Rat neutrophil activation and effects of lipoxygenase and cyclooxygenase inhibitors

Activation (defined as lysosomal enzyme secretion and generation of O(2) of rat neutrophils has been measured with the use of varying doses of soluble stimuli (phorbol myristate acetate (PMA); calcium ionophore A23187; and N-formyl-methionyl-leucyl-phenyl-alanine (FMLP] and particulate agents (immune complexes and zymosan particles). With either the calcium ionophore or the chemotactic peptide (FMLP), substantial enzyme release occurred, but the amount of O(2) produced was very small. Cytochalasin B greatly enhanced the enzyme release response to the chemotactic peptide but had little effect on neutrophil responses to other soluble stimuli. The cell response to PMA resulted in the greatest production of O(2) with significant enzyme secretion. When cell stimulation with insoluble stimuli (immune complexes or zymosan particles) was studied, significant amounts of enzyme release occurred in parallel with the generation of substantial amounts of O(2). The presence of cytochalasin B enhanced the cell responses to immune complexes but had an inhibitory effect on zymosan-induced responses. As expected, the amount of lysozyme secreted by stimulated rat neutrophils tended to exceed the amount of beta-glucuronidase released from the same cells. Neutrophil responses were investigated in the presence of drugs that were demonstrated in the rat neutrophil to inhibit either the lipoxygenase or the cyclooxygenase pathway. Inhibitors of the cyclooxygenase pathway (indomethacin, piroxicam, ibuprofen, BW755C), with few exceptions, consistently enhanced the enzyme secretion response, while effects on O(2) generation were less clear-cut but tended to be predominantly inhibitory. Drugs with inhibitory effects on the lipoxygenase pathway (nordihydroguaiaretic acid and nafazatrom) had significant inhibitory effects on both enzyme secretion as well as generation of O(2). These data suggest that activation responses (enzyme secretion and O(2) generation) of rat neutrophils may be dissociated (ie, one not always accompanying the other). Further, it appears that neutrophil activation, as defined by enzyme secretion, is enhanced by products of the lipoxygenase pathway and suppressed by products of the cyclooxygenase pathway. Generation of O(2) is not affected in such a clear-cut manner. Taken together the data suggest that enzyme release and O(2) production by activated rat neutrophils may be under separate control.     

Therapeutic reduction of ongoing carrageenin-induced inflammation by lipoxygenase,but not cyclooxygenase inhibitors

A variety of steroidal and nonsteroidal antiinflammatory agents (NSAs) were compared for their effectiveness against a developing acute inflammatory reaction. Only BW 755C [3-amino-l-(m-trifiuoromethyl)-phenyI-2-pyrazole] was immediately effective when administered 2 h after carrageenin, as the 3-h swelling was significantly reduced. Dexamethasone, a steroidal antiinflammatory agent, produced a delayed reduction. When administered at 2 h postcarrageenin, there was no effect at 3 h; but at 6 h, dexamethasone significantly reduced the swelling. In contrast, neither of the classic NSAs, aspirin or indomethacin, were effective when administered after the carrageenin. The results demonstrating a postcarrageenin effectiveness by BW 755C and dexamethasone are discussed as a possible reflection of an action on the lipoxygenase pathways of the arachidonic cascade that is not shared by the classic NSA.     

Edema and cell infiltration in the phorbol ester-treated mouse ear are temporally separate and can be differentially modulated by pharmacologic agents

The temporal patterns of edema and accumulation of the PMN marker enzyme, myeloperoxidase (MPO), were examined following application of tetradecanoylphorbol acetate (TPA) to mouse ears. After application of 2.5 g TPA, edema peaked at 6 hr, while MPO activity peaked at 24 hr. Pharmacological agents with defined mechanisms of action, delivered orally or topically, were assessed for effects on these responses. For oral administration, compounds were delivered 1 hr before and 6 hr after TPA and for topical administration compounds were delivered at 15 min and 6 hr after TPA. Topical and oral corticosteroids inhibited both edema and MPO accumulation. Cyclooxygenase and lipoxygenase inhibitors were very effective against MPO accumulation but were either inactive or moderately active vs edema. Anti-histamine/anti-serotonin agents had little effect on edema, but could inhibit or exacerbate MPO accumulation depending on dose and route of administration. Topically applied histamine itself did not effect TPA-induced edema, but markedly suppressed MPO accumulation. Acetone, the vehicle, when topically applied between 0.5 and 2 hr after TPA inhibited MPO accumulation by 60–80%, but had little effect on edema. Acetone applied before 0.5 hr or after 2 hr had no effect on either parameter. These results indicate that in the TPA-induced ear inflammation model the MPO response at 24 hr may be a useful additional indicator of drug activity.     

