Intracellular sequestration of amiodarone: role of vacuolar ATPase and macroautophagic transition of the resulting vacuolar cytopathology.

BACKGROUND AND PURPOSE: Tissue deposits of the anti-arrhythmic drug amiodarone are a major source of side effects (skin discoloration, etc.). We addressed the mechanism of the concentration of amiodarone in cells, and characterized the resulting vacuolar cytopathology and its evolution towards macroautophagy. EXPERIMENTAL APPROACH: Sequestration of amiodarone in human cells (macrophages, smooth muscle cells, HEK 293a cells) was evaluated using its violet fluorescence and cytopathology using GFP-conjugated subcellular markers. Autophagic signalling was probed by immunoblotting for the effector protein LC3. A patient biopsy of amiodarone-induced blue-gray skin discoloration was investigated for the presence of macroautophagy (immunofluorescence for LC3). KEY RESULTS: Most of the amiodarone (1-20 microM, 4-24 h) captured by cultured cells (macrophages were most avid) was present in enlarged vacuoles. The specific vacuolar ATPase (V-ATPase) inhibitors, bafilomycin A1 or FR167356, prevented vacuolization and drug uptake. Vacuoles in HEK 293a cells were positive for markers of late endosomes and lysosomes (GFP-Rab7, -CD63) and for an effector of macroautophagy, GFP-LC3. The vacuoles accumulated endogenous LC3 and filled with lipids (Nile red staining) following longer amiodarone treatments (> or =24 h). The electrophoretic mobility of both GFP-LC3 and endogenous LC3 changed, showing activation in response to amiodarone. Paraffin tissue sections of the pigmented skin exhibited granular LC3 accumulation in superficial dermis macrophages. CONCLUSION AND IMPLICATIONS: Vacuolar sequestration of amiodarone occurs at concentrations close to therapeutic levels, is mediated by V-ATPase and evolves towards persistent macroautophagy and phospholipidosis. This cytopathology is not cell type specific, but tissue macrophages appear to be particularly susceptible.

Two-year effects of alendronate on bone mineral density and vertebral fracture in patients receiving glucocorticoids: a randomized, double-blind, placebo-controlled extension trial.

OBJECTIVE: To evaluate the continued efficacy and safety of alendronate (ALN) for up to 2 years in patients receiving glucocorticoids. METHODS: This is a 12-month extension of a previously completed 1-year trial of daily ALN, performed to evaluate the effects of ALN over a total of 2 years in 66 men and 142 women continuing to receive at least 7.5 mg of prednisone or equivalent daily. All patients received supplemental calcium and vitamin D. The primary end point was the mean percentage change in lumbar spine bone mineral density (BMD) from baseline to 24 months. Other outcomes included changes in hip and total body BMD, biochemical markers of bone turnover, radiographic joint damage of the hands, and vertebral fracture incidence. RESULTS: The mean (+/-SEM) lumbar spine BMD increased by 2.8 +/- 0.6%, 3.9 +/- 0.7%, and 3.7 +/- 0.6%, respectively, in the groups that received 5 mg, 10 mg, and 2.5/10 mg of ALN daily (P < or = 0.001) and decreased by -0.8 +/- 0.6% in the placebo group (P not significant) over 24 months. In patients receiving any dose of ALN, BMD was increased at the trochanter (P < or = 0.05) and maintained at the femoral neck. Total body BMD was increased in patients receiving 5 or 10 mg ALN (P < or = 0.01). These 2 dose levels of ALN were more effective than placebo at all sites (P < or = 0.05). Bone turnover markers (N-telopeptides of type I collagen and bone-specific alkaline phosphatase) decreased 60% and 25%, respectively, during treatment with ALN (P < or = 0.05). There were fewer patients with new vertebral fractures in the ALN group versus the placebo group (0.7% versus 6.8%; P = 0.026). The safety profile was similar between treatment groups. CONCLUSION: Alendronate is an effective, well-tolerated therapy for the prevention and treatment of glucocorticoid-induced osteoporosis, with sustained treatment advantages for up to 2 years.

Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.

Long-term influence of inhaled corticosteroids on bone metabolism and density. Are biological markers predictors of bone loss?

