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M1 and M2 activated macrophages (Mϕs) have different roles in inflammation. Because pathogens may first encounter resting cells, we investigated lipid mediator profiles prior to full activation. Human monocytes were differentiated with granulocyte Mϕ colony-stimulating factor (GM-CSF) or Mϕ colony-stimulating factor (M-CSF), which are known to prime toward M1 or M2 phenotypes, respectively. Lipid mediators released during resting conditions and produced in response to bacterial stimuli (LPS/N-formylmethionyl-leucyl-phenylalanine or peptidoglycan) were quantified by liquid chromatography-mass spectrometry. In resting conditions, both Mϕ phenotypes released primarily proresolving lipid mediators (prostaglandin E2 metabolite, lipoxin A4, and 18-hydroxyeicosapentaenoic acid). A striking shift toward proinflammatory eicosanoids was observed when the same cells were exposed (30 min) to bacterial stimuli: M-CSF Mϕs produced considerably more 5-lipoxygenase products, particularly leukotriene C4, potentially linked to M2 functions in asthma. Prostaglandins were formed by both Mϕ types. In the M-CSF cells, there was also an enhanced release of arachidonic acid and activation of cytosolic phospholipase A2 However, GM-CSF cells expressed higher levels of 5-lipoxygenase and 5-lipoxygenase-activating protein, and in ionophore incubations these cells also produced the highest levels of 5-hydroxyeicosatetraenoic acid. In summary, GM-CSF and M-CSF Mϕs displayed similar proresolving lipid mediator formation in resting conditions but shifted toward different proinflammatory eicosanoids upon bacterial stimuli. This demonstrates that preference for specific eicosanoid pathways is primed by CSFs before full M1/M2 activation.-Lukic, A., Larssen, P., Fauland, A., Samuelsson, B., Wheelock, C. E., Gabrielsson, S., Radmark, O. GM-CSF- and M-CSF-primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures.

Die Arachidonsaure, eine vierfach ungesattigte C20-Fettsaure, hat in den letzten 15 Jahren weit uber den Kreis der Ernahrungsforscher hinaus Interesse erheischt. Sie bildet einen Acylbestandteil der Phospholipide aller tierischen Zellen. Die schon lange bekannte Tatsache, das bestimmte ungesattigte Fettsauren in der Nahrung enthalten sein mussen, damit eine normale Funktion des Organismus gewahrleistet ist, hat mit der Entdeckung einer weit aufgefacherten Palette von biologisch hochwirksamen Oxidationsprodukten der Arachidonsaure, einer Kaskade, wie sie oft genannt werden, eine Erklarung erfahren. Arachidonsaure entsteht im Organismus aus Linol- und Linolensaure, die zu den sogenannten essentiellen Fettsauren gehoren. Ulf von Euler konnte schon 1935 in Samenflussigkeit einen Stoff nachweisen, der glatte Muskulatur zur Kontraktion bringt. Dieser spater rein isolierte Faktor wurde als Prostaglandin bezeichnet. Die Zahl der Prostaglandine nahm dann stark zu, auch die der synthetisch erhaltenen Analoga. Sie entstehen biosynthetisch aus Arachidonsaure und ahnlichen C20-Fettsauren durch enzymatische Oxidation mit einer Cyclooxygenase. Deren Wirkung wird durch Acetylsalicylsaure, Indomethazin und andere Stoffe gehemmt, weshalb diese Mittel Entzundungen lindern oder heilen, die als Folgen der Wirkung bestimmter Prostaglandine auftreten. Nach den Prostaglandinen wurden als weitere Umwandlungsprodukte der Arachidonsaure die Thromboxane entdeckt, die eine starke Zusammenballung (Aggregation) der Blutplattchen bewirken, die fur die Blutgerinnung zustandig sind. In einem wohlausgewogenen Gleichgewicht wirkt den Thromboxanen das bald danach entdeckte Prostacyclin entgegen, das die Aggregate auflost. Beide Wirkstoffe sind Metaboliten der Arachidonsaure, die ebenfalls im Verlauf der cyclisierenden Oxidation entstehen. Deshalb last sich auch das Bild der Blutgerinnung durch Aspirin und ahnlich wirkende Mittel beeinflussen. Es gibt jedoch Kontraktionsvorgange und Entzundungs-erscheinungen, die auf diese Mittel nicht, wohl aber auf Steroidhormone (Cortison) ansprechen. Fur solche Erscheinungen, zu denen auch das Asthma gehort, sind hochstwahrscheinlich die zuletzt entdeckten Umwandlungsprodukte der Arachidonsaure ausschlaggebend, die auf einem anderen enzymatischen Weg, uber ein Hydroperoxid der Arachidonsaure entstehen. Sie werden in Leukozyten gebildet und enthalten das dreifach ungesattigte Strukturelement eines Triens und wurden deshalb als Leukotriene bezeichnet. Ihre erstaunlich starke Histamin-artige Wirkung bei der Kontraktion der Lungenkapillaren, ihre ausgepragte.

