Lipid droplet biogenesis and COX-2 pathway activation are triggered by Barrett's esophagus and adenocarcinoma, but not esophageal squamous cell carcinoma risk factors.

Esophageal cancer (EC) is an aggressive disease, presenting two main histological subtypes: adenocarcinoma (EAC) and squamous cell carcinoma (ESCC). The two EC subtypes widely differ concerning virtually all factors. ESCC development is mainly associated with tobacco and alcohol abuse, whereas obesity and chronic gastroesophageal reflux disease (GERD) are important risk factors not only for EAC, but also for for Barrett's esophagus (BE), an intestinal metaplasia that precedes EAC. Obesity triggers ectopic lipid droplets (LD) accumulation in non-adipose tissues. LD are organelles involved in cell metabolism, signaling, proliferation and production of inflammatory mediators. Therefore, the aim of this work was to investigate LD occurrence and role in EC. This study shows progressive LD levels increase along EAC development, in esophageal samples from non-obese through obese individuals, as well as BE, and EAC patients, whereas no significant changes were observed in ESCC samples, when compared to non-tumor samples. Additionally, in order to mimic BE and EAC risk factors exposure, a non-tumor esophageal cell line was incubated with oleic acid (OA) and acidified medium and/or deoxycholic acid (DCA), revealing a significant increment in LD amount as well as in COX-2 and CXCL-8 expression, and in IL-8 secretion. Further, COX-2 expression and LD amount presented a significant positive correlation and were detected co-localized in EAC, but not in ESCC, suggesting that LD may be the site for eicosanoid production in EAC. In conclusion, this study shows that obesity, and BE- and EAC-associated inflammatory stimuli result in a gradual increase of LD, that may be responsible for orchestrating inflammatory mediators' production and/or action, thus contributing to BE and EAC genesis and progression.

Lipid bodies accumulation in Leishmania infantum-infected C57BL/6 macrophages.

Lipid bodies (LBs) are intracellular accumulations of neutral lipids surrounded by a single membrane. These organelles are involved in the production of eicosanoids, which modulate immunity by either promoting or dampening inflammatory responses. Leishmania infantum, the etiological agent of visceral leishmaniasis in Brazil, is an intracellular parasite that causes disease by suppressing macrophage microbicidal responses. C57BL/6 mouse bone marrow-derived macrophages infected with L. infantum strain LcJ had higher numbers of LB+ cells (P<.0001) and total LBs than noninfected cultures. Large (>3 µm) LBs were present inside parasitophorous vacuoles (PVs). These results contrast with those of L. infantum-infected BALB/c macrophages, in which the only LBs are derived from parasite, not macrophage origin. Increased LBs in C57BL/6 macrophages in close association with parasites would position host LBs where they could modulate L. infantum infection. These results imply a potential influence of the host genetics on the role of LBs in host-pathogen interactions. Overall, our data support a model in which the expression, and the role of LBs upon infection, ultimately depends on the specific combination of host-pathogen interactions.

Involvement of TLR6 in the induction of COX-2, PGE2 and IL-10 in macrophages by lipids from virulent S2P and attenuated R1A Babesia bovis strains.

Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains.

Dengue induces platelet activation, mitochondrial dysfunction and cell death through mechanisms that involve DC-SIGN and caspases.

BACKGROUND: Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self-limiting febrile illness through to a life-threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV-induced platelet activation and thrombocytopenia are incompletely understood. PATIENTS AND METHODS: Freshly isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alteration and activation of cell death pathways. In parallel, we examined direct DENV-induced activation and apoptosis of platelets obtained from healthy subjects. RESULTS: We found that platelets from DENV-infected patients exhibited increased activation by comparison to control subjects. Moreover, platelets from DENV-infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and -3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV-infected patients. Platelet activation, mitochondrial dysfunction and caspase-dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV-induced platelet activation was shown to occur through mechanisms largely dependent on DC-SIGN. CONCLUSIONS: Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, which may contribute to the development of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC-SIGN as a critical receptor in DENV-dependent platelet activation.

PPAR gamma activation protects the brain against microvascular dysfunction in sepsis.

