The role of PGE2-associated inflammatory responses in gastric cancer development

Accumulating evidence indicates that inflammation plays a critical role in cancer development. Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid biosynthesis, including prostaglandin E₂ (PGE₂), and plays a key role in both inflammation and cancer. It has been demonstrated that inhibition of COX-2 and PGE₂ receptor signaling results in the suppression of tumor development in a variety of animal models. However, the molecular mechanisms underlying COX-2/PGE₂-associated inflammation in carcinogenesis have not yet been fully elucidated. In order to study the role of PGE₂-associated inflammatory responses in tumorigenesis, it is important to use in vivo mouse models that recapitulate human cancer development from molecular mechanisms with construction of tumor microenvironment. We have developed a gastritis model (K19-C2mE mice) in which an inflammatory microenvironment is constructed in the stomach via induction of the COX-2/PGE₂ pathway. We also developed a gastric cancer mouse model (Gan mice) in which the mice develop inflammation-associated gastric tumors via activation of both the COX-2/PGE₂ pathway and Wnt signaling. Expression analyses using these in vivo models have revealed novel mechanisms of the inflammatory responses underlying gastric cancer development. PGE₂-associated inflammatory responses activate epidermal growth factor receptor (EGFR) signaling through the induction of EGFR ligands and ADAMs that release EGFR ligands from the cell membrane. In Gan mice, a combination treatment with EGFR and COX-2 inhibitors significantly suppresses gastric tumorigenesis. Moreover, PGE₂-associated inflammation downregulates tumor suppressor microRNA, miR-7, in gastric cancer cells, which suppresses epithelial differentiation. These results indicate that PGE₂-associated inflammatory responses promote in vivo gastric tumorigenesis via several different molecular mechanisms.     

Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia

The activation of microglia plays an important role in a variety of brain disorders by the excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂) and proinflammatory cytokines. We investigated here whether pinoresinol isolated from the fruits of Forsythia koreana Nakai inhibits the inflammatory responses in LPS-activated microglia. Pinoresinol inhibited the production of NO, PGE₂, TNF-α, IL-1β and IL-6 in LPS-activated primary microglia. Also, pinoresinol attenuated mRNA and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and proinflammatory cytokines in LPS-activation. However, most of these inhibitory effects of pinoresinol have been mediated by extracellular-signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) phosphorylation and the NF-κB dependent. The results suggest that pinoresinol attenuates inflammatory responses of microglia and could be potentially useful in modulation of inflammatory status in brain disorders.     

Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

Francisella tularensis induces the synthesis of prostaglandin E₂ (PGE₂) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE₂ synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA₂), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE₂ synthesis, cPLA₂ hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE₂. However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA₂ phosphorylation (a marker of activation) and PGE₂ synthesis. These results suggested to us that cPLA₂ is not responsible for the liberation of AA to be converted into PGE₂ by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE₂ biosynthesis. cPLA₂, on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE₂ synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.     

Immune cells and mediators involved in the inflammatory responses induced by a P-I metalloprotease and a phospholipase A2 from Bothrops atrox venom

Bothrops envenomations can promote severe inflammatory responses by inducing edema, pain, leukocyte recruitment and release of chemical mediators by local cells. In the present study, two toxins from Bothrops atrox venom (the P-I metalloprotease Batroxase and the acidic phospholipase A₂ BatroxPLA₂) were evaluated in relation to their inflammatory effects induced in vivo and in vitro, mainly focusing on the participation of different immune cells and inflammatory mediators. Both toxins mainly promoted acute inflammatory responses with significant recruitment of neutrophils in the early hours (1–4 h) after administration into the peritoneal cavity of C57BL/6 mice, and increased infiltration of mononuclear cells especially after 24 h. Among the mediators induced by both toxins are IL-6, IL-10 and PGE₂, with Batroxase also inducing the release of L-1β, and BatroxPLA₂ of LTB₄ and CysLTs. These responses pointed to possible involvement of immune cells such as macrophages and mast cells, which were then evaluated in vitro. Mice peritoneal macrophages stimulated with Batroxase produced significant levels of IL-6, IL-1β, PGE₂ and LTB₄, whereas stimulus with BatroxPLA₂ induced increases of IL-6, PGE₂ and LTB₄. Furthermore, both toxins were able to stimulate degranulation of RBL-2H3 mast cells, but with distinct concentration-dependent effects. Altogether, these results indicated that Batroxase and BatroxPLA₂ promoted local and acute inflammatory responses related to macrophages and mast cells and to the production of several mediators. Our findings should contribute for better understanding the different mechanisms of toxicity induced by P-I metalloproteases and phospholipases A₂ after snakebite envenomations.     

