Testosterone inhibits tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in human aortic endothelial cells

It was shown that an increase in the bacteriochlorophyll (BChl) c antenna size observed upon lowering growth light intensities led to enhancement of the hyperchromism of the BChl c Q(y) absorption band of the green photosynthetic bacterium Chloroflexus aurantiacus. With femtosecond difference absorption spectroscopy, it was shown that the amplitude of bleaching of the oligomeric BChl c Q(y) band (as compared to that for monomeric BChl a) increased with increasing BChl c content in chlorosomes. This BChl c bleaching amplitude was about doubled as the chlorosomal antenna size was about trebled. Both sets of findings clearly show that a unit BChl c aggregate in the chlorosomal antenna is variable in size and governed by the grow light intensity, thus ensuring the high efficiency of energy transfer within the BChl c antenna regardless of its size.     

Effect of interleukin-1beta and tumor necrosis factor-alpha on gene expression in human endothelial cells

Interleukin-1(IL-1) and tumor necrosis factor- (TNF-) are two majorcytokines that rise to relatively high levels during systemicinflammation, and the endothelial cell (EC) response to these cytokinesmay explain some of the dysfunction that occurs. To better understandthe cytokine-induced responses of EC at the gene expression level,human umbilical vein EC were exposed to IL-1 or TNF- for varioustimes and subjected to cDNA microarray analyses to study alterations intheir mRNA expression. Of ~4,000 genes on the microarray, expressionlevels of 33 and 58 genes appeared to be affected by treatment withIL-1 and TNF-, respectively; 25 of these genes responded to bothtreatments. These results suggest that the effects of IL-1 andTNF- on EC are redundant and that it may be necessary to suppressboth cytokines simultaneously to ameliorate the systemic response.

     

Differential regulation of the human IFN-gamma receptor expression in Raji and IM9 lymphoblastoid cells versus THP-1 monocytic cells by IFN-gamma and phorbol myristate acetate

Expression of the human (hu) IFN-gamma-R has been studied in Raji and IM9 cells (two B lymphoblastoid cell lines) and in THP-1 cells (a monocytic cell line) with respect to IFN-gamma binding sites, receptor protein and mRNA levels. Although, in these three cell lines, the hu-IFN-gamma-R mRNA was expressed to the same extent, the high affinity receptor was expressed differently both in cell surface receptor binding and amount of receptor protein. Various ligands are able to modulate the expression of their own receptor. We investigated the modulation of the hu-IFN-gamma-R by its ligand. Hu-IFN-gamma induced a rapid and dose-dependent decrease of its cell surface receptor number without alteration of receptor affinity, amounts of receptor protein or hu-IFN-gamma-R mRNA accumulation and stability. Thus, in Raji, IM9, and THP-1 cells, the hu-IFN-gamma had no effect on its receptor gene expression and the cell surface decrease was simply due to ligand blocking and receptor internalization rather than true down-regulation. The second messenger in the hu-IFN-gamma signal transduction pathway is not well characterized, but activation of protein kinase C has been reported in some cases. Therefore, the modulation of the hu-IFN-gamma-R expression by PMA, a potent activator of protein kinase C and a modulator of other receptor expression, has been investigated. In Raji and IM9 cells, PMA had no or few effects on the cell surface receptor number and no detectable effect on the receptor protein or on mRNA levels. In contrast, in THP-1 cells, PMA treatment induced a time and dose-dependent five- to sixfold increase of the cell surface receptors due to a rapid and persistent increase of the hu-IFN-gamma-R gene expression in THP-1 cells was specifically inhibited or reversed by hu-IFN-gamma treatment. The modulation of the hu-IFN-gamma-R expression by PMA in THP-1 cells and by hu-IFN-gamma in PMA-treated THP-1 cells seems associated with their effect on monocyte-macrophage differentiation and/or macrophage activation.     