Sepsis is a severe disorder characterized by systemic inflammatory responses in the presence of an infection and may progress to multiple organ dysfunction and death. Alterations in cerebral microcirculation fulfill a crucial role in the pathogenesis of severe sepsis, and include a decrease in capillary density and disturbances in leukocyte movement along capillaries. Nevertheless, the mechanisms involved in sepsis-associated cerebral microcirculatory alterations have so far not been defined. We investigated the effect of the peroxisome proliferator-activated receptor gamma (PPARγ) selective agonist rosiglitazone on leukocyte/endothelial cell interaction and functional capillary density in the brain in the cecal ligation and puncture (CLP) model of sepsis. Anti-inflammatory effects of rosiglitazone on the cerebral microcirculation were marked. Functional capillary density increased and leukocyte rolling and adhesion were decreased in animals submitted to CLP and treated with rosiglitazone. Our data provide evidence for involvement of PPARγ activation in leukocyte-endothelium interactions and alterations in capillary density. Improved cerebral perfusion in animals treated with rosiglitazone, suggests that PPARγ activation is protective against cerebral microvascular dysfunction in sepsis.

Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils.

BACKGROUND AND PURPOSE: Prostaglandin (PG) D(2) has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD(2) induces leukotriene (LT) C(4) secretion from eosinophils. Here, we have characterized how PGD(2) signals to induce LTC(4) synthesis in eosinophils. EXPERIMENTAL APPROACH: Antagonists and agonists of DP(1) and DP(2) prostanoid receptors were used in a model of PGD(2) -induced eosinophilic inflammation in vivo and with PGD(2) -stimulated human eosinophils in vitro, to identify PGD(2) receptor(s) mediating LTC(4) secretion. The signalling pathways involved were also investigated. KEY RESULTS: In vivo and in vitro assays with receptor antagonists showed that PGD(2) -triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP(1) and DP(2) receptors. DP(1) and DP(2) receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC(4) synthesis, but not LTC(4) transport/export, was activated by PGD(2) receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP(1) /DP(2) receptor-driven LTC(4) synthesis. Although not sufficient to trigger LTC(4) synthesis by itself, DP(1) receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD(2) -induced LTC(4) synthesis. Similarly, concurrent DP(2) receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD(2) -induced LTC(4) synthesis. CONCLUSIONS AND IMPLICATIONS: Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD(2) receptors described here, our data suggest that both DP(1) and DP(2) receptor antagonists might be attractive candidates for anti-allergic therapies.

Lipids from attenuated and virulent Babesia bovis strains induce differential TLR2-mediated macrophage activation.

Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.

Early short-term versus prolonged low-dose methylprednisolone therapy in acute lung injury.

The present study compared the effects of early short-term with prolonged low-dose corticosteroid therapy in acute lung injury (ALI). In total, 120 BALB/c mice were randomly divided into five groups. In the control group, saline was intratracheally (i.t.) instilled. In the ALI group, mice received Escherichia coli lipopolysaccharide (10 microg i.t.). ALI animals were further randomised into four subgroups to receive saline (0.1 mL i.v.) or methylprednisolone (2 mg x kg(-1) i.v.) at 6 h, 24 h or daily (for 7 days, beginning at day 1). At 1, 3 and 8 weeks, in vivo and in vitro lung mechanics and histology (light and electron microscopy), collagen and elastic fibre content, cytokines in bronchoalveolar lavage fluid and the expression of matrix metalloproteinase (MMP)-9 and -2 were measured. In vivo (static elastance and viscoelastic pressure) and in vitro (tissue elastance and resistance) lung mechanics, alveolar collapse, cell infiltration, collagen and elastic fibre content and the expression of MMP-9 and MMP-2 were increased in ALI at 1 week. Methylprednisolone led to a complete resolution of lung mechanics, avoided fibroelastogenesis and the increase in the expression of MMP-9 and MMP-2 independent of steroid treatment design. Thus, early short-term, low-dose methylprednisolone is as effective as prolonged therapy in acute lung injury.

Inflammatory response and bacterial dissemination after laparotomy and abdominal CO2 insufflation in a murine model of peritonitis.

