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Prostaglandin E2 production by tissue-fixed macrophages (Mphi) after severe injury contributes to an enhanced susceptibility to infection and sepsis. The purpose of this study was to investigate the effect of cyclic adenosine monophosphate (cAMP) on prostaglandin (PGE2) production and cyclooxygenase II (COX-2) gene activation in LPS-stimulated macrophages (Mphi). RAW264.7 cells, a mouse Mphi cell line, were exposed to various concentrations of dibutyryl cAMP +/- lipopolysaccharide (10 microg/mL) stimulation. Total Mphi ribonucleic acid (RNA) was harvested for the determination of COX-2 messenger RNA (mRNA) with mouse complementary deoxyribonucleic acid (cDNA) by Northern blot assay. Mphi supernatant was collected for the measurement of tumor necrosis factor (TNF) by L929 bioassay and PGE2 by enzyme-linked immunosorbent assay (ELISA), respectively. Mphi NFkappaB activity was determined by electrophoretic mobility shift assays (EMSA). Dibutyryl cAMP significantly inhibited TNF production by LPS-stimulated Mphi. Dibutyryl cAMP (1 mM) alone induced PGE2 production. Dibutyryl cAMP (100 microM and 1 mM) also augmented PGE2 production by LPS-stimulated Mphi. Dibutyryl cAMP had similar effect on Mphi COX-2 mRNA expression and NFkappaB activity. Our data demonstrate that cAMP modulates Mphi TNF production and upregulates COX-2 gene and PGE2 production.

Background: Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects. Although fish oil is readily incorporated into the cell membrane and influences the production of eicosanoids, the exact mechanism is not clear. This study was designed to investigate the effects of eicosapentaenoic acid (EPA), a major component of fish oil, on macrophage (Mphi) cyclooxygenase (COX) gene expression induced by LPS., Methods: RAW 264.7 cells, a mouse Mphi cell line, were grown in EPA-rich media for 24 h. Mphi were washed and exposed to Escherichia coli LPS (10 microg/ml). Membrane lipid profile was determined by gas chromatographic analysis. COX-1 and COX-2 mRNA expressions were determined by Northern blot assays with mouse-specific cDNA probes. PGE(2) production of Mphi was measured by ELISA. Mphi production of COX-2 protein was determined by Western blot assays with an anti-COX-2 antibody., Results: Incubation in EPA-rich media increased membrane EPA and decreased arachidonic acid (AA) composition. COX-2 mRNA expression was induced by EPA and further augmented by LPS stimulation. EPA also augmented Mphi production of COX-2 protein. In comparison, COX-1 mRNA expression was not affected by either LPS stimulation or EPA incubation. EPA reduced PGE(2) production by LPS-stimulated Mphi. To further support that COX-2 mRNA was regulated by COX product, exogenous PGE(2) was added to Mphi prior to LPS stimulation. PGE(2) reduced COX-2 mRNA of LPS-stimulated Mphi., Conclusion: EPA displaces AA and reduces PGE(2) production by LPS-stimulated Mphi. Fish oil inhibition of Mphi PGE(2) production induces COX-2 mRNA expression through a COX-2 product-mediated feedback mechanism., (Copyright 1999 Academic Press.)

Metabolism of macrophage (MO) membrane phospholipids produces key mediators of inflammation and major second messengers that modulate inflammatory responses during sepsis. Sphingomyelin is a major class of phospholipid that releases ceramide and sphingosine. This study was designed to investigate the involvement of sphingomyelin metabolites in MO activation by lipopolysaccharide (LPS). Rabbit alveolar MO were obtained by bronchoalveolar lavage and exposed to C6-ceramide, a cell-permeable analogue of natural ceramide, or sphingosine in the presence of Escherichia coli LPS (100 ng/mL). Tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays. Total nuclear extract was harvested for the measurement of nuclear factor KB (NFkappaB) with electrophoretic mobility shift assays. MO TNF production was measured by L929 bioassays. C-6 ceramide did not have any effects on MO TNF production or TNF mRNA expression with or without LPS stimulation. Inhibition of ceramide metabolism with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), or N-oleoyl-ethanolamine (NOE) also did not induce TNF mRNA or TNF production. In comparison, sphingosine inhibited TNF mRNA expression as well as TNF production of LPS-stimulated MO. LPS-induced MO NFkappaB activity was also reduced by sphingosine. Our data indicate that ceramide alone has no effect on macrophage activity, but its metabolite sphingosine down-regulates MO activation induced by LPS stimulation. Therefore, the sphingomyelin pathway is involved in the regulation of MO activation.