Inhibition of glutathione synthesis of Ascaris suum by buthionine sulfoximine

Abstrac t We investigated the effect of dl-buthionine-S,R-sulfoximine (BSO), a selective glutathione (GSH)-depleting agent, on the GSH synthesis of Ascaris suum. The GSH concentrations of the reproductive and muscle tissues of A. suum were determined to be 8.5±0.3 and 14.3±1.3 (n=3) nmol/mg protein, respectively. After treatment of the parasites with 10 μM BSO for 24 h, the GSH content of the reproductive tissue of A. suum was totally depleted as compared with that of untreated controls. However, the GSH levels of the muscle tissue were reduced to only 50% after treatment of the worms for 24 h with 10 μM BSO. Exogenous GSH had no significant effect on the GSH level of the parasites when the worms were incubated for 4 h in RPMI 1640 medium supplemented with 1 mM GSH. In the presence of exogenous GSH, BSO was less effective in depleting the GSH levels of the parasites, which may indicate that the parasites can replenish their GSH levels. GSH depletion, which has been discussed as being therapeutically effective when normal and tumor cells or parasites have markedly different requirements for GSH, may have applications in the development of drugs against nematode infections. Received: 7 September 1995 / Accepted: 15 November 1995     

Anticonvulsant effects of four neuropeptides in the rat hippocampus during self-sustaining status epilepticus

The plaques in multiple sclerosis (MS) autopsy tissue contain tumor necrosis factor-alpha (TNF-alpha) at high concentrations. Moreover, microglia are able to convert L-tryptophan to quinolinic acid. Thus, TNF-alpha and quinolinic acid are endogenous compounds which may compromise oligodendrocytes during inflammatory demyelination. It is also known that cellular functions depend on adequate concentrations of glutathione (GSH). As some apoptotic oligodendrocytes have been observed in MS plaques, it was therefore logical to determine whether oligodendrocyte apoptosis would occur in response to TNF-alpha, quinolinic acid or GSH depletion. Oligodendrocytes were treated in vitro with TNF-alpha, quinolinic acid and the GSH-depleting agent, buthionine sulfoximine (BSO), respectively, and the numbers of intact and apoptotic cells were counted. TNF-alpha reduced the numbers of mature oligodendrocytes, but not immature oligodendrocytes, without producing apoptosis. Quinolinic acid and BSO each caused oligodendrocyte loss via apoptosis, and GSH ethyl ester partly protected the cells against BSO. The data suggest that oligodendrocytes undergo apoptosis under adverse conditions that result from an endogenous toxicant or depletion of GSH.     

Glutathione deficiency intensifies ischaemia‐reperfusion induced cardiac dysfunction and oxidative stress

The efficacy of glutathione (GSH) in protecting ischaemia‐reperfusion (I‐R) induced cardiac dysfunction and myocardial oxidative stress was studied in open‐chest, stunned rat heart model. Female Sprague–Dawley rats were randomly divided into three experimental groups: (1) GSH‐depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg^–1, i.p.) 24 h prior to I‐R, (2) BSO injection (4 mmol kg^–1, i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg^–1, i.v.) infusion 1 h prior to I‐R, and (3) control (C), receiving saline treatment. Each group was further divided into I‐R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by ?50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia‐reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and –dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I‐R, whereas ±dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I‐R. For BSO + AT125, ±dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I‐R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I‐R. Rate‐pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I‐R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I‐R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole‐body GSH homeostasis.     

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

Objective

Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E₂ (PGE₂) are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE₂ by HGFs were examined.

Methods

HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE₂ levels were evaluated by ELISA.

Results

H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE₂ production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE₂ production. Up to 10 ??g/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE₂ production, but they were significantly increased at 100 ??g/ml. Similarly, 0.01 ??g/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE₂ production, but they were significantly increased at concentrations of 0.1 and 1 ??g/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE₂ production.

Conclusion

These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE₂ production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 ??g/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.     

<Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Induces RANKL in T-cells

Porphyromonas gingivalis is an oral pathogen highly implicated in chronic periodontitis, a disease characterized by inflammatory destruction of the tooth-supporting alveolar bone and eventually, tooth loss. T-cell innate immune responses are actively involved in this pathological process. Receptor activator of NF-κB Ligand (RANKL) is a cytokine that stimulates bone resorption, while its soluble decoy receptor osteoprotegerin (OPG) blocks its action. This study aimed to investigate in Jurkat T-cells the effects of P. gingivalis on the RANKL-OPG system and the major inflammatory mediator of bone resorption prostaglandin E₂ (PGE₂). P. gingivalis caused concentration-dependent up-regulation of RANKL gene expression and protein production, assessed by quantitative PCR and ELISA, respectively. PGE₂ production was also enhanced. However, OPG was not detected. In conclusion, P. gingivalis induces RANKL and PGE₂ in T-cells, potentially favoring bone resorption. These T-cell responses to P. gingivalis may contribute to the pathogenesis of inflammatory alveolar bone destruction occurring in chronic periodontitis.     

Prostaglandin E2 suppresses phytohemagglutin-induced immune responses of normal human monoculear cells by decreasing intracellular glutathione generation,but not due to increased DNA strand breaks or apoptosis

Prostaglandin E₂ (PGE₂) at concentrations more than 1×10^–8 M markedly suppressed the cell proliferation and release of soluble molecules of interleukin-2 receptor (sIL-2R), CD4 (sCD4) and CD8 (sCD8) from phytohemagglutinin (PHA)-stimulated normal human mononuclear cells (MNC) in a dose-related manner. To further elucidate the subcellular mechanism of the inhibitory effect of PGE₂ on PHA-stimulated MNC, intracellular concentration of glutathione (GSH) in PHA-stimulated MNC was sequentially measured from day 1 to day 3 by enzymic method. Furthermore, the effect of PGE₂ on nuclear DNA including DNA strand breaks in alkali treatment and DNA fragmentation (apoptosis) of PHA-stimulated MNC were also measured. We found intracellular GSH levels were significantly decreased in the early stage of lymphocyte activation (day 1), but no evidence of increased DNA stand breaks or apoptotic process appeared in 3-day culture. In addition, butathione sulfoximine (a specific GSH inhibitor) and dibutyryl cyclic AMP also exhibited both proliferation inhibition and GSH-decreasing effect on PHA-stimulated MNC as well as PGE₂. These results suggest that the immunosupressive effect of PGE₂ is mediated by the decreased generation of intracellular GSH, but not by the increased DNA strand breaks or apoptotic mechanism in the cells.     

Staphylococcus aureus induces expression of receptor activator of NF-kappaB ligand and prostaglandin E2 in infected murine osteoblasts

Osteomyelitis is an inflammatory disease of the bone that is characterized by the presence of necrotic bone tissue and increased osteoclast activity. Staphylococcus aureus is responsible for approximately 80% of all cases of human osteomyelitis. While the disease is especially difficult to treat, the pathogenesis of S. aureus-induced osteomyelitis is poorly understood. Elucidating the molecular mechanisms by which S. aureus induces osteomyelitis could lead to a better understanding of the disease and its progression and development of new treatments. Osteoblasts can produce several soluble factors that serve to modulate the activity or formation of osteoclasts. Receptor activator of NF-κB ligand (RANK-L) and prostaglandin E₂ (PGE₂) are two such molecules which can promote osteoclastogenesis and stimulate bone resorption. In addition, previous studies in our laboratory have shown that osteoblasts produce inflammatory cytokines, such as interleukin 6, following infection with S. aureus, which could induce COX-2 and in turn PGE₂, further modulating osteoclast recruitment and differentiation. Therefore, we hypothesized that following infection with S. aureus, osteoblasts will express increased levels of RANK-L and PGE₂. The results presented in this study provide evidence for the first time that RANK-L mRNA and protein and PGE₂ expression are upregulated in S. aureus-infected primary osteoblasts. In addition, through the use of the specific COX-2 inhibitor NS 398, we show that when PGE₂ production is inhibited, RANK-L production is decreased. These data suggest a mechanism whereby osteoblasts regulate the production of RANK-L during infection.     

Effects of captopril on factors affecting gastric mucosal integrity in aspirin-induced gastric lesions in Sprague-Dawley rats

Introduction

Captopril is an angiotensin-converting enzyme inhibitor, which is used as an antihypertensive agent and has shown antioxidant properties. This study aims at determining the effects of captopril on factors affecting gastric mucosal integrity in aspirin-induced gastric lesions.