Long-term effects of high doses of inhaled corticosteroids (ICS) on bone density and metabolism are still uncertain. Fifty-one patients (37 male, 14 female) using beclomethasone or budesonide at a daily dose > 800 microgram/d (high-dose group [Group HD] mean: 983 microgram/d [prescribed dose x estimated compliance]) or no or < 500 microgram/d (control group [Group C] mean: 309 microgram/d) for more than 5 yr were enrolled in this study. Each had, 3 yr ago and at this last evaluation, a clinical evaluation and measurements of expiratory flows and of bone density and bone metabolism markers. Lumbar spine bone density (last visit) was similar in the two groups with respective values of 0.94 +/- 0.03 (HD) and 0.96 +/- 0.03 g/cm2 (C) (p > 0.05). T and Z scores were -1.21 +/- 0.19 and -0.70 +/- 0.18 (HD), -0.95 +/- 0.25 and -0.47 +/- 0.21 (C) respectively (p > 0.05). A correlation was found between the decrease in bone density and the mean daily dose of corticosteroid in Group HD although these changes were quite small, mean bone density being unchanged over the 3-yr period. Serum and urinary parameters were similar in the two groups. Furthermore, neither initial bone density nor any of the biological parameters could predict changes in bone density over a period of 3 yr. In conclusion, bone density was similar in both study groups and not significantly different over a 3-yr period. Neither initial bone density nor biological markers of bone metabolism helped to predict changes in bone mass.

Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils.

Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX-2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM-CSF, TNF-alpha, and fMLP, induced COX-2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX-2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX-2 protein expression was initiated. In LPS-treated neutrophils, efficient induction of COX-2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX-2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX-2, raising the possibility that phospholipase D activation might take part in the process of COX-2 induction. Major COX-2-derived prostanoids synthesized by inflammatory neutrophils were identified by liquid-chromatography and tandem mass-spectrometry as TXA2 and PGE2. The agonist-induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX-2 inhibitor NS-398. These results show that COX-2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX-2 isoform is preeminent over COX-1 for the stimulated-production of prostanoids, and also suggest that neutrophil COX-2 displays a distinct profile of expression among circulatory cells.

Interleukin-1 expression during hyperoxic lung injury in the mouse.

An important component of the pathophysiologic response to hyperoxia (O2) is pulmonary inflammation, although the roles of specific inflammatory mediators during pulmonary O2 toxicity are not completely known. Interleukin-1 (IL-1) is an early inflammatory mediator and is sufficient to elicit many of the responses associated with acute injury. The IL-1 family comprises two bioactive proteins, IL-1alpha and IL-1beta, and their natural antagonist IL-1ra. Here we report studies of IL-1 regulation during hyperoxic lung injury in the adult mouse. When assayed by Northern blot, increases in IL-1beta mRNA were seen after 2 days of hyperoxia. In contrast, IL-1alpha mRNA was barely detectable before 4 days of hyperoxia. To further understand the cellular origin of IL-1beta expression in lungs, in situ hybridization and immunohistochemical analyses were performed. IL-1beta mRNA or protein was not detected in the lungs of unexposed animals. At 3 days, we observed the accumulation of IL-1beta transcripts in pulmonary interstitial macrophages and in a subset of neutrophils, and immunodetectable IL-1beta protein was co-localized in adjacent sections. At 4 days of exposure, IL-1beta transcripts were widespread in lung tissue, but many areas rich in IL-1beta mRNA were devoid of immunodetectable IL-1beta. However, it is not known whether increased synthesis of IL-1beta or the uncoupling of IL-1beta protein and mRNA accumulation has a role in pathophysiology of pulmonary O2 toxicity.

Mechanisms of action of antimalarials in inflammation: induction of apoptosis in human endothelial cells.

Antimalarials are beneficial therapeutic agents in systemic lupus and rheumatoid arthritis. These autoimmune diseases have abnormally low apoptosis of inflammatory cells. Both disorders have an abnormal angiogenesis. In the present report, antimalarials were demonstrated to selectively increase apoptosis of HUVECs in vitro. A 24-h exposure to 50 or 150 microM of the drugs was associated with a significant loss of substrate-adherent cells. Chloroquine exhibited an inhibitory effect on HUVEC proliferation over 7 days. Programmed cell death in HUVECs rendered nonadherent by chloroquine was confirmed by the induction of DNA fragmentation in floating cells. Northern blot analysis revealed a rapidly increased expression of the bcl-x(s) gene without any change in the expression of the bcl-2 gene, indicating that HUVECs under chloroquine were undergoing apoptosis. The onset of the apoptotic cascade in HUVECs appeared shortly after the addition of chloroquine. The effect of chloroquine on apoptosis was distinct from acute cell lysis and was restricted to HUVECs. Antimalarials also induced IL-1alpha production. In parallel, chloroquine alone did not increase the expression of IL-6. Anti-IL-1alpha Ab or IL-1Ra only marginally reversed chloroquine-induced depression of proliferation for the low drug concentration, but not the massive cell death effect at and above 50 microM. Taken together, these data may indicate that antimalarials repress angiogenesis. The autocrine mechanism involving IL-1alpha accounts only for a minor fraction of the full antiendothelial effect of chloroquine, which is mainly dependent on apoptosis.

