In vitro enhanced thromboxane B2 release by polymorphonuclear leukocytes and macrophages after treatment with human recombinant interleukin 1

Macrophages (M phi) and polymorphonuclear leukocytes (PMNs) are cell types that interact with bacterial endotoxin and play key roles in mediating the inflammatory reaction. We examined the ability of rat peritoneal macrophages and human PMNs to synthesize thromboxane A2 (detected as TxB2) in response to human recombinant interleukin 1 (hrIL1). TxB2 levels were measured by radioimmunoassay in the cell-free supernatant of cell suspensions after 1 hr incubation at 37 degrees C. TxB2 release by both PMNs and M phis was increased in a dose-dependent manner by hrIL1.     

PGE2 and PGF2α release by human peritoneal macrophages in endometriosis

Objective: To test for differences in the amount and activity of peritoneal macrophages present in the peritoneal fluid of women with, and without endometriosis using prostaglandin release by macrophages in culture as a marker.Patients: Women of reproductive age undergoing laparoscopy for infertility or chronic pelvic pain with postoperative diagnosis of endometriosis and women undergoing laparoscopy for sterilization.Methods: Peritoneal fluid was aspirated during laparoscopy, volume was recorded, macrophages were isolated via a Ficoll Paque gradient and kept in primary culture. PGE₂ and PGF_2α release of the cells were measured before and after stimulation with zymosan.Results: Women with endometriosis had significantly more peritoneal macrophages than controls. Peritoneal macrophages of women with endometriosis released significantly more PGE₂ than those of the control group: 8.4 ± 2.0 versus 1.4 ± 0.4 ng/ml/10⁶cells (mean ± SEM, p=0.0005) and PGF_2α : 10 ± 4.3 (endometriosis) versus 1.8 ± 0.4 (control) ng/ml/10⁶cells (mean ± SEM, p = 0.045).Conclusion: There is a significant increase in the amount of prostaglandins released by peritoneal macrophages from women with endometriosis. These prostaglandins might alter uterine and tubal contractility, thereby affecting fertility.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2α on human umbilical cord vessels

The effect of six naturally occurring prostaglandins on isolated umbilical arteries and veins has been studied. All six prostaglandins had a constricting effect on the umbilical vessels. On the umbilical artery preparations the potencies in decreasing order were A₂>B₂>F_2α>B₁>E₂>A₁. Prostaglandin B₂ was more potent than PGA₂ on the umbilical vein. Polyphloretin phosphate (PPP) antagonised the constricting effect of all six prostaglandins without altering responses to 5-hydroxytryptamine.     

In vitro and in vivo chemotaxis of guinea pig leukocytes toward leukotriene B4 and its w-oxidation products

Leukotriene B₄ (LTB₄), 20-OH-LTB₄, and 20-COOH-LTB₄ were studied for their relative activities towards guinea pig peritoneal eosinophils and neutrophils during in vitro chemotaxis in modified Boyden chambers. The leukotrienes were also injected into guinea pig skin, and the cellular infiltrate in 4 hour biopsies was evaluated histologically. Eosinophils migrated more actively than neutrophils towards LTB₄in vitro, while in vivo, more neutrophils were observed. 20-OH-LTB₄, was markedly less active than LTB₄in vivo and in vitro, and 20-COOH-LTB₄ was barely active at all. Crude ionophore-stimulated neutrophil supernatants (ECF) were more active towards eosinophils than towards neutrophils, both in vivo and in vitro, compared to the pure leukotrienes. The data confirm the potent chemotactic properties of LTB₄ for eosinophils and neutrophils, with less activity of its w-metabolites.     

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Defective inhibition by dexamethasone of leukotriene B4 and C4 production by leukocytes in patients with cystic fibrosis

To investigate the effects of glucocorticoids on leukotriene (LT) generation in patients with cystic fibrosis (CF), we evaluated calcium ionophore A23187-induced LTB₄ and LTC₄ production by leukocytes with and without pretreatment with dexamethasone. The CF patients were in good condition and did not have acute infection. There were no significant differences in LTB₄ and LTC₄ production without dexamethasone pretreatment between the CF patients and controls. However, the ratios of LTB₄ and LTC₄ production by leukocytes preincubated with dexamethasone to those of leukocytes without dexamethasone pretreatment were significantly higher in the CF patients than in the controls (both p < 0.05). Our data suggest that the response of LTB₄ and LTC₄ production to dexamethasone is disturbed in patients with CF. The generation of LTs may be enhanced due to a disturbance in glucocorticoid suppression.     

