In vitro effect of N-methoxy-3-(3,5-ditert-butyl-4-hydroxybenzylidene)-2-pyrrolidone (E-5110), a novel nonsteroidal anti-inflammatory agent,on generation of some inflammatory mediators

Cultured rat synovial cells generate PGE₂ upon stimulation with a factor derived from rate polymorphonuclear cells (Prostaglandins27, 697, 1984). E-5110 inhibited PGE₂ generation by the synovial cells. The IC₅₀ values (M) for inhibition of PGE₂ generation were 0.026 for E-5110, 0.008 for indomethacin, 0.112 for piroxicam, 0.003 for R-830, 0.667 for BW-755C and 2.05 for benoxaprofen. Calcium ionophore A-23187-stimulated LTB₄ generation by human neutrophils was inhibited by E-5110 with an IC₅₀ value of 0.20 M, which was similar to NDGA. The inhibition of LTB₄ by E-5110 was more potent than that of R-830, BW-755C or benoxaprofen. E-5110 also inhibited superoxide generation by human neutrophils stimulated with opsonized zymosan, f-Met-Leu-Phe and phorbol myristate acetate. These results indicate that E-5110 is a potent dual inhibitor that suppresses superoxide generation.     

The Proinflammatory Cytokines and Arachidonic Acid Metabolites in Human Overnight Tears: Homeostatic Mechanisms

The tear film plays an important role in the defense of the external ocular surface. During sleep a number of changes take place, including increased production and release of various inflammatory mediators. We have studied the hypothesis that closed-eye tears contain proinflammatory cytokines and lipid inflammatory mediators, which serve to recruit polymorphonuclear leukocytes (PMNs) and regulate the function of PMNs and IgA during sleep. We investigated interleukin-1, interleukin-6, interleukin-8, monocyte chemotactic protein 1, granulocyte–macrophage colony stimulating factor (GM-CSF), leukotriene B₄ (LTB₄), and platelet activating factor (PAF) in open and closed-eye tears of normal healthy subjects. Results showed that IL-6, IL-8, GM-CSF, LTB₄, and PAF were present in high levels in closed-eye tears compared to open-eye tears. Closed-eye tears were able to recruit neutrophils, with maximal recruitment after 8 hr of sleep, suggesting that chemokine IL-8 and the lipid chemoattractant LTB₄ were active. Flow cytometric analysis revealed that incubation of neutrophils with closed-eye tears up-regulated the surface expression of IgA receptor, indicating that the GM-CSF in tears was functionally active. Up-regulation of cytokines and the lipid inflammatory mediator LTB₄ during eye closure are noteworthy, as each of these cytokines has an established role in initiation and amplification of the inflammatory response. IL-8 and LTB₄ may act as potent chemoattractants and activators for PMNs, whereas IL-6 and GM-CSF potentiate the secretion and function of IgA and enhance neutrophil responsiveness to proinflammatory agonists.     

Leukotriene B4 production by blood neutrophils in allergic rhinitis—effects of cetirizine

Background: Mucosal inflammatory processes in late phase of allergic diseases involve cytokine production, cell adhesion molecule overexpression and release of inflammatory mediators with chemotactic activity, such as leukolriene B₄ (LTB₄), We had previously observed increased production of LTB₄ by neutrophils in patients with allergic rhinitis and discussed the role of granulocyte macrophage-colony stimulating factor (GM-CSF) priming. Some antihislaminic compounds were shown to diminish the production of leukotrienes by neutrophils. Objective: In a first step, we evaluated in ex vivo and in vitro studies, the effects of cetirizine on LTB₄ production by blood neutrophils from allergic and healthy subjects. In a second step, we studied the in vitro effect of cetirizine on LTB₄ production by neutrophils from healthy subjects during GM-CSF priming of these cells. Methods: Neutrophils from both populations were purified from venous blood and LTB₄ production was measured using high performance liquid cromatography (HPLC) method. Results: In ex vivo studies, cetirizine treatment induced a decreased LTB₄ production by neutrophils in allergic rhinitis. This effect of decreased LTB₄ production was reproduced in vitro with 10^?8–10^-6 cetirizine. Nevertheless, this anti-Hl compound had no effect on neutrophil priming with GM-CSF. Conclusion: As LTB₄ is an important chemotactic factor, Cetirizine could act on inflammatory cell recruitment by inhibiting LTB₄ production by neutrophils.     

