Inhibition of eicosanoid and PAF formation by dexamethasone in rat inflammatory polymorphonuclear neutrophils may implicate lipocortin 's'.

In polymorphonuclear neutrophils, phospholipase A2 activity is the rate-limiting step for platelet-activating factor (PAF) formation and for the biosynthesis of arachidonic acid derivatives, leukotrienes and prostaglandins. Glucocorticosteroids inhibit phospholipase A2 activity by inducing in target cells the synthesis and release of phospholipase A2 inhibitory proteins named 'lipocortins'. Here, we report that rat pleural inflammatory polymorphonuclear neutrophils, treated with dexamethasone, decrease their production of eicosanoids and PAF. Evidence is presented which may implicate lipocortin 's' in these inhibitions since (i) phospholipase A2 inhibitory proteins are found in the supernatant of dexamethasone-treated cells, (ii) this supernatant inhibits the formation of lipid mediators in untreated cells, inhibition being reversed either by incubating the supernatant with a monoclonal antibody against rat lipocortin or by boiling it and (iii) a 36 kDa lipocortin from mice lungs mimics the effects of dexamethasone when added exogenously on untreated cells. Our results favour the hypothesis that the newly formed lipocortin 's' could be responsible for the antiphospholipase A2 activity of glucocorticosteroids.

The presence of lipocortin in human embryonic skin fibroblasts and its regulation by anti-inflammatory steroids.

Human embryonic skin fibroblasts in culture produce pro-inflammatory lipid mediators and all types of prostanoids. When these cells were treated with the anti-inflammatory steroid, dexamethasone, prostaglandin production was inhibited. This phenomenon required glucocorticoid receptor occupancy and mRNA and protein synthesis. The inhibitory effect was prevented by treating the cells with a monoclonal antibody, BF 26, raised against renocortin, a lipocortin-like protein formed in rat kidney medulla interstitial cells in culture. When the proteins present in the supernatants and the cell pellets derived from control and dexamethasone-treated cells were analyzed for their ability to inhibit phospholipase A2, four inhibitory peaks, at 45, 30, 15 kDa and one peak under 12 kDa, were found in the supernatants of control and dexamethasone-treated cells, whereas one single inhibitory peak at 15 kDa was found in the cell pellets. The antiphospholipase activity was much greater in dexamethasone-treated cells than in control cells. These results suggest that preformed lipocortin exists in human cells and that lipocortin is synthesized and released under glucocorticoid treatment.

tRNAs undermethylation in a met-regulatory mutant of Saccharomyces cerevisiae.

A study of in vivo and in vitro methylation of tRNAs in regulatory mutants affected in methionine-mediated repression (eth2, eth3, eth10) has led to the following results: 1) The eth2-2 carrying strain presents a great undermethylation of its tRNAs of the same order of magnitude as observed during methionine starvation of methionine auxotrophs. 2) This undermethylation leads to a shift of the tRNAIII met peak on a BD cellulose column, while tRNAIII met peak is unchanged. 3) The study of a double mutant strain carrying eth2 and met2 mutations has shown that this undermethylation is a consequence of the high internal pool of methionine. 4) Undermethylation unequally affects the different bases and the different tRNA species.

Effect of intracellular oxygen-free radicals on the formation of lipid derived mediators in rat renomedullary interstitial cells.

An intracellular generation of oxygen free radicals was induced by phenazine methosulfate (PMS) in rat renomedullary interstitial cells (RMIC) in culture. This response was associated with an increase in PGE2 and 15 HETE production. The synthesis of cyclooxygenase and lipoxygenase derivatives in PMS-treated cells was inhibited by indomethacin and NDGA respectively. Inhibitors of PLA2 such as mepacrine and dexamethasone were able to inhibit partially the PGE2 synthesis induced by PMS. The formation of lyso-platelet activating factor, a product of membrane-bound phospholipid, by a PLA2 catalyzed reaction was also stimulated in PMS-treated cells. Superoxide dismutase added to the incubation medium enhanced the PMS-dependent PGE2 synthesis whereas catalase decreased it, suggesting the involvement of H2O2 in this process. In addition, a depletion of soluble thiol groups was observed in PMS-treated cells. Treatment of RMIC by the thiol oxidative agent, diamide, mimicked the effect of PMS on PGE2 synthesis, whereas diamide did not increase the formation of lyso-PAF indicating its inability to stimulate PLA2. These results suggest that cyclooxygenase may be involved in this process, indeed added arachidonate, bypassing PLA2, enhanced PGE2 synthesis in PMS-treated cells further supporting the involvement of cyclooxygenase. In conclusion, generation of oxygen free radicals by PMS in RMIC enhanced the synthesis of lipid derived mediators. A decrease in the cellular thiol content is partially involved in cyclooxygenase activation but does not appear to be involved in PLA2 activation.