Expression of TREM-1 is inhibited by PGD2 and PGJ2 in macrophages

TREM-1 is a superimmunoglobulin receptor present on neutrophils and monocytes, which plays an important role in the amplification of inflammation. The natural ligands for TREM-1 have not been identified; however, Toll-like receptor ligands are known to induce the expression of TREM-1. Blockade of TREM-1 has shown to improve survival in animal models of sepsis. In the present studies, we investigated the role of lipid mediators in the expression of TREM-1. In a macrophage cell line, we show that the expression of TREM-1 in response to LPS and bacteria Pseudomonas aeruginosa is inhibited by PGD₂ and cyclopentanone prostaglandins PGJ₂ and 15-dPGJ₂. The inhibition of TREM-1 by these prostaglandins is independent of the PGD₂ receptors and PPARγ but occurs by activation of Nrf2 and inhibition of NF-κB. Our data suggest a novel mechanism by which these prostaglandins exhibit anti-inflammatory effects and a new therapeutic approach to inhibition of TREM-1.     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Production rates of prostaglandin F, 6-keto-PGF1α and thromboxane B2 by perifused human endometrium

Human endometrium obtained from fresh hysterectomy specimens was perifused for 7 hr in 95% O₂/5% CO₂ at 37°C. The phase of the menstrual cycle was determined by histological examination. The concentrations of PGF, 6-keto-PGF_1α and TxB₂ in 20 min fractions of the perifusion medium were measured by radioimmunoassay and production rates were calculated in terms of dry weight of tissue. Biphasic patterns of production were observed; high initial values fell to about 20% at 2 hr and then increased to relatively stable values at about 4 hr which were maintained for the next 2 hr. During this latter period, production rates in endometria taken at different phases of the cycle differed markedly from each other; the production rates of PGF in secretory and early proliferative endometria were low (15.8 ± 2.6, mean ± SEM and 67.2 ± 8.3 ng/min/g respectively) whereas they were high in late proliferative and premenstrual endometria (188.0 ± 16.7 and 196.4 ± 16.9 ng/min/g respectively). The patterns of production of 6-keto-PGF_1α and TxB₂ were similar to those of PGF but the absolute values were much lower (<10%). We conclude that the observed rates of production of prostaglandins by perifused human endometrium are consistent with synthesis being stimulated either by estrogen or withdrawal of hormonal support and being inhibited by progesterone.     

Measurement of thromboxane B2 in human plasma or serum by radioimmunoassay

A radioimmunoassay for thromboxane B₂ (TxB₂), a stable metabolite of thromboxane A₂, is described. The method consists of extraction of TxB₂ into ethyl acetate from acidified plasma or serum samples and saturation analysis using specific antibodies produced in rabbits against TxB₂-BSA conjugate. The 50 % displacement level of the standard curve was 19.1 ± 2.9 pg/tube (mean ± S.D., n = 19). The method blank was 3.4 ± 3.1 pg/ml (n = 15) and the assay sensitivity thus 9.6 pg/ml (mean blank + 2 S.D.). When 100 to 200 pg of TxB₂ were added to plasma, 96.2–103.6 % were recovered. The intra-assay coefficient of variation varied from 6.7 to 9.7 %, and the inter-assay coefficient of variation was 18.6 % (n = 10). The TxB₂ concentration in the plasma of 14 healthy subjects varied from 29.3 to 120.8 pg/ml with a mean ± S.D. of 70.1 ± 26.1 pg/ml, when the blood was collected into tubes containing acetylsalicylic acid (ASA), whereas significantly higher (p < 0.001) TxB₂ concentrations of 68.3 – 285.3 pg/ml with a mean ± S.D. of 151.8 ± 66.6 pg/ml were obtained from the same subjects in the plasma of blood which was collected into tubes containing no ASA. When blood samples from 10 subjects were allowed to clot at 0, +24 or +37°C for 60 min., the TxB₂ concentrations in the sera were 2053 ± 870 pg/ml, 4001 ± 1370 pg/ml and 178557 ± 54000 pg/ml, respectively. The TxB₂ levels in sera which were separated from blood samples incubated at +37°C, correlated significantly (p < 0.001) with the TxB₂ productions in platelet-rich-plasma (PRP) after an induced aggregation. Our results indicate 1) when TxB₂ is measured in plasma, the use of prostaglandin synthesis inhibitor in the collection tubes is necessary and 2) the measurement of TxB₂ in serum of blood which has been kept at +37°C for a strictly standardized period of time could replace the use of PRP in TxB₂ studies.     