The synthesis of a potent competitive LTA4hydrolase inhibitor (A, Ki=1.6 nM) prompted an investigation into the biological activity of A against LTB4 biosynthesis, as well as the design, synthesis, and evaluation of a new series of inhibitors B to further probe the active site of LTA4 hydrolase. On the basis of these results, and previously reported site-directed mutagenesis and inhibition studies, the mechanisms of peptide and epoxide hydrolysis catalyzed by LTA4 hydrolase are discussed.

Differentiation of the human monocytic cell line Mono Mac 6 with calcitriol plus transforming growth factor-beta (TGFbeta) strongly induces 5-lipoxygenase (5-LO) mRNA and protein expression. The mechanism of 5-LO mRNA induction by these agents was investigated. Analysis of mature 5-LO mRNA by reverse-transcriptase-PCR gave a 42-fold induction which was not due to alterations in 5-LO half life and which was only in part due to an induction of gene transcription. There was an up to fivefold increase in 5-LO primary transcripts by TGFbeta and calcitriol, which could be inhibited by cycloheximide. No significant effects on 5-LO transcription were observed with TGFbeta or calcitriol alone. However, treatment of the cells with either calcitriol or TGFbeta and addition of the corresponding second inducer lead to an about fourfold induction of primary transcript levels. Addition of cycloheximide together with the second inducer inhibited only the TGFbeta but not the calcitriol effects, which indicated that there is a direct stimulation of 5-LO transcription by calcitriol in the presence of TGFbeta-induced proteins. In order to investigate the effects of TGFbeta/calcitriol on 5-LO transcript, elongation and maturation, the relative changes in immature and mature 5-LO RNA species were analyzed by reverse-transcription-PCR. Analysis of exons 1-5 indicated an about threefold induction of 5-LO transcripts by calcitriol/TGFbeta, respectively. However, when exons 6-14 were determined, more pronounced increments were found (3.6-12-fold). Selective analysis of polyadenylated and spliced 5-LO mRNA species gave a 42-fold induction. The effects of both TGFbeta and calcitriol on transcript elongation and maturation were inhibited by cycloheximide. Our results show that induction of 5-LO mRNA by calcitriol and TGFbeta is due to a modest increase in 5-LO gene transcription and to the stimulation of transcript elongation and maturation.

5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. Here, we demonstrate that extracellular signal-regulated kinases (ERKs) can phosphorylate 5-LO in vitro. Efficient phosphorylation required the presence of unsaturated fatty acids and was abolished when Ser-663 was mutated to alanine. In intact HeLa cells stimulated with arachidonic acid (AA), impaired 5-LO product formation was evident in cells expressing the S663A-5-LO mutant compared with cells expressing wild-type 5-LO. For Mono Mac 6 cells, priming with phorbol myristate acetate (PMA) before stimulation with ionophore was required for ERK1/2 activation and efficient 5-LO phosphorylation, in parallel with substantial AA release and 5-LO product formation. Inhibition of PKC by GF109203x or MEK1/2 by U0126 (or PD98059) abolished the 5-LO up-regulation effects of PMA. In contrast, these inhibitors failed to suppress 5-LO product formation induced by stimuli such as AA plus ionophore, which apparently do not involve the ERK1/2 pathway. Based on inhibitor studies, ERKs are also involved in AA-stimulated 5-LO product formation in PMNL, whereas a role for ERKs is not apparent in 5-LO activation induced by ionophore or cell stress. Finally, the data suggest that ERKs and p38 MAPK-regulated MAPKAPKs can act in conjunction to stimulate 5-LO by phosphorylation.