Background: Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects, though the exact mechanism(s) are unknown. This study investigated the effects of eicosapentanenoic acid (EPA), a major component of fish oil, on transcriptional regulation of tumor necrosis factor (TNF) gene in lipopolysaccharide (LPS)-stimulated macrophages (MO)., Methods: RAW 264.7 cells, a mouse MO cell line, were grown in EPA-rich media for 24-48 h. MO were washed and exposed to Escherichia coli LPS (1 microg/ml) for 2 h. TNF mRNA expression was measured by Northern blot assays. Total nuclear extracts were harvested for the measurement of NF kappa B with electrophoretic mobility shift assays. Supershift assays were performed with anti-P50 or anti-P65 antibodies to show components of NF kappa B dimers. TNF production was determined by L929 bioassays., Results: LPS stimulated RAW cell TNF mRNA expression and NF kappa B activity. In contrast, RAW cells grown in EPA-rich media had less TNF mRNA expression and an altered composition of the NF kappa B subunits (P65/P50 dimers) in the presence of LPS. TNF production by LPS-stimulated MO was reduced by EPA., Conclusions: The inhibitory effect of EPA on LPS-stimulated MO TNF gene transcription and protein elaboration is, in part, mediated through altering NF kappa B activation by reducing the P65/P50 dimers., (Copyright 1999 Academic Press.)

Background: Adult respiratory distress syndrome (ARDS) causes severe morbidity and mortality in trauma patients. One potential method to attenuate the lung injury is to inhibit alveolar macrophage production of proinflammatory mediators. The purpose of this study was to investigate the cellular mechanism of interleukin 10 (IL-10) inhibition on LPS-stimulated macrophage (Mphi). We hypothesized that IL-10 inhibited phospholipase C signal pathways in Mphi. IL-10 inhibition would be restored by calcium ionophores and protein kinase C (PKC) activation., Methods: Rabbit alveolar Mphi were obtained by bronchoalveolar lavage. Mphi were treated with Escherichia coli LPS (10 ng/ml) in the presence of various concentrations of human IL-10. Cell lysates and supernatant were analyzed for proagulants (PCA) and tumor necrosis factor (TNF), respectively. TNF mRNA expression of alveolar Mphi was also measured by Northern Blot assay. Macrophage PGE2 production was measured by ELISA., Results: IL-10 inhibited the production of both TNF and PCA by LPS-stimulated Mphi. In addition, IL-10 also reduced TNF mRNA expression. Similarly, PGE2 production by LPS-stimulated Mphi was also attenuated by IL-10. An increase in the intracellular [Ca2+] induced by A23187 failed to reverse this IL-10-mediated inhibition. In comparison, phorbol myristate acetate, a protein kinase C (PKC) activator, restored TNF and PCA production despite the presence of IL-10., Conclusions: IL-10 inhibits Mphi production of inflammatory mediators. This inhibition is, at least in part, mediated by modulating the PKC activity., (Copyright 1998 Academic Press.)