Possible non-involvement of lipoxygenase pathway in the cerebral blood flow decrease due to indomethacin.

Indomethacin (10n mg/kg i.p.) induced a marked decrease in local cerebral blood flow (l.CBF), which was measured in the frontal cortex of unanesthetized rats by the hydrogen clearance technique. Brain prostaglandins (PGD2, PGE2, PGF2 alpha,) and 6kPGF1 alpha the stable metabolite of prostacyclin, were significantly decreased. Treatments with inhibitors of lipoxygenase (BW 755C and nordihydroguaiaretic acid) and with FPL 55712, a leukotriene receptor antagonist, did not influence the effect of indomethacin on 1.CBF. The results suggest that the release of vasoconstrictory leukotrienes does not play a major role in the lowering of 1.CBF by indomethacin.

Mechanism of inhibition of tumor necrosis factor production by chlorpromazine and its derivatives in mice.

In previous work, we reported that chlorpromazine inhibits tumor necrosis factor (TNF) production in endotoxin lipopolysaccharide-treated mice, and protects against lipopolysaccharide toxicity. Chlorpromazine is used as an antipsychotic and has several effects on the central nervous system. It acts on different neurotransmitter receptors and has other biochemical activities some of which, like inhibition of phospholipase A2, might be responsible for the inhibitory effect on TNF production. To investigate the role of these actions in the inhibition of TNF production by chlorpromazine, we have synthesized some chlorpromazine derivatives that do not have central activities. Some of these analogs have lost their affinity for various receptors and their phospholipase A2 inhibitory activity, but still inhibit TNF production. No correlation was found between TNF inhibition and the ability to inhibit nitric oxide (NO) synthase, whereas a good correlation was evident between TNF inhibition and antioxidant activity.

Electron capture negative ion chemical ionization analysis of arachidonic acid.

A highly sensitive (subpicomole level) and structural specific method for the analysis of arachidonic acid esterified to complex glycerophospholipids has been developed using combined capillary gas chromatography and mass spectrometry. The methodology is based upon the formation of the pentafluorobenzyl ester of arachidonic acid, which is efficiently ionized using electron capture negative ion chemical ionization conditions to yield an abundant carboxylate anion at m/z 301. Quantification is carried out following hydrolysis of the complex glycerophospholipid in the presence of a known amount of (2H8) arachidonic acid. The use of this method is illustrated by the quantification of arachidonic acid within the glycerophospholipid classes isolated from resident peritoneal macrophage cells isolated from HS mice.

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin.

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).

The effect of ethanol on arachidonic acid metabolism in the murine peritoneal macrophage.

Exposure to ethanol in man has been linked to an alteration of the immune surveillance system and reduced ability of the macrophage to undergo phagocytosis. Since ethanol has been suggested to alter membrane function and inhibit the production of calcium ionophore stimulated synthesis of prostaglandins and leukotrienes by the human neutrophil and transformed murine mast cell, the dose response effect of ethanol on the biosynthesis of icosanoids by the peritoneal macrophage during zymosan phagocytosis was studied. Peritoneal macrophages from two inbred strains of mice derived from a common stock (HS) and selected for sensitivity to ethanol (short sleep [SS]/long sleep [LS]) were studied. Zymosan phagocytosis was found to lead to synthesis of LTC4 (70 ng/10(6) cells), 6-keto-PGF1 alpha (5 ng/10(6) cells) and PGE2 (3 ng/10(6) cells). For the HS macrophage, ethanol caused a dose dependent inhibition of these lipid mediators as well as inhibition of phagocytosis and release of beta-hexosaminidase. However, a difference was observed in arachidonate metabolism stimulated by phagocytosis between the LS and SS mice below 100 mM ethanol. The SS mouse had a 50% inhibition of cyclooxygenase products at 86 mM ethanol with no inhibition of lipoxygenase metabolites. The LS mice had a trend suggesting increased lipoxygenase metabolites below 100 mM ethanol. At these levels of ethanol which can be found in man, these results suggest there may be differential production of lipid mediators under genetic control.

Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression.

DNA methylation has been correlated with reduced gene expression in a number of studies, although evidence for a casual link between the two events has been lacking. Because microinjection of simian virus 40 (SV40) DNA into the nucleus of Xenopus laevis oocytes results in the synthesis of both early and late viral gene products, it was possible to test whether a specific methylation event can affect gene expression. The single SV40 Hpa II site at 0.72 SV40 map units was specifically methylated with Hpa II methylase. When this DNA was injected into oocytes, there was a marked reduction in the synthesis of the major late viral capsid protein VP-1, relative to the synthesis by an unmethylated control. However, production of the early proteins (the large and small tumor antigens) was not affected by Hpa II methylation. Therefore, methylation at a single site on the viral DNA located near the 5' end of the late region can specifically repress late gene expression. The possible mechanisms by which this repression is mediated are discussed.