Pharmacological profile of a novel,orally active leukotriene B4 antagonist,SM-15178

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B₄ (LTB₄). SM-15178 inhibited [³H]LTB₄ binding to its receptors on human neutrophils (IC₅₀=0.30M). It inhibited LTB₄-induced chemotaxis of human neutrophils (IC₅₀ =0.72M) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30M. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10M. LTB₄-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC₅₀ values of 0.55, 0.52, and 0.58 M, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB₄-induced transient leukopenia. It also suppressed LTB₄-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB₄ antagonist and that it might be effective for the treatment of some types of inflammatory diseases.     

Antiinflammatory effects of second-generation leukotriene B4 receptor antagonist,SC-53228: Impact upon Leukotriene B4- and 12(R)-HETE- mediated events

Leukotriene B₄ (LTB₄) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] are proinflammatory products of arachidonic acid metabolism that have been implicated as mediators in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB₄ and 12(R)-HETE elicit a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxyl]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid}, a first-generation LTB₄ receptor antagonist, inhibited the chemotactic actions of LTB₄ when given orally with an ED₅₀ value of 1.7 mg/kg. The second-generation LTB₄ receptor antagonist, SC-53228 [(+)-(S)-7-(3-{2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)carbonyl]phenoxy} propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid], inhibited LTB₄-induced chemotaxis when given intragastrically with an ED₅₀ value of 0.07 mg/kg. Furthermore, SC-53228 inhibited 12(R)-HETE-induced granulocyte chemotaxis with an oral ED₅₀ value of 5.8 mg/kg. When dosed orally over a range of 0.03–100 mg/kg, SC-53228 gaveC _max plasma concentrations of 0.015–41.1g/ml. SC-53228 inhibited LTB₄-primed membrane depolarization of human neutrophils with an IC₅₀ value of 34 nM. As a potent LTB₄ receptor antagonist, SC-53228 may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, in which LTB₄ and/or 12(R)-HETE are implicated as inflammatory mediators.     

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid

Exposure of human neutrophils to 5(S),12(R) dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (leukotriene B₄, LTB₄) resulted in a time- and concentration- (10^–9-10^–6 M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB₄. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB₄ prior to contact with cytochalasin B. LTB₄-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N, N-dietliylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB₄-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) andN-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB₄-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive, Neutrophils pretreated with LTR₄ or 5(S), 12(R), 20-trihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB₄, an -oxidation metabolite of LTB₄) were desensitized to the subsequent exposure to LTB₄. Cross-desensitization was also demonstrated between LTB₄ and 20-OH-LTB₄. The stimulus specific nature of LTB₄-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) orN-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB₄-pretreated neutrophils. Enzyme release from LTB₄-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB₄-induced degranulation.     

Comparative evaluation of arachidonic acid (AA)- and tetradecanoylphorbol acetate (TPA)-induced dermal inflammation

The effects of topical application of arachidonic acid (AA) or phorbol ester, tetradecanoylphorbol 13-acetate (TPA), on edema response, vascular permeability, MPO, NAG, and generation of eicosanoids were studied in two murine models of cutaneous inflammation. AA produced a short-lived edema response with a rapid onset that was associated with marked increases in levels of prostaglandins (PGE₂, 6-keto-PGF₁, PGF₂), thromboxane B₂ (TxB₂) and leukotriene B₄ (LTB₄), with smaller increases in levels of LTC₄. TPA produced a longer-lasting edema that was associated with marked influx of neutrophils and predominant formation of LTB₄ along with significant changes in levels of TxB₂. Circulating T lymphocytes have no apparent role in the acute inflammatory responses induced by either agent. Arachidonic acid-induced vascular permeability preceded the edema response and neutrophil influx, whereas TPA-induced vascular permeability paralleled the edema response and influx of neutrophils. Mast cells appear to be important in the complete expression of inflammatory response, i.e., edema, cellular influx, and vascular permeability induced by either AA or TPA, as these responses were blunted in mast cell-deficient mice. Inhibitors of CO or 5-LO attenuated inflammatory responses in both models. The LTB₄ receptor antagonist, SC-41930, inhibited the inflammatory response to TPA but had little effect on that initiated by AA. This suggests that LTB₄ is an important mediator in the phorbol ester-induced inflammatory response, whereas peptidoleukotrienes and prostaglandins regulate vascular permeability responses in the arachidonate model.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophilsin vitro. Evidence of formation of antiinflammatory compounds

Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B₄ (LTB₄), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB₄ by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 M) and different concentrations (0–100 M) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB₄ was dose dependently inhibited by both LA (IC₅₀=45 M) and DGLA (IC₅₀=40M). This inhibition of LTB₄ formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB₄ formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB₄ formation (IC₅₀=7.5 M and IC₅₀=0.2 M). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB₄ formation (IC₅₀=0.75 M). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB₄ formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB₄ formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

Ebselen is a specific inhibitor of LTB4-mediated migration of human neutrophils

Ebselen is a seleno-organic anti-inflammatory compound with glutathione peroxidase-like activity that has the unique characteristic of mediating the isomerization of 5-HETE and LTB₄ to their biologically inactive trans isomers, both directly in fluid phase and indirectly through metabolic pathways in stimulated peripheral blood leukocytes. LTB₄ is an inflammatory mediator with potent chemotactic activity for neutrophilic leukocytes. We studied the effects of ebselen on the chemotactic and chemokinetic responses with human-blood-derived neutrophils. With the use of 120-m-thick 5-m-pore durapore filters and low BSA concentrations (0.05%) in the chemotaxis buffers, ebselen was evaluated for its effect on both chemotactic and chemokinetic responses to LTB₄, C5a, and fMLP. Ebselen at 3–20 M concentrations inhibited both chemotactic and chemokinetic responses to optimal concentrations of LTB₄ without altering chemotactic responses to C5a or fMLP. Likewise, ebselen at 20 M specifically inhibited LTB₄-stimulated transendothelial migration of neutrophils, while not altering responses to C5a nor fMLP.     

Activation of human neutrophil LFA-1 (CD11a) by leukotriene B4

Homotypic aggregation (HA) of human neutrophils by the potent leukotactic factor, leukotriene B₄ (LTB₄), and phorbol myristate acetate (PMA) was evaluated by recording the net decrease in absorbency at 650 nm of suspensions of 10⁷ neutrophils/ml in a microtiter plate reader, which was found to correlate with microscopic evidence of aggregation. LTB₄-elicited HA was increased maximally by approximately one third above HA in buffer at 30 min, whereas PMA-induced HA reached a maximal level more than 21–fold higher than buffer control at 60 min. The involvement of LFA-1 in LTB₄-induced HA of neutrophils was suggested initially by the inhibitory effect of monoclonal anti-CD 18 and anti-CD11a antibodies. The binding to neutrophils of a monoclonal anti-LFA-1 antibody (NK1-L16) specific for an activation epitope of CD11a was increased a maximum of 28-fold and sixfold, respectively, after 1 and 5 min of preincubation with 10 nM LTB₄and fivefold after 5 min with PMA. Thus, both LTB₄ and PMA induce an activating conformational change in the CD11a adherence receptor of human neutrophils.     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB₄ formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipiddependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B₄ (LTB₄) in FMLP-stimulated neutrophils, the ID₅₀ was approximately 4.5 g/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB₄ nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB₄ production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

Arachidonic Acid (AA) and Tetradecanoylphorbol Acetate (TPA) Exert Systemic Effects When Applied Topically in the Mouse