Background: Prostaglandin E2 (PGE2) is a major contributor to the production and maintenance of immunosuppression after overwhelming injury, leading to increased infectious morbidity and mortality in trauma patients. Elucidation of the cellular pathways involved in PGE2 production could lead to potential therapeutic interventions. The purpose of this study was to determine the role of cyclooxygenase II (COX-2) in PGE2 production by Mphi and to investigate the cellular mechanism of COX-2 gene activation., Methods: Mouse macrophages (Mphi), RAW 264.7, were exposed to Escherichia coli lipopolysaccharide (LPS) in the presence of cyclooxygenase inhibitors (ibuprofen or NS398). COX-1 and COX-2 mRNA expression and PGE2 production were measured by Northern blot assay and enzyme-linked immunosorbent assay, respectively. Nuclear factor kappaB (NFkappaB) activity was measured by electrophoretic mobility shift assay. To elucidate the role of NFkappaB in LPS-induced COX-2 gene activation, Mphi were exposed to LPS in the presence of an NFkappaB inhibitor, TPCK., Results: LPS increased Mphi COX-2 mRNA expression but had no effect on COX-1 mRNA expression. Both ibuprofen and NS398 inhibited COX-2 mRNA as well as PGE2 production by LPS-stimulated Mphi. In addition, LPS-induced NFkappaB activity was attenuated by these agents. Inhibition of NFkappaB with TPCK reduced COX-2 but not COX-1 gene expression and decreased PGE2 production by LPS-stimulated Mphi., Conclusion: Our data indicate that COX-2 gene expression by LPS-stimulated Mphi is dependent on NFkappaB. Cyclooxygenase inhibitors reduced PGE2 production by inhibiting both COX-2 mRNA expression and preventing NFkappaB activation.

Objective: Vascular endothelial cells (EC) play a pivotal role in diffuse organ injury seen in ARDS and MOFS. On exposure to cytokines or endotoxin (LPS) EC are stimulated to express adhesion molecules as well as proinflammatory and procoagulant activity. However, the potential feedback control of EC on macrophages (M-theta) is not clear. We studied the cellular mechanism of iloprost, a PGI2 analogue, in regulation of TNF production by LPS-stimulated M-theta., Methods: Rabbit alveolar M-theta and mouse M-theta RAW 264.7 cells were exposed to Escherichia coli LPS in the presence of various concentrations of iloprost. TNF production was measured by L929 bioassays. To further study the cellular mechanism of iloprost on M-theta activation, RAW 264.7 cells were stimulated by LPS (10 micrograms/ml) in the presence of either iloprost or specific mitogen-activated protein kinase (MAPK) inhibitors, either PD98059 or SB202190. P44/P42 and P38 MAPK activation were evaluated by Western blot assays with anti-phospho MAPK antibodies., Results: LPS induced M-theta TNF production, which was inhibited by iloprost. Iloprost also attenuated the activation of P44/P42 and P38 induced LPS. Inhibition of P44/P42 with PD98059 or P38 with SB202190 significantly reduced TNF production by LPS-stimulated RAW cells., Conclusions: The regulatory mechanism of EC on M-theta activation is dependent on PGI2. The effect of PGI2 on M-theta is, at least in part, mediated through inhibiting MAPKs.

Background: The development of multiple organ failure in septic patients is due to a systemic inflammation orchestrated by macrophages (Mphi). Elucidation and control of the mechanism involved in Mphi activation in sepsis is crucial to improving survival. An early event of Mphi activation involves the hydrolysis of membrane phospholipid by phospholipase A2 (PLA2) and subsequent generation of platelet-activating factor (PAF)., Objective: We designed this study to test the hypothesis that Mphi gene expression depends on PAF., Design: Rabbit alveolar Mphi were obtained by bronchoalveolar lavage and were stimulated with 10 ng/mL of Escherichia coli endotoxin lipopolysaccharide (LPS), PAF (1 micromol/L), LPS+/-CV3988 (10 micromol/L), a PAF receptor antagonist, or LPS+/-PLA2 inhibitors: AACOCF3 (50 micromol/L) or manoalide (10 micromol/L). After 4 hours of incubation, Mphi tumor necrosis factor (TNF) messenger RNA (mRNA) expression was assessed by Northern blot analyses. The TNF production in the Mphi supernatant was measured by L929 bioassays., Results: The LPS-stimulated Mphi expressed increased levels of TNF mRNA and produced an enormous amount of TNF. CV3988, a PAF antagonist, inhibited LPS-induced TNF mRNA. Furthermore, inhibiting PAF production with AACOCF3, or manoalide, also inhibited LPS-induced Mphi TNF mRNA expression. The effect of PAF depends on changes in intracellular calcium concentration. Inhibitors of calcium flux attenuated the PAF effects on LPS-stimulated Mphi., Conclusions: Our data suggest that LPS-induced Mphi gene expression is mediated by PAF. It is likely that modulation of PAF production or activity may be beneficial in down-regulating the overactivity of Mphi in sepsis.