BACKGROUND: The immunologic repercussions due to cavity insufflation are the focus of great discussion. The aim of this study was to compare the inflammatory response and bacterial dissemination after laparotomy and abdominal CO2 insufflation in a murine model of peritonitis. METHODS: Swiss mice were inoculated intraperitoneally with 0.5 ml of a solution containing 1 x 10(8) colony-forming units (CFU)/ml of Escherichia coli and were divided into three groups as follow: control (anesthesia for 30 min), laparotomy (2.5-cm midline incision for 30 min), and CO2 pneumoperitoneum (CO2 cavity insufflation for 30 min). The number of leukocytes, CFU/ml counting, and the levels of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha), and IL-10 were evaluated in blood, peritoneal, and pleural fluid samples obtained at 90 min and 18 h after the procedures. RESULTS: The laparotomy group showed a greater bacterial dissemination to the blood, peritoneum, and pleural cavity and also greater neutrophil migration to the peritoneal cavity compared to the CO2 insufflated and control groups. The 24-h mortality was also significantly higher in the laparotomy group. The IL-6 levels showed a precocious rise in all groups submitted to bacterial inoculation at the 90-min time point. At the 18-h time point, IL-6 levels in the peritoneum were significantly higher in the laparotomy group than in the control or CO2 insufflated groups. At the same time, TNF-alpha levels were higher in the laparotomy and CO2 insufflated groups than in controls; IL-10 levels showed no differences among the groups. CONCLUSIONS: Our results suggest that cavity insufflation with CO2 is a more effective method of access, inducing less bacterial dissemination and also a less intense inflammatory response. Cavity insufflation with CO2 may present a good option for the surgical treatment of patients with bacterial peritonitis.

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU.

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.

Antinociceptive effect of Nidularium procerum: a Bromeliaceae from the Brazilian coastal rain forest.

Nidularium procerum, a common plant of the Brazilian flora, has not yet been studied for its pharmacological properties. We report here that extracts of N. procerum show both analgesic and anti-inflammatory properties. Oral (p.o.) or intraperitoneal (i.p.) administration of an aqueous crude extract from leaves of N. procerum (LAE) inhibited the writhing reaction induced by acetic acid (ED50 value = 0.2 mg/kg body weight, i.p.) in a dose-dependent manner. This analgesic property was confirmed in rats using two different models of bradykinin-induced hyperalgesia; there was 75% inhibition of pain in the modified Hargreaves assay, and 100% inhibition in the classical Hargreaves assay. This potent analgesic effect was not blocked by naloxone, nor was it observed in the hot plate model, indicating that the analgesic effect is not associated with the activation of opioid receptors in the central nervous system. By contrast, we found that LAE (0.02 microg/ml) selectively inhibited prostaglandin E2 production by cyclooxygenase (COX)-2, but not COX-1, which is a plausible mechanism for the analgesic effect. A crude methanol extract from the leaves also showed similar analgesic activity. An identical extract from the roots of N. procerum did not, however, block acetic acid-induced writhes, indicating that the analgesic compounds are concentrated in the leaves. Finally, we found that LAE inhibited an inflammatory reaction induced by lipopolysaccharide in the pleural cavity of mice.

Macrophage lipid body induction by Chagas disease in vivo: putative intracellular domains for eicosanoid formation during infection.

Lipid bodies (LB), lipid-rich inclusions abundantly present in cells engaged in inflammation, are specialized intracellular domains involved in generating inflammatory mediators, the eicosanoids. Since the acute Trypanosoma cruzi infection triggers a potent inflammatory reaction characterized by a great increase of peripheral blood monocyte (PBM) and macrophage numbers, we investigated the LB occurrence in these cells. The experimental rat infection by T. cruzi (Y strain) induced significant increase of the LB numbers in peritoneal macrophages at day 6 and 12, accompanied by significant enhancement of Prostaglandin E(2) (PGE(2)) production, as measured by EIA. At day 12, ultrastructural analysis of the heart, a target organ of the disease, showed numerous macrophages with LB prominently increased in number (mean of 8.3 per section view, range of 1-25) compared to controls (mean of 2.6 per section view, range of 0-3) and size. PBM from all groups rarely showed LB. Our results demonstrate, for the first time, that T. cruzi infection in rats elicits important LB formation in inflammatory macrophages but not in PBM. The increase in LB numbers during infection positively correlates with increased generation of PGE(2), suggesting that LB may have a role in the heightened eicosanoid production observed during T. cruzi infection.

NS-398: cyclooxygenase-2 independent inhibition of leukocyte priming for lipid body formation and enhanced leukotriene generation.