We studied the systemic actions of topically applied arachidonic acid (AA) and tetradecanoylphorbol acetate (TPA) in the mouse. AA or TPA-induced ear edema, increases in vascular permeability, eicosanoid levels, neutrophil and mononuclear influx were determined in both phlogogen-treated and contralateral vehicle-treated ear of each mouse and were compared with vehicle-treated ears from control mice. Edema and vascular permeability increases appeared only in AA- or TPA-applied ears. Moreover, in contralateral ears from AA-treated mice an increase in 6-keto-PGF₁ and LTB₄ was found. Only LTB₄ increased in the contralateral ear after TPA. Contralateral ears from AA- or TPA-treated mice also showed a significant increase in MPO levels. The increased levels of 6-keto-PGF₁ but not those of LTB₄ in contralateral ears were reduced by indomethacin applied simultaneously with the phlogogen. AA also increased plasma and serum levels of LTB₄, but not those of 6-keto-PGF₁. In contrast, TPA increased plasma and serum levels of 6-keto-PGF₁ and LTB₄. The results show that both AA and TPA applied topically exert systemic effects.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 M). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.     

Priming effects of leukotriene B4 on endothelial cell injury induced by tpa-activated leukocytes

Among arachidonic acid metabolites, leukotriene B₄ (LTB₄) plays an important role in inflammation, such as in the activation, adhesion, chemotaxis, and invasion of leukocytes. In this paper, we examined the effect of LTB₄ on endothelial cell injury induced by polymorphonuclear leukocytes (PMNLs).⁵¹Crrelease, a marker of cellular injury, was elicited from prelabeled endothelial cells when the cells were cocultured with PMNLs activated by phorbol ester (TPA, 12-0-tetradecanoyl-phorbol-13-acetate). Under this condition, pretreatment of PMNLs with LTB₄ enhanced their injury in a dose-dependent manner (0.2–2 M). However, LTB₄ alone at any dose could not induce any cellular injury. We also determined the amount of active oxygen species produced by PMNLs in response to TPA. The intensity of luminoldependent chemiluminescence, a marker of active oxygen production, in PMNLs was also increased by pretreatment with 1 M LTB₄. These data suggest that LTB₄ enhances endothelial cell injury by the priming effect on active oxygen production in activated PMNLs.     

Peripheral blood neutrophil leukotriene B4 release and migration in rheumatoid arthritis

The present study was designed to compare peripheral blood neutrophil migration and leukotriene (LT) release between patients with rheumatoid arthritis (RA) and healthy controls and to correlate the neutrophil functions with clinical disease activity. Nineteen patients with moderately active RA and 19 age and sex matched healthy volunteers participated in this study. Isolated peripheral blood neutrophils from RA patients released equal amounts of LTB₄ but their random migration was enhanced as compared with neutrophils from healthy controls. LTB₄ release in whole blood was significantly lower in samples from RA patients than in those from the healthy volunteers (13.5±1.4 and 19.1±1.4 ng/10⁶ neutrophils respectively; P<0.001). LTB₄ release from isolated RA neutrophils correlated with the levels of C-reactive protein, duration of morning stiffness and Ritchie articular swelling index. Concentrations of hyaluronate, cyclic AMP and 13,14-dihydro-15-keto-prostaglandin were not different between patients with RA and healthy volunteers. Neither was there any difference in TXB₂ production by platelets during blood clotting. In conclusion, peripheral blood neutrophils of RA patients seem to be primed and/or activated as their random migration is enhanced as compared with those of healthy volunteers. In RA, LTB₄ release from peripheral blood neutrophils seems to reflect the clinical activity of the disease. However, RA neutrophils released smaller (in whole blood) or equal (isolated cells) amounts of LTB₄ as compared with the respective controls. These contradictory findings suggest that LTB₄ release from peripheral blood neutrophils has no major role in the regulation of disease activity in rheumatoid arthritis.     

Inhibition of leukotriene B4 in neutrophils by lipoxins A4 and B4

Lipoxins are derived from the oxygenation products of arachidonic acid in human leukocytes. They have exhibited selective biological effects different from those of other eicosanoids. We have examined the effect of lipoxin A₄ and B₄ (LXA₄, LXB₄) on the production of leukotriene B₄ (LTB₄) in human neutrophils. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit LTB₄ generation was assessed after incubation with calcium ionophore A23187. We found that the pretreatment of neutrophils with lipoxins inhibit the release of LTB₄ by A23187 stimulated PMNs. Our data suggests that LXA₄ and B₄ can contribute to immunosuppression in an inflammatory state via the inhibition of LTB₄ synthesis.     