Liver ischemia and reperfusion injury is mediated by oxygen free radicals, cytokines, and prostanoids produced by Kupffer cells and infiltrating neutrophils. Fish oil-supplemented diets alter membrane phospholipid composition and modify prostanoids and cytokine production in response to ischemia and reperfusion. This study tested the hypothesis that a fish oil-supplemented diet would attenuate warm liver ischemia and reperfusion injury in the rat. Male Sprague-Dawley rats were fed Vital HN supplemented with either fish oil (FO) or corn oil (CO) by the continuous duodenal infusion for 5 days. Total dietary fat (26% of total calories), caloric intake (70 cal/day), and volume (60 ml/day) were identical between two groups. Plasma eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels increased significantly in rats fed fish oil (0 to 16.3% for EPA and 2 to 12% for DHA). Liver histology was similar in both groups before ischemia. On Day 6, rats were subjected to 60 min of reversible hepatic ischemia. Plasma TNF levels, 1 and 24 hr after reperfusion, were not different between FO and CO rats. Liver injury assessed by bile flow, histology, plasma ALT, and bile glutathione efflux did not differ between groups. We conclude that our fish oil-supplemented enteral diet does not attenuate warm liver ischemia and reperfusion injury in rats.

Background: Nuclear factor kappa B (NF kappa B) is an important transcriptional activator protein and is a crucial component of the host's response to infection. The activation of NF kappa B is correlated with the phosphorylation of inhibitory kappa B (I kappa B) and its subsequent degradation. We hypothesized that protease inhibitors which prevented I kappa B degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines., Methods: Rabbit alveolar macrophages (M phi) were obtained by bronchoalveolar lavage. M phi were exposed to Escherichia coli lipopolysaccharide (LPS) (10 ng/ml) in the presence of various concentrations of protease inhibitors, either N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) or N-benzoyl-L-tyrosine ethyl ester (BTEE). Total RNA was extracted for Northern blot assay of tumor necrosis factor (TNF) mRNA expression using a rabbit genomic DNA probe. Total nuclear extracts were also obtained for the measurement of the NF kappa B activity with the electrophoretic mobility shift assay. The TNF production in the M phi supernatant was measured by L929 bio-assays., Results: NF kappa B activity induced by LPS was inhibited by either BTEE or TPCK. Inhibition of NF kappa B activity by these agents also prevented TNF mRNA expression and TNF production induced by LPS. The cellular mechanism leading to NF kappa B activation was further studied. TNF mRNA expression and NF kappa B activation were inhibited by D609, a phospholipase C (PLC) inhibitor, as well as by protein kinase C (PKC) inhibitors. In addition, direct stimulation of PKC led to NF kappa B activation and TNF mRNA expression., Conclusions: These data suggest that TNF mRNA expression of LPS-stimulated M phi is mediated through NF kappa B, NF kappa B activation is intimately regulated by the PLC signaling pathway.