Role of protein kinase C alpha, Arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells.

The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins. A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD. The time courses of the two responses were nearly identical. The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium. The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA, suggesting that PLD activation was, at least in part, a calcium-regulated event. Phorbol 12-myristate 13-acetate (PMA) also stimulated PLD activity in human bone cells. Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PMA reduced concomitantly the amounts of PKC alpha, but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators. The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha. Therefore, PKC-alpha-induced PLD activation is unlikely to mediate the effect of NaF. Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells. This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation. Membrane Arf and RhoA contents were assessed by Western immunoblot analyses. Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells. Translocation of the small GTPases was calcium-independent. We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes. The results also indicate that the NaF-induced Arf/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.

The IL-1 and IL-1 receptor antagonist (IL-1Ra) response of human neutrophils to EBV stimulation. Preponderance of IL-Ra detection.

The present study investigated the effect of EBV on gene expression and protein synthesis of IL-1 and its natural IL-1R antagonist (IL-1Ra) in human peripheral blood neutrophils. EBV induced a rapid accumulation of IL-1 and IL-lRa mRNA in neutrophils that was associated with the later appearance of considerable amounts of IL-1alpha, IL-1beta, and IL-Ra proteins. Approximately 3200 and 610 times more IL-Ra than IL-1alpha a or IL-1beta, respectively, was secreted by neutrophils in response to EBV. The effect induced by EBV cannot reflect an overall metabolic activity of neutrophils, since EBV failed to induce granulocyte-macrophage OF synthesis. Heat-inactivated virus was unable to stimulate cytokine synthesis, whereas UV-irradiated virus retained the full IL-1- and IL-1Ra-inducing potential of the native particle. Furthermore, pretreatment of cells with cycloheximide or phosphonoacetic acid did not abrogate the effect of EBV, suggesting that EBV does not penetrate the cell, but that a virion's structural molecule is required to induce such an effect. In this respect, neutralization of the viral particles with the mAb 72A1, which is known to react with glycoprotein gp350 of the viral envelope, inhibits the production of IL-1 and IL-1Ra, suggesting that gp350 could be involved in this process. Thus, the elevated levels of IL-1Ra detected for EBV-stimulated neutrophils might be part of a mechanism used by the virus to evade the immune system.

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.

Pathologic leukocyte infiltration of the rabbit aorta confers a vasomotor effect to chemotactic peptides through cyclooxygenase-derived metabolites.

We determined the effect of chemotactic peptides FMLP and C5a, postulated to be relatively selective activators of phagocytes, on the thoracic aorta of rabbits subjected to experimental pathologies that allowed infiltration by leukocytes, i.e., dietary atherosclerosis and serum sickness. Aortic ring tissues isolated from hypercholesterolemic rabbits, precontracted or not by phenylephrine, exhibited a rapid and relatively sustained (10 to 20 min) contractile response when challenged by FMLP (10 nM and above); precontracted tissues also responded to C5a (2.5 nM and above). Aortic rings from rabbits with serum sickness (13 days post-BSA injection) exhibited brief contractions that were often followed by a relaxation in phenylephrine-precontracted tissues. In both models, tissues from normal weight-matched animals were not consistently responsive to these peptides. The cyclooxygenase inhibitor indomethacin extensively reduced the contractile effect of either peptide on precontracted aortic rings in both models. Chemotactic peptide-induced increased prostanoid secretion was evident only in the fluid bathing atherosclerotic aortic rings. Morphologic correlations included the demonstration of cells positive for the RAM-11 macrophage marker and the C5a receptors in tissues from rabbits with hypercholesterolemia (numerous clusters of cells) or serum sickness (modest infiltration). Control aortic rings responded to FMLP by a significant contraction if cultured for 2 h in the presence of resident peritoneal cells (84% macrophages), but not in the presence of a high density of PBL (less than 0.5% monocytes). Infiltrating or adherent macrophages in the blood vessel wall confer to some phagocyte activating peptides the role of eicosanoid-dependent vasoconstrictor agents.

Monosodium urate crystals promote neutrophil adhesion via a CD18-independent and selectin-independent mechanism.

