Walter C. Pickett

Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid

Two groups of six adults with persistent asthma, who were identical clinically, received 0.1 or 4 g of purified eicosapentaenoic acid ethyl ester (EPA) daily for 8 weeks. Both doses increased significantly the generation of leukotriene B5 (LTB5) from EPA by polymorphonuclear (PMN) and mononuclear leukocytes, while only the high dose decreased leukocyte arachidonic acid (AA) and the generation of LTB4 and prostaglandin E2 from AA. Only the high dose led to inhibition of PMN leukocyte chemotaxis to multiple stimuli by a mean of 57–70% (P<0.01), without altering monocyte chemotaxis, the production of plateletactivating factor by mononuclear leukocytes, or the IgE-dependent release of histamine from basophils. Both doses of EPA increased the responses of T lymphocytes to phytohemagglutinin by a mean of 73% or more (P<0.01) without modifying the numbers of helper and suppressor T lymphocytes. EPA affects the functions of several types of leukocytes critical to inflammation and immunity.

Antigen-induced sulfidopeptide leukotriene release from the guinea pig superfused trachea.

The effect of antigen (ovalbumin) challenge on smooth muscle contraction and release of sulfidopeptide leukotrienes and histamine from superfused, actively sensitized guinea pig trachea was examined. Maximum concentrations of ovalbumin caused the release of 16 +/- 4 ng/g immunoreactive sulfidopeptide leukotriene (i-LT) and 27 +/- 3% of the endogenous histamine (x +/- S.E.M., n = 19). High performance liquid chromatography combined with a sulfidopeptide leukotriene radioimmunoassay was used to demonstrate that on a molar basis, approximately 10% of the leukotriene immunoreactivity recovered was LTC4, 45% LTD4 and 45% LTE4. Indomethacin slightly increased ovalbumin-induced histamine release and substantially enhanced (3-fold) i-LT release from the trachea. Neither the profile nor rate of sulfidopeptide leukotriene release was altered by indomethacin. Indomethacin had no effect on the maximum amplitude of the antigen-induced contraction but significantly enhanced the magnitude of contraction observed after 10 min of antigen exposure. These results demonstrate that actively sensitized airways synthesize and release sulfidopeptide leukotrienes upon challenge with specific antigen and that endogenously formed LTC4 is efficiently metabolized to LTD4 and LTE4. The results with indomethacin support the hypothesis that indomethacin potentiates antigen-induced airway contraction in vitro by enhancing the release of mast cell associated mediators.

Sensitive method for the analysis of phospholipid subclasses and molecular species as 1-anthroyl derivatives of their diglycerides

A sensitive high-performance liquid chromatographic (HPLC) method for the separation and quantitation of phospholipid subclasses and molecular species has been developed. Phospholipids for analysis are hydrolyzed to the diradyl glycerols (DGs) with phospholipase C and the resulting DGs reacted with a molar excess of 1-anthroyl nitrile in the presence of quinuclidine or 4-dimethylaminopyridine to form a stable adduct. The anthroyl-DGs were separated into alkenylacyl, alkylacyl, and diacyl subclasses either by using normal-phase HPLC or by thin-layer chromatography on silica gel G plates. Molecular species within alkenylacyl, alkylacyl, and diacyl subclasses were separated using reversed-phase HPLC. Separation of the individual subclasses was achieved for ethanolamine phosphoglycerides from bovine brain, as well as choline and ethanolamine phosphoglycerides from human neutrophils. Separation and quantitation of individual molecular species were carried out for alkenylacyl, alkylacyl, and diacyl subclasses of bovine brain ethanolamine phosphoglycerides by their absorbance at 254 nm with correction for recoveries as normalized to the internal standard 1,2-dipentadecanoyl-3-phosphatidylcholine added before the hydrolysis of phospholipids with phospholipase C or 1,2-dipentadecanoyl-3-anthroyl glycerol added after complete derivatization. The extinction coefficient of the 1-anthroyl derivatives were greater than 68,000 permitting the generation of concentration-dependent determinations which were linear to less than 1 pmol when monitored at 254 nm. Thus, this procedure provides a new and very sensitive method for the quantitation of picomole quantities of phospholipids or DGs by HPLC techniques.