Intercellular adhesion molecule-1 and vascular endothelial growth factor expression kinetics in macrophage-derived foam cells

Macrophage-derived foam cells seem to play an important role during inflammatory response of atherosclerosis, in which the overexpression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF) are associated with the early and later pathological changes in foam cell formation. In this study, we investigated the expression kinetics of ICAM-1 and VEGF in macrophage-derived foam cells. The foam cell model was established through incubating the human monocyte line (U937 cells) with oxidized-low density lipoprotein (ox-LDL). Up-regulated expressions of ICAM-1 and VEGF were analyzed in protein and mRNA levels in U937 foam cells by flow cytometry, ELISA, and Northern blot. Kinetic studies showed the deferent kinds of expression curves in dose response and time course. The expression dose-kinetics demonstrated that the ICAM-1 showed the peak expression induced by ox-LDL 50 mg/L, while VEGF levels increased in a dose-dependent manner with the maximum level induced by ox-LDL 200 mg/L. Time-kinetic studies revealed that the ICAM-1 levels showed the peak expression in 12 h while VEGF expression increased in a time-dependent manner with the maximum level in 48 h. These results proved that both ICAM-1 and VEGF expressions were enhanced in the macrophage-derived foam cells, but ICAM-1 expression increased earlier than the up-regulation of VEGF; low dose of ox-LDL mainly up regulated ICAM-1 expression, while high dose mainly increased the VEGF expression.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-alpha -induced NF-kappa B activation and intercellular adhesion molecule-1 expression in endothelial cells.

In this study, we explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Studies were made in mice lacking the p47(phox) subunit of NAD(P)H oxidase as well as in cultured mouse lung vascular endothelial cells (MLVEC) from these mice. In response to TNFalpha challenge, NF-kappaB activation and ICAM-1 expression were significantly attenuated in lungs of p47(phox)(-/-) mice as compared with wild-type (WT) mice. The attenuated NF-kappaB activation in p47(phox)(-/-) mice was secondary to inhibition of NIK activity and subsequent IkappaBalpha degradation. Induction of neutropenia using anti-PMN serum prevented the initial TNFalpha-induced NF-kappaB activation and ICAM-1 expression in WT mice, indicating the involvement of PMN NAD(P)H oxidase in signaling these responses. Moreover, the responses were restored upon repletion with PMN obtained from WT mice but not with PMN from p47(phox)(-/-) mice. These findings were recapitulated in MLVEC co-cultured with PMN, suggesting that NF-kappaB activation and resultant ICAM-1 expression in endothelial cells occurred secondary to oxidants generated by the PMN NAD(P)H oxidase complex. The functional relevance of the PMN NAD(P)H oxidase in mediating TNFalpha-induced ICAM-1-dependent endothelial adhesivity was evident by markedly reduced adhesion of p47(phox)(-/-) PMN in co-culture experiments. Thus, oxidant signaling by the PMN NAD(P)H oxidase complex is an important determinant of TNFalpha-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.     

High-density lipoprotein subfraction 3 decreases ADAMTS-1 expression induced by lipopolysaccharide and tumor necrosis factor-alpha in human endothelial cells.

Endothelial expression of matrix metalloproteinases has been implicated in angiogenesis and endothelial cell proliferation. Recently, it has been shown that high-density lipoproteins (HDLs) promote angiogenesis. In the present study, we investigated the effects of native HDLs on the expression of several proteases and their inhibitors in human umbilical vein endothelial cells. We show that ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif) was potently induced by incubation with lipopolysaccharide or tumor necrosis factor-alpha and that the expression was significantly reduced in the presence of HDL subfraction 3. Since ADAMTS-1 has recently been shown to inhibit endothelial cell proliferation, the result of the present work may represent a new mechanism by which HDL could have a positive effect on endothelial cell and vascular wall function.     

Histamine and cis-urocanic acid augment tumor necrosis factor-alpha mediated induction of keratinocyte intercellular adhesion molecule-1 expression