A cDNA encoding an arachidonate 12-lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12-lipoxygenase cDNA were used to isolate the initial polymerase-chain-reaction product (395 bp). The final sequence of the rat 12-lipoxygenase cDNA coding region (1989 bp) was verified by analysis of several separate polymerase-chain-reaction products. The open reading frame corresponded to a protein of 662 amino acid residues, with a calculated molecular mass of 75,305 Da. Also the rat 12-lipoxygenase contained the six conserved histidines, characteristic for all cloned lipoxygenases. It displayed the highest degree of identity to porcine leukocyte 12-lipoxygenase (71%) and to human 15-lipoxygenase (75%), with less resemblance to human platelet 12-lipoxygenase (59%) or rat leukocyte 5-lipoxygenase (41%). The recombinant enzyme was expressed in Escherichia coli and incubated with arachidonic acid. Primarily 12-lipoxygenase (but also some 15-lipoxygenase) enzyme activity was obtained. A part of the brain 12-lipoxygenase cDNA was used as probe in Northern blots. A 2.7-kb mRNA was more abundant in RNA from rat leukocytes, lung, and aorta, than in RNA from rat brain. Sequencing of parts of the corresponding cDNAs (from leukocytes and lung), and comparison to the brain 12-lipoxygenase sequence, indicated that these mRNAs from the different rat tissues were identical.

and adhesion to post- capillary venules [3]. In [4-61 LTB., was suggested to be a weak stimulator of polymorphonuclear leuko- cyte (PMNL) degranulation. However, it is now clear that previous purification procedures for biosyntheti- tally prepared LTB4 were inadequate. The newly discovered LTB4 isomer, SQ,12Q-DHETE, co-chro- matographs with LTB4 in the reverse phase high- pressure liquid chromatography (HPLC) systems commonly used [7]. As a consequence, earlier studies probably tested preparations which contained a mix- ture of both isomers with the non-leukotriene product predominating. Therefore, we have investigated the role of LTB4, SQ,12(S)-DHETE and their o-oxida- tion products (fig.1) [8] in leukocyte degranulation in vitro. In addition, the activity of the non-enzymati- tally formed LTB4 isomers was investigated. Cytochalasin B, phenolphthalein glucuronic acid, and dried

Biological activities of Lipoxin A include lung strip contraction and dilation of arterioles in vivo. Acta Physiol Scand130, 643–647. Received 7 December 1986, accepted 27 February 1987. ISSN 0001–6772. Departments of Physiology and Physiological Chemistry, Karolinska Institute, and Department of Experimental Medicine, Pharmacia AB, Sweden. Lipoxin A ([5s,6r,15s]-5,6,15–trihydroxy-7,9,13–trans-II-cis-eicosatetraenoic acid), a recently characterized lipoxygenation product of arachidonic acid, in submicromolar concentrations elicited long-lasting contractions of the guinea-pig lung strip. The response to lipoxin A was not due to release of acetylcholine, histamine, noradrenaline or cyclo-oxygenase products. 15–hydroperoxyeicosatetraenoic acid (15–HPETE), one precursor of lipoxin A, also contracted the lung strip, but 15–HPETE was less potent and the response was comparatively short-lived. Furthermore, lipoxin A was inactive on the guinea-pig trachea whereas 15–HPETE relaxed this preparation. Lipoxin A was also inactive on the guinea-pig ileum. Intravital microscopy of the hamster cheek pouch disclosed that lipoxin A, as well as 15–HPETE, induced arteriolar dilation but had no effects on microvascular permeability or leucocyte adherence to venular endothelium. Taken together, the leucocyte product lipoxin A displayed a pattern of activity in spasmogenic assays and on the microvasculature that was distinct from those known for prostaglandins, thromboxanes and leukotrienes. The findings indicate that lipoxin A is an additional arachidonic acid derived autacoid with biological actions on smooth muscle in vatro and in vivo.

The effects on the isolated human umbilical artery (HUA) of the recently isolated endoperoxide intermediates in prostaglandin (PG) biosynthesis, PGG2 and PGH2, were studied. Both endoperoxides were potent contractors of the artery strips, the threshold concentrations being 3 (1-4) ng/ml for PGG2 and 1 (1-12) ng/ml for PGH2, as compared with 200 (40-400) ng/ml for PGE2. The hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (thromboxane B2), a metabolite of PGG2, appeared in the bath medium indicating PG and thromboxane generation in the isolated HUA. The formation of thromboxane B2 was inhibited by indomethacin (8-40 mug/ml) and by eicosa-5,8,11,14-tetraynoic acid (ETA) (25 mug/ml), ETA also inhibited the contractile responses to both endoperoxides. The results support the view that local generation of PGs might be involved in the closure of the HUA at birth.