Automated extraction and HPLC resolution of lipoxygenase and cyclooxygenase products utilizing high pressure column switching

An automatable column switching technique for extraction of cyclooxygenase and lipoxygenase products has been developed. The extraction column is a 0.4 cm X 3 cm stainless steel column slurry packed with Polygosil (C18, 25-40 mu particle diameter). Supernatants of incubations terminated by the addition of 1 volume MeOH, bath sonicated and centrifuged were pumped onto the extraction column. The non-adherent polar material was eluted to waste with up to five times the sample volume with a high polarity mobile phase. Following this, the extraction column was then eluted to an analytical column with a mobile phase of decreased polarity.

Suppression by ingested eicosapentaenoic acid of the increases in nasal mucosal blood flow and eosinophilia of ryegrass-allergic reactions

Nasal mucosal blood flow, assessed by a laser Doppler probe technique, and the concentration of eosinophils in nasal secretions were quantified during challenge of one nostril with ryegrass-pollen antigen and the other nostril with diluent alone in seven patients with ryegrass-allergic rhinitis. The identical studies were repeated after an 8-week course of 3.5 gm/day of eicosapentaenoic acid (EPA). Ryegrass antigen evoked mean rises in nasal blood flow of 30% to 100% after 10 and 30 minutes that were significant, relative to prechallenge levels and to levels after diluent challenge, both before and after EPA. Antigen-induced increases in nasal blood flow were significantly less after than before EPA at 10 minutes, and at 180 minutes increases were significant only before EPA. In ryegrass-allergic patients with rhinitis who did not take EPA between the two studies, the increases in blood flow after antigen challenge were the same on both occasions. Similarly, the nasal eosinophilia elicited by antigen was significant at 180 minutes only before EPA. Both a composite index of signs and symptoms and the constituent variables, reflecting the clinical response to antigen challenge, were unaffected by EPA. The suppression by EPA of responses of nasal blood flow and nasal eosinophils to antigen challenge supports a role for fatty acid and phospholipid mediators in allergic rhinitis, but the clinical assessment did not provide evidence for any symptomatic benefit from EPA.

Modulation of Eicosanoid Biosynthesis by Novel Pyridinylpyrimidines

A simple but effective strategy for drug development has been controlling those molecules that cause disease either through specific antagonists or synthesis inhibitors. Accordingly, structural elucidation of key mediators profoundly affects this approach by providing clear targets. The 1911 discovery of histamine is a classic example, eventually leading to a number of specific antagonists that remain therapeutically useful today. More recently, the discovery of the slow-reacting substance of anaphylaxis (SRS-A) as the sulfidopeptidyl leukotrienes (LT),’ and the rabbit aorta-constricting substance as a mixture of thromboxane Az (TXA2), prostaglandin Hz (PGHz), and PGGz,2 for example, have led to the synthesis of a number of specific inhibitors and antagonists with therapeutic potential. Although specific inhibition or antagonism is advantageous with respect to toxicity by minimally affecting other homeostatic processes, it is often insufficient due to redundancy of mediators of diverse origin and structure such as histamine and LTC. Because of this, an alternative to specific inhibitors and antagonists is the design of cell “stabilizing” agents or mediator release inhibitors. These agents prevent the release of both preformed mediators and the biosynthesis of other mediators such as eicosanoids. The demonstration that chromolyn sodium-a useful drug for the management of asthma-inhibits mediator release from antigen-stimulated mast cells’ greatly bolstered the mast cell-based search for mediator release inhibitors, despite clear evidence that this was chromolyn’s mechanism of action in asthma. A new generation of mediator release inhibitors containing a pyridinylpyrimidine structure is described below: but unlike chromolyn, the pyridinylpyrimidines also inhibit basophils and phagocytes. This series of compounds inhibits eicosanoid biosynthesis primarily at levels proximal to the phospholipase Az (PLA,), but to a lesser extent at distal sites such as the thromboxane synthetase. From in vivo and in vitro data, the pyridinylpyrimidines appear to be a promising new class of compounds for development as therapy for allergic and inflammatory diseases where eicosanoids and granule release are essential components of the etiology.

Stimulation of prostaglandin E2 synthesis in chondrocytes by a factor derived from activated macrophages

The serum-free spent medium of lipopolysaccharide-activated rabbit peritoneal macrophages contains a proteinaceous factor that stimulates the synthesis of PGE2 in rabbit articular chondrocytes. Synthesis of this factor by macrophages is inhibited by cycloheximide. Stimulation of PGE2 in chondrocytes is detected after a four-hour exposure to the macrophage factor and is completely abolished by the addition of either cycloheximide or indomethacin to the chondrocyte cultures. The macrophage derived factor has an apparent molecular weight of 30,000, is heat stable and not inactivated upon reductive alkylation or on treatment with phenylglyoxal. Activity is partially destroyed upon treatment with acid (pH 2.0) and upon trypsin treatment.