Because the induction of new lipid body formation in leukocytes correlates with and likely contributes to their enhanced 'primed' prostaglandin and leukotriene formation, we evaluated two selective cyclooxygenase (COX)-2 inhibitors. Three types of stimuli, cis -unsaturated fatty acids, platelet activating factor and protein kinase C activators, stimulate lipid body formation. NS-398 (0.1-10 microM), but not another COX-2 inhibitor, SC58125 (0.1- 10 microM), blocked leukocyte lipid body formation elicited by all three types of stimuli and also blocked priming for enhanced LTB(4) production and PGE(2) production. The effect of NS-398 on lipid body formation was independent of its inhibitory effects on COX-2 since arachidonate-induced lipid body formation in COX-2-deficient mouse leukocytes was also inhibited by NS-398. By means of its ability to inhibit leukocyte lipid body formation, NS-398 may exert actions independent of its COX-2 inhibition and more broadly contribute to the suppression of formation of COX-1 and lipoxygenase-derived eicosanoids.

Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity.

Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth.

LPS induces eosinophil migration via CCR3 signaling through a mechanism independent of RANTES and Eotaxin.

Mounting evidence suggests that lipopolysaccharide (LPS) modulates bronchoconstriction and eosinophil function in asthma. We have investigated the role of different chemokines in the eosinophil influx to the pleural cavity after LPS stimulation. Expression of mRNA for eotaxin, regulated on activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, and monocyte chemotactic protein (MCP)-1 was increased in cells recovered from the mouse pleural cavity 6 h after LPS administration. Eotaxin and RANTES, but not MIP-1alpha, protein levels were also increased in cell-free pleural washes recovered 6 h after LPS stimulation (LPW). Antimurine eotaxin and antimurine RANTES antibodies (Abs) failed to inhibit LPS-induced eosinophil influx into mouse pleural cavity in vivo. Pertussis toxin inhibited LPW-induced eosinophil shape change in vitro, suggesting the involvement of G protein-coupled receptors in LPW signaling. Blockade of CCR3 receptors diminished eosinophil shape change induced by LPW fractions in vitro and LPS-induced eosinophil accumulation in vivo. To investigate further contribution of CC chemokines, we administered a 35-kD CC chemokine neutralizing protein (vCKBP) in vivo. vCKBP inhibited the eosinophil accumulation induced by eotaxin and ovalbumin, but did not block that induced by LPS or LPW. Our data suggest that LPS-induced eosinophil accumulation depends on G protein-coupled CCR3 receptor activation, through a mechanism independent of eotaxin, RANTES, or other vCKBP-inhibitable CC chemokines.

Mechanisms of allergen- and LPS-induced bone marrow eosinophil mobilization and eosinophil accumulation into the pleural cavity: a role for CD11b/CD18 complex.

OBJECTIVE: The mechanisms involved in bone marrow eosinophil emigration and recruitment to inflammatory sites are not fully understood. The involvement of CD11b/CD18 in marrow eosinophil release induced by lipopolysaccharide (LPS) or allergen was investigated in mice. METHODS: Eosinophil and neutrophil counts in the pleural cavity, blood and bone marrow were performed at different time intervals after the intrathoracic injection of LPS (250 ng/cavity) or ovalbumin (OVA, 12 microg/cavity; into actively sensitized mice) and compared to anti-CD11b/CD 18 (5C6, 1 mg/mouse) or anti-IL-5 (TRFK-5, 500 microg/kg) treated mice. RESULTS: LPS induced local eosinophil influx, that peaked within 24 h and that was preceded by a decrease in marrow eosinophils at 4 h. Antigenic challenge induced a decrease in marrow eosinophils within 4 h, followed by a long lasting pleural eosinophil accumulation and a persistent increase in marrow eosinophil numbers. Pretreatment with anti-CD11b/CD18 abolished LPS-induced neutrophil and eosinophil accumulation in the pleural cavity at 4 and 24 h, respectively. This pretreatment failed to modify neutrophil emigration from bone marrow, but significantly inhibited marrow eosinophil release at 4 h post-LPS or OVA challenge. Anti-IL-5 pretreatment failed to inhibit LPS-induced pleural eosinophil accumulation and mobilization from bone marrow, but it abolished allergen-induced effects, indicating a role for IL-5 in marrow eosinophil mobilization induced by antigen, but not by LPS challenge. CONCLUSIONS: Our results suggest that eosinophil migration induced by antigen or LPS into the pleural cavity is preceded by bone marrow eosinophil release through a mechanism that depends on CD11b/CD18.

TSG-14 transgenic mice have improved survival to endotoxemia and to CLP-induced sepsis.