Background: Eicosanoids play an important role in many aspects of systemic inflammatory responses and host defense. Although the synthesis of eicosanoids by different enzymes has been elucidated, the regulatory mechanism of eicosanoid production is not clear. We designed this study to investigate the hypothesis that PGE2 production by endotoxin (lipopolysaccharide; LPS)-stimulated macrophages (MO) is dependent on phospholipase C (PLC) signaling pathways., Methods: Rabbit alveolar macrophages (MO) were obtained by bronchoalveolar lavage. MO were suspended in RPMI-1640 medium at 1 x 10(6)/mL and were exposed to Escherichia coli LPS (10 ng/mL) +/- various agonists and antagonists of PLC and its secondary messengers. After 24 hours of incubation, prostaglandin E2 (PGE2) production was measured by ELISA., Results: LPS-activated MO produced four times as much PGE2 as did control unstimulated MO. The increase in PGE2 production was inhibited by PLC inhibitors (U73122 or D609) and a low-molecular-weight PLA2 inhibitor, manoalide. An increase in intracellular calcium and activation of both the calmodulin and protein kinase C kinase pathways increase PGE2 production., Conclusions: PGE2 production is intimately dependent on several phospholipases. Production is not only dependent on low-molecular-weight PLA2 cleavage of arachidonic acid from membrane phospholipids, but also by-products of PLC activation. PLC-dependent intracellular Ca-calmodulin signaling and protein kinase C activation provide significant modulation of PGE2 production.

A new method, the motif identification neural design (MOTIFIND), has been developed for rapid and sensitive protein family identification. The method is an extension of our previous gene classification artificial neural system and employs new designs to enhance the detection of distant relationships. The new designs include an n-gram term weighting algorithm for extracting local motif patterns, an enhanced n-gram method for extracting residues of long-range correlation, and integrated neural networks for combining global and motif sequence information. The system has been tested and compared with several existing methods using three protein families, the cytochrome c, cytochrome b and flavodoxin. Overall it achieves 100% sensitivity and > 99.6% specificity, an accuracy comparable to BLAST, but at a speed of approximately 20 times faster. The system is much more robust than the PROSITE search which is based on simple signature patterns. MOTIFIND also compares favorably with BLIMPS, the Hidden Markov Model and PROFILESEARCH in detecting fragmentary sequences lacking complete motif regions and in detecting distant relationships, especially for members of under-represented subgroups within a family. MOTIFIND may be generally applicable to other proteins and has the potential to become a full-scale database search and sequence analysis tool.

Background: Alterations in macrophage (M phi) function are responsible, in part, for adult respiratory distress syndrome and multiple organ failure developing in patients with sepsis. Elucidation and control of these M phi mechanisms during sepsis are crucial to our understanding of this disease and, ultimately, to improving survival of these patients., Objective: To investigate the involvement of calcium flux in endotoxin-induced alveolar M phi production of tumor necrosis factor (TNF) and procoagulant (PC) activity., Design: Rabbit alveolar M phi obtained by bronchoalveolar lavage were exposed to endotoxin in the form of lipopolysaccharide (LPS) extracted from Escherichia coli 0111:B4 in the presence of different specific calcium agonists and antagonists. The TNF expression was measured in the supernatant by L929 bioassays. The PC activity was determined in cell lysates by a one-step coagulation assay., Results: Macrophages activated by LPS produce enormous levels of TNF and PC. Either W7 (20 mumol/L), a calmodulin antagonist, or TMB-8 (50 mumol/L), which prevents calcium release from the endoplasmic reticulum, inhibited production of both TNF and PC activity. Verapamil (50 mumol/L) alone or combined with TMB-8 significantly inhibited both TNF and PC production by LPS-stimulated M phi. Elevating intracellular calcium ([Ca2+]i), using the calcium ionophore, A23187, or thapsigargin alone, did not induce M phi production of TNF but significantly augmented LPS-stimulated TNF production., Conclusion: Our results indicate that increased intracellular calcium causing signal transduction activation through the calmodulin pathway is a necessary, but insufficient, component of the LPS signaling in M phi.