Charles S. T. Hii

Bacterial Lipopolysaccharide and Tumor Necrosis Factor Alpha Synergistically Increase Expression of Human Endothelial Adhesion Molecules through Activation of NF-κB and p38 Mitogen-Activated Protein Kinase Signaling Pathways

ABSTRACT One of the recognized associations of bacterial infection with cardiovascular events is the activation of endothelium and upregulation of adhesion molecules. The two major proinflammatory mediators implicated in the causation of cardiovascular events, bacterial lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF), were found to cooperate to enhance the adhesive properties of endothelial cells. These caused synergistic upregulation of intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells as determined by flow cytometry analysis and enzyme-linked immunosorbent assay. This synergism was not due to TNF causing an upregulation of CD14 expression. Treatment with both LPS and TNF resulted in a marked increase in the translocation of NF-κB into the nucleus. The activity of p38 mitogen-activated protein kinase was also synergistically enhanced, while the activity of c-jun N-terminal kinase was increased in an additive manner. The results demonstrate that LPS and TNF act synergistically to upregulate the expression of endothelial cell adhesion molecules, possibly by amplification of signaling pathways upstream of transcription. These findings have implications for the understanding of the acceleration of atherosclerotic events seen in low-grade infections with gram-negative organisms.

Synthesis and Surface Expression of CD14 by Human Endothelial Cells

ABSTRACT Previous studies have reported that human vascular endothelial cells lack the membrane-bound lipopolysaccharide (LPS) receptor, CD14 (mCD14). By optimizing assay conditions, including the selection of anti-CD14 monoclonal antibody, we now demonstrate that human umbilical vein endothelial cells (HUVEC) express CD14 on the cell surface. Single-passage HUVEC showed approximately 20 times less expression of CD14 than monocytes. Interestingly, there was significant loss of surface CD14 expression with increasing numbers of culture passages. Evidence for synthesis of CD14 by HUVEC was provided by the finding that l-[35S]methionine was incorporated into CD14. In addition, the expression of CD14 on HUVEC was upregulated by LPS, lysophosphatidic acid, and tissue culture supplements, and this upregulation was dependent on protein synthesis. Furthermore, the results imply that mCD14 is required for LPS-induced activation of endothelial cells in the absence of serum and that it acts in concert with serum factors (soluble CD14). Our results provide evidence that CD14 is expressed by endothelial cells and suggest that the previous inability to observe expression of this molecule has been due to culture and staining conditions. This finding has important implications for the understanding of the mechanisms by which LPS stimulates endothelial cells and the management of sepsis caused by gram-negative bacteria.

Activation of neutral sphingomyelinase in human neutrophils by polyunsaturated fatty acids.

Although unesterified polyunsaturated fatty acids (PUFA) have been shown to elicit marked changes in neutrophil function, the associated signal transduction processes require clarification. In this study we examined the effect of PUFA on the sphingomyelin (SM)‐signalling cycle in human neutrophils. Treatment of neutrophils with eicosatetraenoic acid [ arachidonic acid, 20:4(n‐6)] caused a decrease in the mass of cellular SM and an increase in the level of ceramide. 20:4(n‐6)‐stimulated neutral sphingomyelinase (SMase) activity of the leucocytes in a time‐ and concentration‐dependent manner. Other unsaturated fatty acids, docosahexaenoic [22:6(n‐3)], eicosapentaenoic [20:5(n‐3)], octadecenoic [oleic, 18:1(n‐9)] and octadecadienoic [linoleic, 18:2(n‐6)] acids also had the capacity to activate neutral SMase; however, certain 20:4(n‐6) derivatives {20:4(n‐6) methyl ester [20:4(n‐6)ME], 15‐hydroperoxyeicosatetraenoic (15‐HPETE) and 15‐hydroxyeicosatetraenoic (15‐HETE) acids}, very‐long‐chain PUFA {tetracosatetraenoic [24:4(n‐6)] and octacosatetraenoic [28:4(n‐6)] acids} and saturated fatty acids [octadecanoic (stearic, 18:0) and eicosanoic (arachidic, 20:0) acids] had no significant effect. Activation of neutral SMase by 20:4(n‐6) appeared to involve metabolism via 20:4(n‐6)CoA (arachidonoyl CoA) and was not dependent on prostaglandin and leukotriene synthesis. All of the fatty acids and derivatives tested failed to activate acidic SMase of neutrophils. Ceramide was found to inhibit 20:4(n‐6)‐induced superoxide generation by the cells. It is envisaged that the PUFA‐induced ceramide production in neutrophils plays a role in the regulation of biological responses.