Tumor necrosis factor-stimulated gene 14 (TSG-14)/PTX3 was identified originally as a TNF-alpha and IL-1beta-stimulated gene from normal, human foreskin fibroblasts and vascular endothelial cells, respectively. TSG-14 gene encodes a 42-kDa-secreted glycoprotein with a carboxy-terminal half that shares homology with the entire sequence of C-reactive protein (CRP) and serum amyloid P component (SAP), acute-phase proteins of the pentraxin family. Some experimental evidence suggests that TSG-14 plays a role in inflammation, yet its function and mechanism of action remain unclear. We have generated transgenic mice that overexpress the murine TSG-14 gene under the control of its own promoter. From eight transgenic founders, two lineages were derived and better characterized: Tg2 and Tg4, carrying two and four copies of the transgene, respectively. TSG-14 transgenic mice were found to be more resistant to the endotoxic shock induced by LPS and to the polymicrobial sepsis caused by cecal ligation and puncture (CLP). Moreover, macrophages derived from the transgenic mice produced higher amounts of nitric oxide in response to IFN-gamma, TNF-alpha, and LPS as compared with macrophages from wild-type animals, and the augmented response appears to be the consequence of a higher responsiveness of transgenic macrophages to IFN-gamma. The data shown here are the first in vivo evidence of the involvement of TSG-14 in the inflammatory process and suggest a role for TSG-14 in the defense against bacterial infections.

Arachidonyl trifluoromethyl ketone induces lipid body formation in leukocytes.

Leukocyte lipid bodies, abundant in cells associated with inflammation, can be induced to form in response to stimuli that include cis -unsaturated, but not saturated, fatty acids. Arachidonyl trifluoromethyl ketone (AACOCF(3)), a non-esterifiable arachidonate analog and an inhibitor of cytosolic phospholipase A(2)enzymes (PLA(2)), dose-dependently (0-20 microM) stimulated neutrophil lipid body formation, but this stimulation was not attributable to PLA(2)inhibition. Palmitoyl trifluoromethyl ketone, also a PLA(2)inhibitor, failed to stimulate lipid body formation, like palmitic acid itself, and did not inhibit stimulated lipid body formation. Moreover, aspirin, indomethacin and ibuprofen, which inhibit cis -unsaturated fatty acid-induced lipid body formation, inhibited AACOCF(3)-induced lipid body formation. Lipid body induction with AACOCF(3)reflected its structural basis as a cis -unsaturated fatty acid analog. These results indicate that cytosolic PLA(2)enzymes are not active in lipid body induction and cis -fatty acid stimulation of lipid body formation does not require esterification of cis -fatty acids into glycerolipids.

A role for adrenoceptors in the regulation of pleural neutrophilia induced by LPS.

The role of catecholamines in regulating pleural neutrophilia evoked by intrathoracic (i.t.) injection of lipopolysaccharide (LPS) was investigated in Wistar rats by means of surgical adrenalectomy, depletion of catecholamine stores or adrenoceptor blockade. Treatment of animals with a single dose of LPS evoked a dramatic increase in the number of pleural neutrophils concomitant with an increase in the number of these cells in blood at 4 h. Although blood neutrophilia was drastically reduced when catecholamine stores were depleted with intraperitoneal (i.p.) injection of reserpine, pleural neutrophilia was not modified. However, the i.t. injection of reserpine reduced the increase in pleural neutrophils after LPS stimulation. Adrenalectomy failed to inhibit the increase in neutrophil counts in the blood or pleural cavity after LPS challenge. Pretreatment with intravenous (i.v.) injection of prazosin, an alpha(1)/alpha(2B) antagonist, reduced LPS-induced blood but not pleural neutrophilia. On the other hand, although pleural neutrophilia was not affected by systemic pretreatment with the alpha(2)-adrenoceptor antagonist, yohimbine, the local treatment (i. t. injection) with this antagonist markedly reduced the increase in pleural neutrophil counts observed after stimulation by LPS. In contrast, pleural neutrophilia induced by i.t injection of formyl-methionyl-leucyl-phenylalanine (fMLP) was not modified by local treatment with yohimbine. Taken together, our results suggest that catecholamines, through activation of alpha(1) and alpha(2)-adrenoceptors, play a role in the regulation of blood and pleural neutrophilia observed during the inflammatory response evoked by LPS in the pleural cavity.