Material and methods

Eighteen male Sprague-Dawley (200-250 g) rats that were given aspirin (40 mg/100 g body weight) were divided into three groups: the control, captopril (1 mg/100 g body weight daily) and ranitidine (2.5 mg/100 g body weight twice daily) groups. Ranitidine and captopril were given orally for 28 days. Rats in all groups were sacrificed and the parameters measured.

Results

Captopril reduced gastric acidity, and increased gastric glutathione (GSH) and prostaglandin E₂ (PGE₂) significantly in comparison to the control group. Captopril also reduced malondialdehyde (MDA) and gastric lesions insignificantly compared to the control group. Ranitidine healed the lesions significantly compared to the control group. There was no difference between ranitidine and captopril on the severity of lesions, gastric acidity, MDA and GSH. Captopril increased PGE₂ compared to ranitidine (p < 0.05).

Conclusions

Captopril has desirable effects on the factors affecting gastric mucosal integrity (acidity, PGE₂ and GSH) and is comparable to ranitidine in ulcer healing.     

Time-course of phospholipase A2, eicosanoid release and cellular accumulation in rat immunological air pouch inflammation

Phospholipase A₂ activity in the rat air pouch cavity was determined after induction of a reverse passive Arthus reaction. Time-course of phospholipase A₂ activity appeared to correlate with increased prostaglandin E₂ levels in inflammatory exudate and with the influx of mononuclear inflammatory cells.Local administration of anti-inflammatory drugs such as dexamethasone, indomethacin, or a PLA₂ inhibitor such as p-bromophenacyl bromide significantly inhibited exudate volume, cellular influx, granuloma formation, exudate PGE₂ levels and PLA₂ activity, to varying degrees. Dexamethasone treatment significantly reduced all parameters determined, whereas p-bromophenacyl bromide had a significant inhibitory effect on PLA₂ activity and PGE₂ release, and indomethacin only restored PGE₂ levels.These results show that PLA₂ is neither the only nor the most important factor involved in the development of subchronic inflammation.     

Lansoprazole inhibits nitric oxide and prostaglandin E(2) production in murine macrophage RAW 264.7 cells

Aberrantly activated macrophages, which overproduce inflammatory mediators, are involved in the pathogenesis of many inflammatory diseases. We analyzed the anti-inflammatory activity of lansoprazole (LPZ), a typical proton pump (P-ATPase) inhibitor, on RAW264.7 murine macrophages. Treatment of lipopolysaccharide (LPS)-stimulated RAW264.7 cells with LPZ inhibited the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). Since P-ATPase expression was not observed in RAW264.7 cells, the anti-inflammatory effect of LPZ was independent of ATPase. In contrast, diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, decreased NO but not PGE₂ levels. LPZ suppressed the LPS-stimulated production by RAW264.7 cells of reactive oxygen species (ROS), which plays an important role in inflammatory responses. ROS elevation in these cells was associated with NO but not PGE₂ production, suggesting that LPZ inhibits NO production by suppressing NADPH oxidase activity. These findings suggest that LPZ may be useful in the treatment of many inflammatory diseases associated with activated macrophages.     

Positive feedback effect of PGE2 on cyclooxygenase-2 expression is mediated by inhibition of Akt phosphorylation in human follicular dendritic cell-like cells

Prostaglandins (PGs) are bioactive lipid mediators generated from the phospholipids of cell membrane in response to various inflammatory signals. To understand the potential role of PGs in PG production itself during immune inflammatory responses, we examined the effect of PGE₂, PGF_2α, and beraprost on COX-2 expression using follicular dendritic cell (FDC)-like HK cells isolated from human tonsils. Those three PGs specifically augmented COX-2 protein expression in a dose-dependent manner after 4 or 8 h of treatment. The enhancing effect was also reflected in the actual production of PGs and the viable cell recovery of germinal center B-cells. To investigate the underlying molecular mechanism, we examined the impact of PI3K inhibitors on PG-induced COX-2 expression. Interestingly, COX-2 induction by PGE₂ and beraprost, but not PGF_2α, was enhanced by wortmannin and LY294002. In line with this result, Akt phosphorylation was inhibited by PGE₂ and beraprost but not by PGF_2α. The distinct effect of PGE₂ and beraprost from PGF_2α was reproduced in Akt-knockdowned HK cells. Our current findings imply that PGE₂ and PGI₂ stimulate COX-2 expression in FDC by inhibiting Akt phosphorylation. Additional studies are warranted to determine the potential role of Akt as a therapeutic target in patients with inflammatory disorders.     