Kinetics of the induction of three translation-regulatory enzymes by interferon.

Three enzymes that cause inhibition of mRNA translation, eukaryotic initiation factor 2 protein kinase PK-i, oligoisoadenylate synthetase E, and phosphodiesterase 2'-PDi, have been recently isolated from interferon-treated cells. We show that the rise in these three enzyme activities may be used to study the response of uninfected cells to interferon. For each enzyme, a specific microassay that can be carried out on extracts from 2-5 x 10(4) monolayer cells from mouse, monkey, or man was developed. With these assays, the kinetics of induction of the three enzymes in mouse L cells are compared. The dose dependence for protein kinase PK-i induction is shown to be similar to that for the development of the antiviral state. Actinomycin D and anti-interferon serum block enzyme induction if added to the cells early after interferon treatment. The quantitative measurements of the intracellular level of these enzymes provide a new and convenient model to study the cell's response to interferon.

Effects of agonists of peroxisome proliferator-activated receptor gamma on proteoglycan degradation and matrix metalloproteinase production in rat cartilage in vitro.

OBJECTIVE: To examine the effects of agonists of peroxisome proliferator-activated receptor (PPAR) gamma on proteoglycan degradation induced by interleukin (IL)-1beta or tumor necrosis factor (TNF)alpha in cartilage in vitro. DESIGN: Proteoglycan degradation was measured as release of radioactivity from rat cartilage explants previously labeled with (35)SO2-4. Western blots were used to examine tissue levels of aggrecan neoepitopes NITEGE and VDIPEN, generated by aggrecanases and matrix metalloproteinases (MMP), respectively. Production of MMP-2, -3 and -9 by cultured rat chondrocytes was measured by zymography and by fluorimetric assay. RESULTS: IL-1beta-induced proteoglycan degradation was likely due to aggrecanase, since it was associated with a strong increase of NITEGE signal. MMP-dependent VDIPEN signal increased only after further incubation with pro-MMP activator APMA. PPAR agonists 15d-PGJ(2) and GI262570 (10 microM) inhibited IL-1beta- and TNFalpha-induced proteoglycan degradation measured both before and after addition of APMA. The agonists also inhibited cytokine-induced MMP production by isolated chondrocytes. CONCLUSION: This study shows that PPARgamma agonists inhibit cytokine-induced proteoglycan degradation mediated by both aggrecanase and MMP. This effect is associated with inhibition of production of MMP-3 and -9. These results support the interest for PPARgamma agonists as candidate inhibitors of pathological cartilage degradation.

Comparison of collagenase-cleaved articular cartilage collagen in mice in the naturally occurring STR/ort model of osteoarthritis and in collagen-induced arthritis.

OBJECTIVE: The STR/ort mouse develops a naturally occurring osteoarthritis of the femorotibial joint that provides a model with which to establish the time course of biochemical changes taking place in articular cartilage in the disease. Our objective was to define the onset, location and progression of type II collagen cleavage by collagenase in the tibial cartilage of the STR/ort mouse. For comparison, cartilage collagen cleavage was also studied in collagen-induced arthritis in DBA mice. DESIGN: STR and control CBA mice aged 6-45 weeks were examined. DBA/1 mice were studied 2 and 3 weeks after initiating collagen-induced arthritis. Collagen cleavage was detected by immunolocalization using the antibody COL2-3/4Cshort which recognizes a carboxy terminal neoepitope created by collagenase cleavage of type I and II collagens. RESULTS: No COL 2-3/4Cshort immunostaining was observed in the intact cartilage of healthy young or old mice. The earliest detectable collagen degradation occurred at the cartilage surface coincident with the appearance of surface roughening. As fibrillations developed, further collagen degradation was evident around the edge of the lesion and in adjacent extracellular matrix. In contrast, staining was observed throughout the cartilage matrix in type II collagen-induced arthritis prior to the development of histopathological lesions. CONCLUSION: No evidence was found for collagen cleavage in intact/pre-lesional cartilage from STR/ort mice. Local collagen cleavage was, however, clearly associated with very early histopathological lesions and immunostaining with COL 2-3/4Cshort increased with progression of the latter. In contrast, type II collagen cleavage occurs throughout the articular cartilage at an early stage in collagen-induced arthritis.

Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs.