Characterization of arachidonic acid metabolism,superoxide production,and bacterial killing by bovine alveolar neutrophils elicited with leukotriene B4 and zymo-san-activated plasma

Leukotriene B₄ (LTB₄) and zymosan-activated plasma (ZAP) were each instilled into the lungs of steers to elicit alveolar neutrophils for subsequent functional analysis. Prior to instillation of either agent, bronchoalveolar lavage cell populations consisted of 95.8 ± 0.4% macrophages (mean ±SEM). Four hours after instillation of LTB₄ or ZAP, the lavage cell populations consisted of 75.0 ± 8.8% and 90.7 ± 0.7% neutrophils, respectively. Alveolar neutrophils elicited with LTB₄ and stimulated with the calcium ionophore A23187 released diminished amounts of LTB₄ and increased amounts of 5-hydroxyeicosatetraenoic acid (5-HETE) as compared to circulating neutrophils. Release of superoxide anion was decreased for LTB₄-elicited alveolar neutrophils as compared to circulating cells, while bacterial killing was unchanged. ZAP-elicited alveolar neutrophils released diminished amounts of LTB₄ when stimulated with A23187 as compared to circulating neutrophils. There were no differences observed in 5-HETE levels between the two cell populations. In addition, release of superoxide anion was diminished among ZAP-elicited alveolar cells, while bacterial killing was unchanged. Incubation of circulating neutrophils with LTB₄ did not influence the release of arachidonate metabolites, superoxide anion, or bacterial killing. However, incubation of circulating neutrophils with ZAP, followed by A23187 resulted in a reduction in the release of LTB₄, as compared to control cells. Prior exposure to ZAP did not influence the release of superoxide anion or bacterial killing by the circulating neutrophils.     

A comparison of the anti-inflammatory activity of selective 5-lipoxygenase inhibitors with dexamethasone and colchicine in a model of zymosan induced inflammation in the rat knee joint and peritoneal cavity

Intraperitoneal and intra-articular (knee joint) injection of zymosan in the rat caused two phases of increased vascular permeability, a rapid increase (0.25–0.5 h) and a secondary increase (2–3 h) which was temporally associated with the onset of leukocyte infiltration. Intraperitoneal injection of zymosan led to a single peak of eicosanoid production (LTB₄, C₄, D₄, E₄ and 6-oxo-PGF₁ ) which was maximal at 0.125–0.25 h. Intra-articular injection led to an initial peak of LTB₄ production (maximal at 0.25 h) and a secondary peak of LTB₄ and PGE₂ production (maximal at 3 h). Oral administration of the 5-lipoxygenase (5-LO_ inhibitors phenidone, BW A4C (N-hydroxy-N-[3-(3-phenoxyphenyl)-2-propenyl] acetamide), A63162 (N-hydroxy-N-[1-(4-(phenylmethoxy) phenyl)ethyl] acetamide and ICI 207 968 (2-[3-pyridylmethyl]-indazolinone inhibited LTB₄ production in A23187 stimulation bloodex vivo. The glucocorticosteroid dexamethasone had no effect in this model. The initial phase of increased vascular permeability in the peritoneal cavity and LTB₄ production was dose dependently inhibited by the 5-LO inhibitors phenidone, BW A4C, A63162, and ICI 207 968 but not by dexamethasone or colchicine. The initial phase of increased permeability in the joint was unaffected by phenidone, BW A4C, dexamethasone or colchicine. However the latter two drugs inhibited the later phase of increased permeability and leukocyte infiltration in the joint and peritoneal cavity. These results demonstrate that zymosan induces eicosanoid productionin vivo but the relative importance of these mediators varies depending on the inflammatory site. Dexamethasone had a similar profile of anti-inflammatory activity to colchicine suggesting that inhibition of cell infiltration is the major mechanism of its therapeutic effect. No evidence for dexamethasone induced inhibition of eicosanoid production could be found suggesting that inhibition of the production or activity of other mediators is more important.