TRPM2 channel protective properties of N-acetylcysteine on cytosolic glutathione depletion dependent oxidative stress and Ca2+ influx in rat dorsal root ganglion

N-acetylcysteine (NAC) is a thiol-containing (sulphydryl donor) antioxidant, which contributes to regeneration of glutathione (GSH) and also acts through a direct reaction with free radicals. Thiol depletion has been implicated in the neurobiology of sensory neurons and pain. We reported recently an activator role of intracellular GSH depletion on calcium influx through transient receptor potential melastatin-like 2 (TRPM2) channels in rat dorsal root ganglion (DRG). NAC may have a protective role on calcium influx through regulation of TRPM2 channels in the neurons. Therefore, we tested the effects of NAC on TRPM2 channel currents in cytosolic GSH depleted DRG in rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with buthionine sulfoximine (BSO). In whole-cell patch clamp experiments, TRPM2 currents in the DRG incubated with BSO were gated by H(2)O(2). TRPM2 channels current densities, cytosolic free Ca(2+) content, and lipid peroxidation values in the neurons were higher in H(2)O(2) and BSO + H(2)O(2) group than in controls; however GSH and GSH peroxidase (GSH-Px) values were decreased. BSO + H(2)O(2)-induced TRPM2 channel gating was totally inhibited by extracellular NAC and partially inhibited by 2-aminoethyl diphenylborinate. GSH-Px activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by NAC. In conclusion, we observed a modulator role of NAC on Ca(2+) influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. NAC incubation before BSO exposure appears to be more protective than NAC incubation after BSO exposure. Since cytosolic thiol group depletion is a common feature of neuropathic pain, our findings are relevant to the etiology and treatment of pain neuropathology in DRG neurons.     

Cholera Toxin Induces a Shift from Inactive to Active Cyclooxygenase 2 in Alveolar Macrophages Activated by Mycobacterium bovis BCG

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E₂ (PGE₂) by lung cells, including alveolar macrophages. PGE₂ plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE₂ by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE₂ release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE₂ release in the lungs and NE-associated COX-2 in the majority of COX-2⁺ macrophages. These COX-2⁺ macrophages were the primary source of PGE₂ release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE₂ release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE₂ release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.     

High cytokine levels in perforated acute otitis media exudates containing live bacteria

Acute otitis media (AOM) is an inflammatory response to microbes in the middle ear, sometimes associated with rupture of the tympanic membrane. Human leukocytes produce different patterns of inflammatory mediators in vitro when stimulated with Gram-positive and Gram-negative bacteria, respectively. Here, we investigated the cytokine and prostaglandin E₂ (PGE₂) responses in middle ear fluids (MEFs) from children with spontaneously perforated AOM, and related the mediator levels to the presence of pathogens detected by culture (live) or PCR (live or dead). Furthermore, the in vivo cytokine pattern was compared with that induced in leukocytes stimulated by dead bacteria in vitro. MEFs with culturable pathogenic bacteria contained more interleukin (IL)-1β (median: 110 μg/L vs. <7.5 μg/L), tumour necrosis factor (TNF) (6.3 μg/L vs. <2.5 μg/L), IL-8 (410 μg/L vs. 38 μg/L) and IL-10 (0.48 μg/L vs. <0.30 μg/L) than culture-negative fluids, irrespective of PCR findings. IL-6 and PGE₂ were equally abundant (69–110 μg/L) in effusions with live, dead or undetectable bacteria. Cytokine levels were unrelated to bacterial species and to the presence or absence of virus. Similar levels of TNF and IL-6 as found in the MEFs were obtained by in vitro stimulation of leukocytes, whereas 11 times more IL-1β and 3.5 times more IL-8 were produced in vivo, and 22 times more IL-10 was produced in vitro. Vigorous production of proinflammatory cytokines accompanies AOM with membrane rupture, regardless of the causative agent, but the production seems to cease rapidly once the bacteria are killed and fragmented. IL-6 and PGE₂, however, remain after bacterial disintegration, and may play a role in the resolution phase.