Direct evidence that ERK regulates the production/secretion of interleukin‐2 in PHA/PMA‐stimulated T lymphocytes

Although p21ras, raf‐1 and MEK have been shown to regulate directly the transcriptional activity of NFAT (nuclear factor of activated T cells) and/or the interleukin‐2 (IL‐2) promoter, direct evidence that the extracellular signal‐regulated protein kinase (ERK) is involved in regulating IL‐2 production is still lacking. Here, we demonstrate that transfection of Jurkat cells with a dominant negative mutant of ERK1 (Erk1‐K71R) resulted in the suppression of mitogen‐stimulated production/secretion of IL‐2. This was accompanied by a parallel inhibition of mitogen‐stimulated ERK activity. These data provide direct evidence, for the first time, that ERK plays a vital role in regulating the production/secretion of IL‐2.

Neutrophil migration inhibitory properties of polyunsaturated fatty acids. The role of fatty acid structure, metabolism, and possible second messenger systems.

The n-3 polyunsaturated fatty acids (PUFA) appear to have antiinflammatory properties that can be partly explained by their biological activity on leukocytes. Since leukocyte emigration is an essential component of the inflammatory response, we have examined the effects of the n-3 PUFA (eicosapentaenoic and docosahexaenoic acids) on neutrophil random and chemotactic movement. Preexposure of neutrophils for 15-30 min to 1-10 micrograms/ml PUFA reduced the random and chemotactic migration to both FMLP- and fungi-activated complement. The inhibitory effect diminished with increasing saturation and carbon chain length, and methylation abolished this activity. Arachidonic and docosahexaenoic acids were the most active fatty acids. The PUFA concentration required to inhibit migration was dependent on cell number, suggesting that the fatty acid effects on leukocyte migration in vivo may be governed by the stage of the inflammatory response. It was concluded that the PUFA rather than their metabolites were responsible for the inhibition since: (a) antioxidants did not prevent the PUFA-induced migration inhibition and the hydroxylated intermediates were less active, and (b) inhibitors of the cyclooxygenase and lipoxygenase pathways were without effect. Inhibitors of protein kinases and calmodulin-dependent enzyme system did not prevent the PUFA-induced migration inhibition, which was also independent of phospholipase D-catalyzed hydrolysis of phospholipids. It is also shown that PUFA decrease the FMLP-induced Ca2+ mobilization.

Inhibition of Stimulus-Induced Endothelial Cell Intercellular Adhesion Molecule-1, E-Selectin, and Vascular Cellular Adhesion Molecule-1 Expression by Arachidonic Acid and Its Hydroxy and Hydroperoxy Derivatives

Localized adhesion of peripheral blood leukocytes to the endothelial lining is essential for their exit from the blood under both physiological and pathological conditions. The establishment, development, and resolution of the inflammatory response is regulated by an array of mediators, many of which remain to be categorized. These include arachidonic acid (20:4n-6) and its hydroperoxy (HPETE) and hydroxy (HETE) derivatives, which are released during inflammation. The data show that human umbilical vein endothelial cells, pretreated with these fatty acids, have a reduced ability to be stimulated by tumor necrosis factor-alpha (TNF-alpha) for enhanced neutrophil and monocyte adhesion; the order of inhibitory activity being 15-HPETE > 15-HETE > 20:4 (n-6). This fatty acid-induced inhibitory activity was reflected in the ability of the mediators to decrease the TNF-alpha-induced expression of the following endothelial adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1), measured by both enzyme-linked immunosorbent assay and flow cytometric analysis. TNF-alpha-induced increased expression of ICAM-1, E-selectin, and VCAM-1 mRNA was significantly depressed by 15-HPETE. Constitutively expressed ICAM-1 and ICAM-1 mRNAs were unchanged by the fatty acids. The saturated fatty acid 20:0 and the methyl ester of 20:4(n-6) had no inhibitory activity. The binding of TNF-alpha to its receptors was not altered by these fatty acids. The fatty acids also inhibited the expression of ICAM-1 and E-selectin induced by phorbol 12-myristate 13-acetate, showing that inhibition occurred at a post-TNF-alpha receptor binding level. The 15-HPETE was found to inhibit the TNF-alpha-induced increase in adhesion molecule expression in the early stage of the incubation, but expression returned to normal after 18 hours. An effect of 15-HPETE on the early cell signaling system was demonstrated by the ability of this fatty acid to inhibit agonist-induced protein kinase C translocation.