Early cellular and molecular events in inflamed skin include the active participation of epidermal keratinocytes (KCs) and dermal mast cells which can produce diffusible mediators such as tumor necrosis factor-alpha (TNF-α), histamine, and urocanic acid (UCA). Rapid induction of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) by KCs is observed following a highly diverse array of stimuli which can provoke both irritant, inflammatory, as well as allergic and immune reactions. To determine if the aforementioned mediators could interact in either an additive or synergistic fashion with each other, cultured KCs were exposed to these mediators alone and in combination, and the degree of ICAM-1 mRNA and protein quantitated. Whereas histamine or cis-UCA alone only weakly induced KC ICAM-1, when they were combined with TNF-α, significant augmentation was observed by Northern blot hybridization studies, immunostaining, and FACS analysis. Other histamine derivatives such as L-histidine, 1-methylhistidine, 3-methylhistidine, or all-trans-UCA had no effect. Histamine pretreatment did not affect cell surface high affinity TNF-α receptors, as determined by ligand binding and immunodetection, and did not induce KC TNF-α production. The KC histamine receptor was also characterized and found not to be influenced by TNF-α, cis-UCA, all-trans-UCA, or diphenyhydramine (an H₁ antagonist), but it was inhibited by cimetidine (an H₂ antagonist). These results demonstrate that 1) KCs can be induced to express ICAM-1 by exposure to histamine and cis-UCA, 2) histamine and cis-UCA can also augment TNF-α inducible ICAM-1 mRNA and cell surface protein expression, 3) this augmentation does not directly involve changes in KC TNF-α receptor number, affinity, or TNF-α production and, 4) KCs possess a type 2 histamine receptor which is not the photoreceptor for UCA. These findings highlight the potential for cross-talk between molecules produced by resident cutaneous cell types above (i.e., KCs) and below (i.e., mast cells) the epidermal basement membrane zone. These cells and their mediators can cooperate to respond to either exogenous or endogenous stimuli leading to rapid and strong KC ICAM-1 expression. Such induction of this important adhesion molecule by KCs ensures the retention of T lymphocytes necessary to participate in the maintenance of cutaneous immunohomeostasis. © 1993 Wiley-Liss, Inc.     

Zymosan-stimulated tumor necrosis factor-alpha production by human monocytes. Down-modulation by phorbol ester.

In this study, we showed that human monocytes produced TNF-alpha in response to zymosan, a particulate agonist. Protein kinase C (PKC) seems to play a regulatory role in zymosan-induced TNF-alpha secretion. The pretreatment of monocytes with PMA induced a dose-dependent inhibition of zymosan-stimulated TNF production. This inhibition was likely due to an activation of PKC because it was prevented by inhibitors of PKC, sphingosine, and staurosporine. Moreover, PMA elicited a profound down-modulation of zymosan binding to monocytes. The inhibition of zymosan binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, PMA did not modify the expression of CD11b/CD18 receptor that is involved in zymosan recognition. In view of these findings, qualitative changes of CD11b/CD18 molecules might account for the inhibition of zymosan binding and TNF production. Thus, PMA specifically increased the association of CD11b/CD18 with the detergent-insoluble cytoskeleton. Cytochalasin B but not microtubule disrupters, nocodazole and colchicine, partially prevented the inhibition of zymosan binding. Hence, the inhibitory action of PMA on zymosan binding seems to be mediated by an increase in attachment of zymosan receptor to cytoskeleton and more likely to microfilaments. The regulatory activity of PKC might represent a first way of limiting cytokine over-production in response to pathogens which interact with monocytes via CD11/CD18 molecules.     

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.     

Disparate effects of tumor necrosis factor-alpha/cachectin and tumor necrosis factor-beta/lymphotoxin on hematopoietic growth factor production and neutrophil adhesion molecule expression by cultured human endothelial cells

Tumor necrosis factor-alpha/cachectin (TNF-alpha) and tumor necrosis factor-beta/lymphotoxin (TNF-beta) are inflammatory mediators with similar spectrums of cytotoxic activity against tumors in vitro and in vivo. We compared the effect of purified recombinant human TNF-alpha and TNF-beta on neutrophil adhesion molecule expression and hematopoietic growth factor production by cultured human umbilical vein endothelial cells. Endothelial cells acquired adhesive properties for neutrophils after a 4-hr incubation with as little as 5 U/ml TNF-alpha. TNF-alpha stimulated a dose-dependent increase in endothelial cell adhesiveness for neutrophils, with a maximal effect at 250 U/ml. In contrast, TNF-beta did not enhance endothelial-dependent neutrophil adherence until a concentration of 600 to 1200 U/ml was reached. Endothelial cells cultured for 24 hr with TNF-alpha, 10 to 1,000 U/ml, released hematopoietic colony-stimulating activity. TNF-beta failed to augment growth factor production by endothelial cells at any concentration tested. Inhibitor assays showed that the absence of detectable colony-stimulating activity was not due to direct inhibition of colony growth by TNF-beta or to release of hematopoietic inhibitors by the TNF-beta-stimulated endothelial cells. Purified natural TNF-beta was similar to recombinant TNF-beta in its effect on neutrophil adhesion molecule expression and growth factor production by endothelial cells. These results indicate that the two immunomodulatory proteins TNF-alpha and TNF-beta differ in their effects on a common target tissue. TNF-beta, which retains tumoricidal properties, shows fewer proinflammatory activities on cultured endothelial cells than TNF-alpha in vitro.