The primary objective of this study was to determine whether monosodium urate (MSU) crystals induced neutrophil adhesion to cellular substrata and, if so, then to elucidate the molecular mechanisms involved. Human umbilical vein endothelial cells (HUVEC), as well as various other cellular substrata, were treated with various sized MSU crystals, washed, and then coincubated in the presence of neutrophils for 60 min. HUVEC exposed to MSU crystals but not to silica crystals or uric acid promoted neutrophil adhesion in a dose- and size-dependent manner, an event also observed with monolayers of rabbit synovial cells and rat intestinal epithelial cells. The increased neutrophil adhesion could not be attenuated by anti-CD18, anti-intracellular adhesion molecule-1, or various anti-selectin antibodies, despite the fact that scanning electron microscopy revealed that neutrophils were adhering primarily to the endothelial cells rather than to exposed crystals. CD18-deficient neutrophils adhered to MSU crystal-treated HUVEC as effectively as their CD18-positive counterparts. The neutrophil adhesion was temperature dependent but did not require protein synthesis. Additionally, HUVEC phagocytosis of crystals was necessary for subsequent neutrophil-endothelial cell interactions to transpire. Pretreatment of endothelial cells and neutrophils with colchicine significantly reduced the adhesive interaction. Our data demonstrate that exposure of endothelial and other cells to MSU crystals promotes neutrophil adhesion that occurs by a firm CD18-independent and selectin-independent adhesive mechanism.

Activation of Lyn is a common element of the stimulation of human neutrophils by soluble and particulate agonists.

The functional responsiveness of human neutrophils is known to be initiated and modulated by protein tyrosine phosphorylation. The regulation of the levels of tyrosine phosphorylation is most likely the result of the coordinated actions of tyrosine kinases and phosphatases, which have so far been only very partially characterized. In the present study, we present evidence demonstrating that the stimulation of neutrophils by a variety of agonists (soluble as well as particulate) leads to the activation of the src-related tyrosine kinase lyn. The stimulation of tyrosine kinase activity of lyn was detected using an immune kinase assay as well as an in situ labeling technique. Phosphoaminoacid analysis of lyn indicated that the autophosphorylation of the kinase was exclusively on tyrosine residues. The time course of the activation of lyn is consistent with its playing a role in the early tyrosine phosphorylation responses of neutrophils. The ability of agonists with widely varying functional end responses to stimulate the activity of lyn indicates that this event plays a key and central role in the control of the activation of human neutrophils.

Superantigen-induced collagenase gene expression in human IFN-gamma-treated fibroblast-like synoviocytes involves prostaglandin E2. Evidence for a role of cyclooxygenase-2 and cytosolic phospholipase A2.

MHC class II molecules expressed in lymphoid and nonlymphoid cells act as signal-transducer molecules. We demonstrate that engagement of MHC class II molecules on human IFN-gamma-treated fibroblast-like synoviocytes by their natural ligand, the staphylococcal enterotoxin A (SEA), selectively induces the production of interstitial collagenase over the expression of the tissue inhibitor of metalloproteinase (TIMP). Collagenase gene expression required de novo protein synthesis and was accompanied by high levels of PGE2 production, suggesting its implication in this response. Two inhibitors that affect prostaglandin biosynthesis, indomethacin and arachidonyl-trifluoromethyl-ketone, inhibited both PGE2 production and collagenase gene expression. The addition of exogenous PGE2 to inhibitor-treated cells partially restored the SEA-induced collagenase, indicating a role for PGE2 in this response. As cyclooxygenases (COX-1 and -2), cytosolic phospholipase A2 (cPLA2), and secreted PLA2 (sPLA2) are the enzymes potentially implicated in prostaglandin synthesis, their involvement in SEA-induced collagenase was investigated. The mRNA levels of COX-2 and cPLA2 rapidly increased following ligation of MHC class II molecules, while COX-1 and sPLA2 mRNA levels were unchanged and transiently depressed, respectively. SEA-induced COX-2 mRNA was translated adequately to protein, whereas cPLA2 protein level was not enhanced, but rapidly phosphorylated, a process previously linked to the enzyme activation. In conclusion, this work demonstrates a selective induction of collagenase gene expression over its natural inhibitor TIMP in human IFN-gamma-treated fibroblast-like synoviocytes mediated, at least in part, by PGE2, and provides evidence that signaling via MHC class II molecules induces the production of PGE2 through enhanced production of COX-2 and possibly activation of the cPLA2.