The regional distribution of ionophore A23187-induced synthesis of leukotrienes and mono-hydroxyeicosatetraenoic acids in the rat brain in vitro was investigated. Pronounced differences in leukotriene C4 formation were observed, with the highest synthetic capacity in the hypothalamus. The formation of leukotriene C4 was about 12-times higher in the hypothalamus as compared to the cerebellum. This finding is in agreement with a possible neuroendocrine role for leukotriene C4. In contrast, the activity of leukotriene B4 synthesis was widely distributed without pronounced regional differences in the rat brain. Formation of 5-, 9-, 11-, 12- and 15-monohydroxyeicosatetraenoic acid was detected in all regions. The major lipoxygenase product in the hypothalamus and thalamus was 5-hydroxyeicosatetraenoic acid, while other monohydroxyeicosatetraenoic acids predominated in the remaining regions tested.

Thromboxane (TX) synthesis was investigated in some characterized platelet disorders and correlated with release of ADP or [14C]serotonin and platelet aggregation. In a case with Bernard-Soulier syndrome TX-synthesis as well as ADP-[14C]serotonin release and platelet aggregation were found to be essentially normal when platelets were incubated with collagen, ADP, arachidonic acid or prostaglandin G2 (PGG2). In a case with Hermansky-Pudlak syndrome aggregation was normal with ADP, whereas aggregation with collagen and PGG2 was markedly decreased. Release of ADP and [14C]serotonin was found to be decreased with all investigated inducers in this platelet disorder, whereas TX-synthesis was normal with arachidonic acid and PGG2. In three cases with Glanzmann's thrombasthenia almost no aggregation was observed with any of these compounds. However, in this disorder platelet TX-formation from arachidonic acid or PGG2 was also normal whereas ADP and collagen both induced less TX-formation than in normal platelets. These results indicate that none of the investigated platelet disorders was associated with abnormal TX-synthesis from arachidonic acid as found in platelet cyclo-oxygenase deficiency. These observations provide further evidence for the hypothesis that the Bernard-Soulier syndrome and Glanzmann's thrombasthenia are primarily associated with glycoprotein abnormalities at receptor level (Nurden & Caen, 1974, 1975, 1976). A hypothetical model for the interrelationship of two specific glycoproteins and thromboxane-synthesis is discussed.

Arachidonic acid, a fourfold unsaturated C20-fatty acid, has attracted interest over the last 15 years far beyond the bounds of nutritional research. It forms an essential acyl component of the phospholopids in all animal cells. The long know fact that certain unsaturated fatty acids must be contained in the diet in order to ensure normal function of the organism has been explained by the discovery of a broad palette of biologically highly active oxidation products of arachidonic acid, a cascade, as it is often referred to. Arachidonic acid is formed in organisms from linoleic and linolenic acids, which belong to the so-called essential fatty acids. As early as 1935 Ulf von Euler was able to detect a substance in semen which caused smooth muscles to contract. This factor, which was later isolated in pure form, was termed prostaglandin. The number of prostaglandins, including the synthetically obtained analogues, then rapidly increased. Prostaglandins are biosynthesized from arachidonic acid and similar C20-fatty acids by enzymatic oxidation with a cyclooxygenase. Its activity is inhibited by acetylsalicylic acid, indomethazine, and other substances, because these relieve or cure inflammations arising from the action of certain prostaglandins. After the prostaglandins, the thromboxanes, which promote aggregation of blood platelets and thus cause thrombosis, were discoverd as further metabolic products of arachidonic acid. In a well-balanced equilibrium the thromboxanes have a biological effect completely opposite to that of prostacyclin, discovered soon after this, which inhibits platelet aggregation. Both of these biologically active substances are metabolites of arachidonic acid, and are formed in the course of the cyclizing oxidation. Aspirin and substances having a similar mode of action, therefore have an influence on thrombosis. There are, however, contraction processes and inflammation symptoms which do not respond to these substances, but to steroid hormones (cortisone). For symptoms of this type, to which asthma also belongs, the most recently discovered metabolic products of arachidonic acid are probably highly decisive; they are formed by another enzymatic route via a hydroperoxide of arachidonic acid. These compounds are formed in leukocytes and contain the triply unsaturated structural element of a triene and were, therefore, termed leukotrienes. Their astonishingly high histamine-like activity in producing contraction of lung capillaries, their marked property of making capillary vessels permeable to blood plasma, i.e. of generating oedema, the ability of leukotriene LTB4 to cause adherence of human leukocytes and their adhesion to the endothelium, make the leukotrienes chemical mediators in allergies and inflammations, and produce biological effects when present at minute concentrations. In the following article one of the three scientists who were awarded this years Nobel prize for medicine and physiology, and who provided an explanation for this extraordinarily interesting class of natural products, gives an account of the conversion of arachidonic acid into the labile epoxide LTA4 and further into the dihydroxy compound LTB4, or the glutathione cysteinylglycine and cysteine conjugates LTC4. LTD4, and LTE4, respectively, as well as of the physiological-pathological implications. The two other prize winners in this area of biologically active compounds, which are often referred to as eicosanoids, are Samuelsson's teacher Sune Bergstom and the Britain John Vane.