Modulation of mouse macrophage receptors by various inflammatory agents

The expression and alterations in the expression of various receptors including Fc (IgG_2b), complement and lectin-like receptors and other surface determinants including I-A antigens and surface mannosyl groups has been described on resident-, glycogen- andCorynebacterium parvum-elicited mouse peritoneal macrophages. The influence of chemotactic factors on the expression of these membrane constituents has also been studied.     

Dual inhibition of cyclooxygenase and lipoxygenase enzymes by human cerebrospinal fluid

Thromboxane A₂ (TXA₂) formed in damaged brain tissue and after thromboembolism and subarachnoid haemorrhage is responsible for cerebral vasospasm. In the present study, we examined the effect of human cerebrospinal fluid (CSF) on the production of thromboxane-A₂ (TXA₂) and 12-hydroxy-eicosatetraenoic acid (12-HETE) by human blood platelets. CSF was drawn by lumbar puncture from normal healthy volunteers (n = 17) and samples judged to be normal after routine examination in the clinical laboratories and were used fresh. We found that CSF inhibited the production of TXA₂ and 12-HETE by blood platelets incubated with C¹⁴ labelled arachidonic acid (AA) in a concentration-related manner. Further biochemical analysis using proteolytic enzymes, gel filtration and membrane partition chromatography showed that the inhibitory activity was peptidic in nature and associated with a peptide of low molecular weight (1,400 Da). This study is the first to demonstrate that human CSF contains a dual inhibitor of cyclooxygenase (COX) and lipoxygenase enzymes in CSF.     

Effect of CGS 25019C and other LTB4 antagonists in the mouse ear edema and rat neutropenia models

Inflammation Research -     

Stimulus-specific production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by bovine alveolar macrophages

Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators. Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing. In this study bovine AMs were prelabeled with [³H]arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187. The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection. All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B₂ and HHT being the major metabolites. Lesser amounts of PGF₂, PGE₂, and PGD₂ were produced. Only stimulation with A23187 resulted in production of LTB₄ and 5-HETE, products of the 5-lipoxygenase pathway. This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated. Such an effect could have important consequences in the development of pulmonary inflammation. Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs.     

Effects of (d-Ala2) methionine enkephalinamide in PMA-induced mouse ear edema

The synthetic opioid peptide (d-Ala²) methionine enkephalinamide (DAMEA) was reported to be an extremely potent inhibitor of the respiratory burst of polymorphonuclear neutrophils (PMNs). In the present paper the activity of DAMEA was investigated in the PMA-induced mouse ear edema since there is strong evidence that reactive oxygen species (ROS) are involved in the pathogenesis of this inflammatory reaction. DAMEA inhibited PMA-induced mouse ear edema in a dose-dependent manner between 10^–6 and 10^–9 mol/ear. The well-known anti-inflammatory activity of narcotic analgesics could be explained partly by an inhibition of ROS production.     

Effects of indole-3-acetic acid on croton oil- and arachidonic acid-induced mouse ear edema

The indole-3-acetic acid (IAA) is a plant growth hormone (auxin) being considered as a tryptophan metabolite in animals. The main purpose of this work was to verify IAA's topical anti-inflammatory action using croton oil- or arachidonic acid-induced mouse ear edema, in comparison to known anti-inflammatory agents. IAA antioxidant activity was also verified by measuring the inhibition of brain homogenate lipid peroxidation with the thiobarbituric acid reactive substances (TBARS) test. IAA inhibited the action of both croton oil- and arachidonic acid-induced edema in a dose-dependent manner (4.0 µmoles IAA inhibited 75.8% in croton oil and 82.5% in arachidonic acid induced ear edema). Both IAA (5.3 mM) and indomethacin (8.0 mM) inhibited TBARS formation. Data suggest that IAA exhibits antiinflammatory effect possibly by its antioxidant activity.     