The metabolism of leukotriene B4 by peripheral blood polymorphonuclear leukocytes in psoriasis

The formation of leukotriene B₄ and its ω-oxidised metabolites has been compared in calcium ionophore-stimulated polymorphonuclear leukocytes, in the absence of exogenous substrate, from fourteen psoriatic subjects and thirteen healthy controls. Although there was no significant difference in the levels of leukotriene B₄, the psoriatic cells synthesised significantly greater amounts of ω-oxidation products than control cells. This difference was confirmed in an experiment comparing the time course of formation of the ω-oxidation products of leukotriene B₄, under similar conditions, in polymorphonuclear leukocytes from four psoriatic subjects and three healthy controls. The kinetic constants for the metabolism of exogenous leukotriene B₄ by 20-hydroxylase were determined by a radiochromatographic enzyme assay in polymorphonuclear leukocytes from three patients with psoriasis and three healthy controls. No significant differences were found in the apparent K_m and V_max values. It is concluded that the increased formation of ω-oxidation products in psoriatic cells may be secondary to increased synthesis of leukotriene B₄ by these cells, with consequent increased metabolism, rather than to an inherent abnormality of the 20-hydroxylase system. Further work is needed to determine the kinetics of the enzymes involved in leukotriene B₄ synthesis in the psoriatic polymorphonuclear leukocyte, and also to assess the contribution of the leukotriene B₄ and ω-oxidation products from polymorphonuclear leukocytes infiltrating the skin to the pathogenesis of the psoriatic lesion.     

Synthesis of leukotriene B4, and prostanoids by human alveolar macrophages: analysis by gas chromatography/ mass spectrometry

Human alveolar macrophages, obtained during diagnostic bronchoscoy, were maintained in monolayer culture. Challenge of these cells (>95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B₄ into the medium, 7.28 ± 5.99 ng/mg cell protein at 2 h mean ± S.D⁴, n = 4). Leukotriene B₄ was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B₂, prostaglandins D₂, E₂, F_2α and the lysosomal enzyme N-acetyl-β-D- glucosaminidase was also measured. Thromboxane B₂ was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 ± 14.8 ng/mg cell protein 2 h after the addition of zymosanA, n = 4), and the synthesis of thromboxane B₂ was inhibited by >90% in 1 μM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B₄ synthesis.     

Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat

Leukocyte numbers and Leukotriene B₄- (LTB₄-) and LTC₄-immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containig 1% uric acid, carragennan or zymosan were implanted for 5h and compared to saline sponges. Increases in leukocyte numbers and LTB₄-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB₄ was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltrations was found to coincide with the maximal levels of LTB₄-immunoreactivity. The LTC₄-immunoreactivity was low and following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB₄-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionphore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with indomethacin; 3 and 10 mg/kg p.o.). Impregnation of sponges with 3 × 10⁻⁶M LTB₄ but not 3 × 10⁻⁷M LTB₄ induced a significant leukocyte migration. It was concluded that LTB₄ can induced leukocyte migration into sponge exudates in the rat but that measurements of LTB₄ in such exudates can not be correlated with the degree of leukocyte infiltration.     

Rb2[B12(OH)12]·2H2O and Rb2[B12(OH)12]·2H2O2: Hydrate and perhydrolate of rubidium dodecahydroxo-closo-dodecaborate

The orthorhombically crystallizing salts Rb₂[B₁₂(OH)₁₂]·2H₂O (a = 1576.81(9), b = 813.08(5), c = 1245.32(7) pm) and Rb₂[B₁₂(OH)₁₂]·2H₂O₂ (a = 1616.54(9), b = 814.29(5), c = 1260.12(7) pm) could be prepared from Rb₂[B₁₂H₁₂] and hydrogen peroxide. Both crystal structures were determined by X-ray single crystal diffraction and refined in the space group Cmce. They are not isostructural to the other compounds containing icosahedral dodecahydroxo-closo-dodecaborate dianions [B₁₂(OH)₁₂]²⁻ and potassium, rubidium or cesium cations already known to literature, but both title compounds crystallize quasi-isotypically exhibiting Rb⁺ cations in 10-fold oxygen coordination. The hydrogen peroxide adduct (Rb₂[B₁₂(OH)₁₂]·2H₂O₂) is explosive on shock and heat, while the hydrate (Rb₂[B₁₂(OH)₁₂]·2H₂O) is not.