Recombinant bovine GM-CSF primes superoxide production but not degranulation induced by recombinant bovine interleukin-1 beta in bovine neutrophils.

Bovine neutrophil activation, superoxide production, and β‐glucosaminadase release induced by various biological stimuli were examined. Plateletactivating factor (PAF) and recombinant bovine interleukin‐1 β (r‐BoIL‐1β) induced superoxide production and β‐glucosaminadase release in bovine neutrophils. When these two responses were compared, the dose requirement for maximum activation was similar for PAF (1 × 10−6 M). However, the concentration of r‐BoIL‐Ι β required for the maximum degranulation (2.5 × 10−7 M) was 100‐fold higher than that for the maximum superoxide production (2.5 × 10‐9 M). Furthermore, pretreatment of cells with recombinant bovine granulocyte‐ macrophage colony‐stimulating factor (r‐BoGM‐CSF) enhanced both superoxide production and β ‐glucosaminidase release induced by PAF. In contrast, whereas superoxide production induced by r‐BoIL‐1β was enhanced by r‐ BoGM‐CSF priming, β ‐glucosaminidase release induced by r‐BoIL‐lβ was significantly reduced by pretreatment with r‐BoGM‐CSF. CL 184,005, a PAF antagonist, inhibited PAF‐induced glucosaminidase release and superoxide production but did not inhibit r‐BoIL‐lβ ‐in‐ duced superoxide production and degranulation. In addition, it did not inhibit the priming effect of r‐BoGM‐ GSF on r‐BoIL‐lβ ‐induced superoxide production. These results suggest that (1) PAF and r‐BoIL‐1β activate bovine neutrophils by different mechanisms, (2) r‐BoGM‐CSF primes superoxide production and degranulation induced by PAF, (3) r‐BoGM‐CSF primes superoxide production but not degranulation induced by r‐BoIL‐lβ, and (4) the priming effect of r‐BoGM‐CSF is not mediated by PAF.

Human neutrophil platelet-activating factor: molecular heterogeneity in unstimulated and ionophore-stimulated cells

The molecular heterogeneity of platelet-activating factor (PAF) in resting and ionophore (A23187) -stimulated human neutrophils was measured by a very sensitive gas chromatography-negative ion chemical ionization mass spectrometric method. The molecular species compositions of PAF, which are due to variations in the 1-O-alkyl chain length, were significantly different between resting and ionophore-stimulated polymorphonuclear leukocytes. The major species of PAF produced by unstimulated polymorphonuclear leukocytes were 16:0, 17:0, 18:1 and 18:0, representing 55, 14, 8 and 10%, respectively, of the total PAF; 16:0 was the predominant PAF (74%) in A23187-stimulated polymorphonuclear leukocytes. The PAF molecular species from unstimulated polymorphonuclear leukocytes was similar to compositions from those of the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, whereas those from the ionophore-stimulated polymorphonuclear leukocytes differed from the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, thus indicating a very high degree of substrate selectivity for PAF synthesis. Although the physiological implications of the variations in PAF composition are not known, these studies indicate that the PAF produced by resting polymorphonuclear leukocytes are significantly different from those produced in response to ionophore.

Studies of the effect of a platelet-activating factor antagonist, CL 184,005, in animal models of gram-negative bacterial sepsis.

The effect of CL 184,005, a potent and specific platelet-activating factor antagonist, has been examined in a variety of animal models relevant to gram-negative bacterial sepsis. Pretreatment of mice with CL 184,005 protected them from the lethal effects of platelet-activating factor. When rats or primates rendered hypotensive with endotoxin were treated with CL 184,005, blood pressure was normalized. Pretreatment of rats with CL 184,005 protected them from the gastrointestinal lesions induced by endotoxin. Pretreatment of rats and mice with CL 184,005 protected them from the lethal effects of endotoxin. Plasma tumor necrosis factor levels in endotoxin-treated mice were lower when the mice were pretreated with CL 184,005. These observations suggest that CL 184,005 may be potentially useful in the treatment of gram-negative bacterial sepsis, and the agent is undergoing clinical evaluation.