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

The Non-Genomic Actions of Vitamin D.

Since its discovery in 1920, a great deal of effort has gone into investigating the physiological actions of vitamin D and the impact its deficiency has on human health. Despite this intense interest, there is still disagreement on what constitutes the lower boundary of adequacy and on the Recommended Dietary Allowance. There has also been a major push to elucidate the biochemistry of vitamin D, its metabolic pathways and the mechanisms that mediate its action. Originally thought to act by altering the expression of target genes, it was realized in the mid-1980s that some of the actions of vitamin D were too rapid to be accounted for by changes at the genomic level. These rapid non-genomic actions have attracted as much interest as the genomic actions and they have spawned additional questions in an already busy field. This mini-review attempts to summarise the in vitro and in vivo work that has been conducted to characterise the rapid non-genomic actions, the mechanisms that give rise to these properties and the roles that these play in the overall action of vitamin D at the cellular level. Understanding the effects of vitamin D at the cellular level should enable the design of elegant human studies to extract the full potential of vitamin D to benefit human health.

Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response.

The role of the extracellular signal-regulated kinase (ERK) 1 and ERK2 in the neutrophil chemotactic response remains to be identified since a previously used specific inhibitor of MEK1 and MEK2, PD98059, that was used to provide evidence for a role of ERK1 and ERK2 in regulating chemotaxis, has recently been reported to also inhibit MEK5. This issue is made more critical by our present finding that human neutrophils express mitogen-activated protein (MAP) kinase/ERK kinase (MEK)5 and ERK5 (Big MAP kinase), and that their activities were stimulated by the bacterial tripeptide, formyl methionyl-leucyl-phenylalanine (fMLP). Dose response studies demonstrated a bell-shaped profile of fMLP-stimulated MEK5 and ERK5 activation, but this was left-shifted when compared with the profile of fMLP-stimulated chemotaxis. Kinetics studies demonstrated increases in kinase activity within 2 min, peaking at 3–5 min, and MEK5 activation was more persistent than that of ERK5. There were some similarities as well as differences in the pattern of activation between fMLP-stimulated ERK1 and ERK2, and MEK5-ERK5 activation. The up-regulation of MEK5-ERK5 activities was dependent on phosphatidylinositol 3-kinase. Studies with the recently described specific MEK inhibitor, PD184352, at concentrations that inhibited ERK1 and ERK2 but not ERK5 activity demonstrate that the ERK1 and ERK2 modules were involved in regulating fMLP-stimulated chemotaxis and chemokinesis. Our data suggest that the MEK5-ERK5 module is likely to regulate neutrophil responses at very low chemoattractant concentrations whereas at higher concentrations, a shift to the ERK1/ERK2 and p38 modules is apparent.

The Immunomodulatory Effects of Novel β-Oxa, β-Thia, and γ-Thia Polyunsaturated Fatty Acids on Human T Lymphocyte Proliferation, Cytokine Production, and Activation of Protein Kinase C and MAPKs

We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (β-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other novel PUFA consisting of β-oxa, β-thia, and γ-thia compounds. All three types displayed anti-inflammatory activity. Each of the unsaturated β-oxa fatty acids showed similar inhibition of PHA-PMA-induced T cell proliferation with a parallel inhibition of TNF-β production. However, β-oxa 25:6n-3 and β-oxa 21:4n-3 displayed lower inhibitory action on IFN-γ production. Surprisingly, β-oxa 23:4n-6 and β-oxa 21:3n-6 had marginal effect on IL-2 production. Thus, structural variation can generate selectivity for different immunological parameters. The β-thia compounds 23:4n-6, 21:3n-6, and 21:3n-3 were highly effective in inhibiting all immunological responses. Of the two γ-thia PUFA tested, γ-thia 24:4n-6 was a strong inhibitor of all responses apart from IL-2, but γ-thia 22:3n-6 had very little inhibitory effect. Two of the most active compounds, β-thia 23:4n-6 and β-thia 21:3n-6, were studied in more detail and shown to have an IC50 of 1–2 μM under optimal conditions. Thus, these PUFA retain the immunosuppressive properties of the n-3 PUFAs, 20:5n-3 and 22:6n-3, but not the neutrophil-stimulating properties. Their action on T lymphocytes is independent of cyclooxygenase or lipoxygenase activity, and they act at a postreceptor-binding level by inhibiting the activation of protein kinase C and ERK1/ERK2 kinases.