An interleukin 1 receptor antagonist blocks the IL-1-induced IL-6 paracrine production through a prostaglandin E2-related mechanism in multiple myeloma.

By analogy with the model of pristane-induced mouse plasmacytomas, we have wondered about the putative role of prostaglandin E2 (PGE2) in the human multiple myeloma (MM) cytokine network, involving interleukin 6 (IL-6) and interleukin 1 (IL-1) as essential myeloma cell growth factors and inducing cofactors respectively. We show that PGE2 is produced in short-term cultures of bone marrow cells of patients with MM, concomitantly with both IL-6 and IL-1. Indomethacin, a potent inhibitor of cyclo-oxygenase and of PGE2 synthesis, significantly inhibits IL-6 production (but not IL-1 production) by 35% to 90% depending on the different MM patients studied and concurrently to that of PGE2. Exogenous PGE2 reverses this inhibition or even stimulates IL-6 production. An IL-1 receptor antagonist (IL-1RA) also significantly inhibits PGE2, IL-6 production and myeloma cell growth. The inhibition of IL-6 production is reversed by adding exogenous PGE2. These results show that induction of IL-6 by IL-1 is related to PGE2 in the bone marrow of patients with MM. Inhibition of PGE2 synthesis (as obtained with indomethacin and the IL-1RA) might be helpful to inhibit myeloma cell proliferation by reducing IL-1-induced endogenous IL-6 production not only in vitro (as demonstrated here) but also in vivo.

Metabolic disposition of leukotriene B4 (LTB4) and oxidation-resistant analogues of LTB4 in conscious rabbits.

1. The kinetics of leukotriene B4 (LTB4), after single i.v. injections of doses of 0.1 to 1 micrograms kg-1, were investigated in conscious rabbits and compared with those of the omega- and beta-oxidation resistant bioactive analogues, 20, 20, 20-trifluoro-LTB4 (20-F3-LTB4) and 3-thio-LTB4, respectively. 2. Immunoreactive LTB4 (IR-LTB4) elimination was first-order, as shown by a constant systemic clearance (ClLTB4) and a proportional increase in the area under the curve (AUC) of the plasma concentration versus time curve over the dose-range studied. Our results showed a good correlation between observed steady-state plasma concentrations (Css) of IR-LTB4 after continuous infusion of LTB4 and those predicted by using the mean estimated ClLTB4 of 93 +/- 4 ml min-1 kg-1, further confirming the linearity of IR-LTB4 elimination. 3. The half-life (t1/2) or IR-LTB4 increased from 0.47 +/- 0.02 to 0.63 +/- 0.04 min as a consequence of a change in the apparent volume of distribution (Vd) from 72 +/- 5 to 109 +/- 13 ml kg-1, for the 0.1 and 1 micrograms kg-1 doses injected, respectively. 4. Single i.v. injections of [3H]-LTB4 (4.7 ng kg-1) were administered, and the decay of plasma [3H]-LTB4 following h.p.l.c. purification was used to estimate the kinetic parameters. The kinetic parameters of [3H]-LTB4 were characterized by a mean systemic clearance (Cl) of 96 +/- 11 ml min-1 kg-1, a t1/2 of 0.53 +/- 0.03 min, and an apparent Vd of 85 +/- 9 ml kg-1, similar to the parameters obtained after LTB4 boluses. 5. The disposition of LTB4 analogues, whether resistant to Omega- or to Beta-oxidation in vitro, did not differ significantly from the disposition of the LTB4 molecule. The half-lives of 20-F3-LTB4 and 3-thio-LTB4 in the circulation were 0.52 +/- 0.07 min and 0.70 +/- 0.11 min, respectively.6. In summary, our results showed that LTB4, as well as Omega-oxidation- and Beta-oxidation-resistant analogues were cleared very rapidly from the rabbit circulation and indicate that in situ, metabolism in blood is not a rate-limiting factor for the elimination of LTB4.

Signalling via MHC class II molecules selectively induces IL-1 beta over IL-1 receptor antagonist gene expression.

Activation of human monocytes or human monocytic cell lines by several types of stimuli coordinately induces IL-1 beta and its antagonist (IL-1Ra) gene expression; alterations in their balance seem to mediate the inflammatory response. Using the human monocytic cell line THP-1, we report that superantigens, such as staphylococcal enterotoxin A (SEA) and Mycoplasma arthritidis -derived superantigen (MAM) induce an increase in the level of IL-1 beta mRNA without any detectable effect on IL-Ra mRNA. Unlike MAM-induced IL-1 beta mRNA, SEA-induced IL-1 beta mRNA was adequately translated into protein. Superantigen-induced gene expression is mediated by signalling, via their receptors, the MHC class II molecules. Thus, it appears that this mode of signalling selectively induces the proinflammatory cytokine IL-1 beta gene expression which, by itself, can have major importance in disease pathology especially in autoimmune diseases.