Recent studies of arachidonic acid metabolism in polymorphonuclear leukocytes have led to the discovery of the biologically active leukotrienes [ 11. The synthesis of these compounds is initiated by oxygenation of arachidonic acid at C-5 forming 5hydroperoxyeicosatetraenoic acid [2], which is further converted to the unstable 5 ,6-epoxy-7,9,11 ,14-eicosatetraenoic acid (leukotriene &, LTA4) [3,4]. The epoxide is hydrolysed either enzymatically to SS,12R-dihydroxy6_cis,8-rruns,lO-trans,l4_cis-eicosatetraenoic acid (LTB,) [S-7] or non-enzymatically to additional isomeric S,12and 5,6-dihydroxy eicosatetraenoic acids [8]. LT& is also converted by addition of glutathione into LTC4 [9,10]. This compound and two metabolites, LTD4 and LTE4, are responsible for the biological activity of most preparations of slowreacting substance of anaphylaxis (SRS-A) [ 11,121. We have reported that a new group of leukotrienes can also be formed by initial oxygenation at C-l 5 [13,14]. Here, we describe the formation of 5S,12Sdihydroxy_6,8,10,14eicosatetraenoic acid and 5S,12S,20-trihydroxy-6,8,10,14-eicosatetraenoic acid in preparations of human leukocytes. These novel metabolites are not formed via the leukotriene pathway. Instead arachidonic acid is transformed by a double oxygenation to the dihydroxy acid and further w oxidized to the trihydroxy acid.

The synthesis of an isomer of prostaglandin F 2alpha,9alpha,11alpha,15(S)-trihydroxyprosta-4-cis,13-transdienoic acid is described. The metabolism of this compound in the rat has been investigated. The rate of degradation by beta-oxidation was slowed down considerably. Thus 10-20% of the injected isomer was excreted in the urine unchanged indicating a longer half-life in the circulation than for prostaglandin F 2alpha. More over 2% was excreted as C20 metabolites, 11-18% as C18 metabolites and 8-15% as C16 metabolites. This relative resistance to degradation by beta-oxidation is of considerable biochemical and pharmacological interest.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.

Arachidonic acid plays a central role in a biological control system where such oxygenated derivatives as prostaglandins, thromboxanes, and leukotrienes are mediators. The leukotrienes are formed by transformation of arachidonic acid into an unstable epoxide intermediate, leukotriene A4, which can be converted enzymatically by hydration to leukotriene B4, and by addition of glutathione to leukotriene C4. This last compound is metabolized to leukotrienes D4 and E4 by successive elimination of a gamma-glutamyl residue and glycine. Slow-reacting substance of anaphylaxis consists of leukotrienes C4, D4, and E4. The cysteinyl-containing leukotrienes are potent bronchoconstrictors, increase vascular permeability in postcapillary venules, and stimulate mucus secretion. Leukotriene B4 causes adhesion and chemotactic movement of leukocytes and stimulates aggregation, enzyme release, and generation of superoxide in neutrophils. Leukotrienes C4, D4, and E4, which are released from the lung tissue of asthmatic subjects exposed to specific allergens, seem to play a pathophysiological role in immediate hypersensitivity reactions. These leukotrienes, as well as leukotriene B4, have pro-inflammatory effects.