrophages, whereas 5-LO was predominantly cytosolic in human neutrophils and resting peritoneal macrophages (10, 11, 12). In addition to the nuclear environment, we have demonstrated the compartmentalization of the key enzyme for LT production, 5-LO, within human eosinophil lipid bodies (17, 21, 26). Accordingly, in this study we observed a significant correlation between lipid body formation induced by LPS and priming for PGE2 and LTB4 production in leukocytes stimulated by submaximal concentrations of the ionophore A23187, suggesting that lipid bodies are early response structures involved in the production of lipid mediators of inflammation. Recently, direct evidence for LT synthesis derived from eosinophil lipid bodies has been provided by using intracellular immunofluorescent localization of LTC4 (26, 29). Together, our findings support a role for lipid bodies to function as specific sites for eicosanoid formation. Of special interest for inflammatory cells where increased eicosanoid synthesis is observed, lipid body arachidonyl phospholipids might provide a source of substrate AA without requiring the perturbation of the integrity of membranes and could be replenished from the arachidonyl triglycerides abundant in lipid bodies (59, 60).
Interestingly, the proinflammatory cytokine involved in the
pathogenesis of sepsis, TNF-, were also shown to colocalize within
leukocyte lipid bodies formed after LPS stimulation in vivo (Fig. 3)
and from septic patients (not shown). Accordingly, the lipid body
localization of TNF- in eosinophils from Crohn’s disease has been
shown previously (61). Accumulating evidence indicate that
TNF- are involved with AA release and synthesis of its metabolites;
conversely, AA and eicosanoids have been implicated in TNF-
synthesis and function(s) (62, 63, 64, 65, 66, 67). The colocalization of
eicosanoid-forming enzymes and TNF- within lipid bodies might be of
importance for intracellular signaling in sepsis.
In conclusion, our studies indicate that leukocyte lipid bodies formed in response to LPS stimulation or sepsis are sites for eicosanoid-forming enzymes and cytokine localization and may develop and function as structurally distinct, intracellular sites for paracrine eicosanoid synthesis during inflammatory conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patricia T. Bozza, Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, RJ, Brazil 21045-900. E-mail address: pbozza{at}gene.dbbm.fiocruz.br
3 Abbreviations used in this paper: PAF, platelet-activating factor; AA, arachidonic acid; LT, leukotriene; PG, prostaglandin; COX, cyclooxygenase; LO, lipoxygenase; PL, phospholipase; i.t., intrathoracic.
Received for publication May 31, 2002. Accepted for publication October 2, 2002.
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 H.-J. van Manen, Y. M. Kraan, D. Roos, and C. Otto Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes PNAS, July 19, 2005; 102(29): 10159 - 10164. [Abstract] [Full Text] [PDF]
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 M. Ogata, K. Nandate, T. Kawasaki, C. Kawasaki, M. Ozaki, and A. Shigematsu A Platelet Activating Factor Receptor Antagonist Inhibits Cytokine Production in Human Whole Blood by Bacterial Toxins and Live Bacteria Anesth. Analg., June 1, 2004; 98(6): 1767 - 1772. [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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, LPS; 
,
vehicle. Lipid bodies were enumerated using light microscopy after
osmium staining. Each bar represents the mean ± SEM lipid bodies
per cell in 50 consecutively counted cells from at least 6 animals.
*, Statistically significant increases in lipid body numbers elicited
by LPS compared with controls. Lipid bodies are sparse in normal cells
(e) but very prominent in leukocytes after LPS
administration in vivo (d), using appropriate lipid
fixation and staining with osmium tetroxide. c, Common
alcohol-based hematological stains cause dissolution of lipid body
formation, which precludes their recognition. Cytocentrifuged
leukocytes were fixed in 3.7% formaldehyde in HBSS and stained with
1.5% osmium tetroxide (d and e), or
fixed in methanol and stained by May-Grünwald-Giemsa
(c).
, Macrophages.
), or vehicle (