Epidermal growth factor-induced rapid relaxation of the isolated rabbit mesenteric artery.

Epidermal growth factor (EGF) (10-100 ng ml-1) induced a rapidly developing relaxation of precontracted rabbit mesenteric artery rings within 30 min of exposure. Indomethacin or protein synthesis inhibitors prevented or acutely reversed the effect of EGF on the preparation and an erbstatin analogue significantly reduced it. It is concluded that the EGF-mediated relaxation may be induced by the rapid production of prostanoids via a cascade of biochemical events initiated by the tyrosine-kinase activity of the receptors for EGF. One or more proteins rapidly produced and rapidly depleted appear to be involved in the activation of arachidonate metabolism.

Crystal-induced neutrophil activation. V. Differential production of biologically active IL-1 and IL-1 receptor antagonist.

Neutrophils produce IL-1 when stimulated by monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals. Neutrophils also generate the IL-1R antagonist (IL-1Ra), especially when incubated with granulocyte-macrophage CSF (GM-CSF) or TNF-alpha. We studied the simultaneous expression of IL-1 and IL-1Ra by GM-CSF- or TNF-alpha-treated neutrophils activated by MSU or CPPD. Neutrophils incubated with GM-CSF or TNF-alpha produced approximately 300 or 200 times more IL-1Ra than IL-1, respectively. Suboptimal concentrations of MSU or CPPD induced low amounts of IL-1 without affecting IL-1Ra. Interaction of GM-CSF- and TNF-alpha-treated neutrophils with MSU or CPPD up-regulated IL-1 while simultaneously down-regulating IL-1Ra. As a result, the bioactivity of IL-1 secreted was enhanced. Synergistic increases of IL-1 (but not IL-1Ra) mRNA levels were noted in GM-CSF- or TNF-alpha-treated neutrophils exposed to CPPD. Treatment of neutrophils with colchicine before incubation with GM-CSF or TNF alpha, inhibited crystal-induced IL-1 by 50 to 55%, but failed to significantly affect IL-1Ra. The IL-1Ra to IL-1 ratio was significantly increased by 185 to 220%. These results demonstrate that IL-1 and IL-1Ra production by human neutrophils are differentially regulated, that the combined presence of GM-CSF or TNF-alpha and microcrystals favor the production of biologically active IL-1 over that of IL-1Ra, and that colchicine selectively inhibits IL-1 without affecting IL-1Ra production.

Rapid protein synthesis and turnover is involved in interleukin-1-induced relaxation of the rabbit isolated mesenteric artery. Analysis of the arachidonate cascade.

The rabbit isolated superior mesenteric artery, when precontracted with phenylephrine, relaxes in a sustained manner in less than 30 min when exposed to recombinant interleukin-1 (IL-1), and this is a prostaglandin (PG)-dependent, endothelium- and 5-lipoxygenase-independent process. We have studied the dependency of IL-1-induced relaxation on protein synthesis and trafficking using isolated rings of rabbit mesenteric arteries. Three chemically unrelated protein synthesis inhibitors (PSIs) (cycloheximide, anisomycin and puromycin) completely prevented the response to IL-1. Moreover, the PSIs reversed IL-1-induced relaxations within 15 to 30 min of application. The amplitude and/or the duration of the relaxation induced by arachidonic acid and iloprost (a PGI2 mimetic) were partially inhibited by cycloheximide treatment, but not those induced by nitroglycerin or by cromakalim. However, tissues initially relaxed by IL-1 and then recontracted by a PSI are still able to relax when challenged with either arachidonic acid or iloprost, suggesting that cyclooxygenase is not depleted and that the responsiveness to PGs is intact under these conditions. Manoalide, thioether amide glycerophosphocholine (type II phospholipase A2 inhibitors) or brefeldin A (an inhibitor of intracellular protein trafficking) did not inhibit IL-1-induced relaxation. The data suggest the involvement of newly synthesized protein(s) with a very rapid turnover in the vascular response to IL-1. PSIs probably act at several levels to inhibit the relaxant response to IL-1, but depletion of tissue cyclooxygenase does not appear to be the limiting mechanism for the PSI effect on IL-1-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)