To examine the role of small nuclear ribonucleoproteins (snRNPs) in mRNA splicing, we have injected SV40 DNA, in the presence or absence of anti-Sm or anti-(U1)RNP antibodies, into the nucleus of X. laevis oocytes, and analyzed the viral specific RNAs and proteins that were synthesized. In the absence of antibodies, the majority of the viral mRNAs were spliced, giving rise to transcripts and proteins analogous to those found in infected monkey cells. However, the relative efficiencies with which the various splice sites were utilized were different in the two cell types. When sera from systemic lupus erythematosus (SLE) patients containing anti-Sm or anti-(U1)RNP antibodies were coinjected with the viral DNA, splicing of L-strand-specific (late) mRNA was dramatically inhibited. Cleavage at both 5' and 3' splice sites was blocked, leading to an accumulation of unspliced primary transcripts. Neither the total amount of late RNA synthesized nor the formation of mature polyadenylated late mRNA 3' ends was affected. These results indicate that U1 snRNPs play a crucial role in mRNA splicing in vivo. Unexpectedly, the effects of the sera on E-strand-specific (early) viral mRNA splicing were different. All anti-Sm or -(U1)RNP sera tested had no detectable effect on the splicing of the mRNA coding for the small tumor antigen. A subset of these sera, however, inhibited large tumor antigen mRNA splicing. On the basis of these data it is suggested that different pre-mRNAs, or even different splice sites within the same pre-mRNA, have dissimilar interactions with snRNP particles in the splicing reaction.

Platelet activation--a role for a 40K anti-phospholipase A2 protein indistinguishable from lipocortin.

Stimulus-response (S-R) coupling in platelets requires an intermediary other than an elevation in cytosolic free calcium ([Ca2+]i). While an increase in [Ca2+]i is essential in S-R coupling, effecting phosphorylation of myosin of relative molecular mass (Mr) 20,000 (20 K), platelet activation is also associated with phosphorylation of a 40K protein, which can occur in the absence of changes in [Ca2+]i. The 40K protein is the substrate for protein kinase C (PKC). Mounting evidence suggests that activation of PKC by diacylglycerol is the other signal involved in S-R coupling. Although phosphorylation of the 40K protein is associated with certain platelet functional responses, no precise role has been accredited to it. Recently, we and others have described several proteins (collectively known as lipocortin) which inhibit phospholipase A2 (PLA2). One of the most conspicuous proteins of this group is a 40K peptide whose inhibitory activity can be suppressed by prior phosphorylation. We hypothesized that the 40K protein described in platelets may possess anti-PLA2 activity and that phosphorylation by PKC, suppressing its inhibitory activity, may represent the mechanism underlying mobilization of arachidonic acid, the precursor of prostaglandins. The results of the present study strongly support this hypothesis.

Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism.

As a model to perhaps better indicate potential in vivo tissue inflammatory events, the generation of leukotriene (LT)B4, 20-OH-LTB4, sulfidopeptide LT, and platelet-activating factor (PAF) from human whole blood stimulated with zymosan was compared with that produced by isolated human neutrophils suspended either in buffer or plasma. Several reports have shown that substantial LTB4 biosynthesis could be induced after addition of zymosan to whole blood, but little was known concerning the generation of other important lipid mediators, or the cellular source of these. We have shown that, in spite of some subject variation, the zymosan-induced production of 20-OH-LTB4, LTB4, and LTE4 reached maxima within 30 to 60 min with 1.1, 2.8, and 0.60 ng/10(6) neutrophils, respectively. These concentrations would be sufficient to induce significant biologic effects. Studies with isolated cell mixtures suggested that the neutrophil was the primary source of the lipid mediators or their precursors in this system, although a number of other cell types contributed as accessory cells to the final amounts and mix of mediators produced. The ratio of neutrophils to accessory cells in mixed cell experiments dramatically modified the metabolic pattern of leukotriene generation. The concentration of LTB4 was increased in the presence of RBC and that of LTE4 when platelets were present. These results suggested that cellular cooperation and transcellular biosynthesis played a key role in the overall production of eicosanoids such as LTB4 and LTC4. The concomitant synthesis of PAF in isolated cells and in whole blood was also determined as another member of the complex lipid mediator network. Maximal production of cell-associated PAF was observed within 30 min after the initiation of phagocytosis and reached levels of 3 to 5 ng PAF/10(6) neutrophils. When other cells were present in a coincubation system, the time course for production of PAF was not altered, but maximal concentration of PAF was lower, perhaps as a result of enhanced PAF metabolism. Study of eicosanoids and other lipid mediator production in mixed cell populations provides insight into those events occurring within tissues, where cross-cell signaling and transcellular biosynthesis may occur.