Metabolism of cyclooxygenase and lipoxygenase products by 15-prostaglandin dehydrogenase from human HL-60 leukemia cells

A number of prostaglandins and mono-hyroxylated fatty acids were investigated as substrates for NAD-dependent 15-prostaglandin dehydrogenase (15-PGDH) obtained from human HL-60 cells. Lipoxygenase and cyclooxygenase-derived substrates possessing an w-6 hydroxyl moiety (15-HETE, 13-HODD and HHT) were metabolized to corresponding keto derivatives at a rate comparable to that for prostaglandins E₂ and F₂. 12-HETE was a poor substrate and 5-HETE was not metabolized. The w-6 hydroxy fatty acids inhibited the metabolism of PGE₂ and therefore they may play a role in modulating bioactive prostaglandin levels.     

Effect of selected antiinflammatory agents and other drugs on zymosan,arachidonic acid,PAF and carrageenan induced paw edema in the mouse

Zymosan, carrageenan, arachidonic acid and platelet activating factor (PAF) were used to induce inflammation (edema) in the paws of mice. Antiinflammatory drugs (e.g., BW755C and indomethacin) as well as cyproheptadine (mediator antagonist), theophylline (phosphodiesterase inhibitor) and guanabenz ( adrenoceptor agonist) showed the greatest efficacy in the carrageenan and zymosan models. Nonsteroidal antiinflammatory (NSAIDs) agents showed greater activity in the arachidonic acid (AA) paw edema model than the dual 5-lipoxygenase (LO)/cyclooxygenase (CO) inhibitors. The PAF model was insensitive to NSAIDs but showed some activity with drugs possessing inhibitory 5-LO activity (e.g., phenidone, BW755C) and the mediator antagonist, cyproheptadine. Suramin, a complement inhibitor, as expected, was active only against zymosan-induced edema. In conclusion, the inhibitory activities of dual 5-LO/CO inhibitors and NSAIDs were not different in the zymosan, carrageenan and AA edema models in the mouse; however, some selectivity for 5-LO inhibitors was observed in the PAF model.     

Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood

We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB₄, 20-OH-LTB₄, and 20-COOH-LTB₄ are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB₄, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.     

Inhibition of histamine secretion from mast cells by lipoxygenase- and cyclooxygenase inhibitors

We have investigated the effect of inhibitors of lipoxygenase (LOX), cyclooxygenase (COX) and dual inhibitors of both enzymes on the degranulation of peritoneal rat mast cells (pRMC) activated by different mechanisms. COX inhibitors weakly affected histamine secretion induced by A23187 and did not influence the histamine secretion induced by protamine in an isotonic medium but blocked protamine-induced release in a hypertonic medium. LOX- and dual-inhibitor inhibited secretion induced by A23187 and protamine under all conditions. As A23187 activates pRMC in an isotonic medium by Ca influx, and protamine acts in a hypertonic medium by mobilization of intracellular Ca and in an isotonic medium by both processes (this paper), LOX but not COX inhibitors, may block Ca influx, and both prevent Ca mobilization.     

Modulation of carrageenan-induced hind paw edema by substance P

Substance P has been implicated as a mediator of inflammation. The involvement of this neuropeptide in carrageenan-induced hind paw edema in the rat was assessed. Subcutaneous injection of carrageenan into the rat paw caused a significant increase in substance P levels, which preceded the onset of inflammation. While injection of substance P alone caused mild edema, coadministration of submaximal doses of carrageenan and substance P resulted in a synergistic exacerbation in the degree of inflammation. This synergistic response was not detected when the nonamidated precursor of substance P was coinjected with carrageenan. The effects of substance P depletion on inflammation were also evaluated. In animals pretreated with capsaicin followed by injection with carrageenan, no significant increase in either the levels of substance P or the extent of edema was observed when compared to capsaicin-treated controls. These results indicate that substance P may play an important role in the early stages of carrageenan-induced paw edema and that a reduction in the biosynthesis of substance P may lessen the severity of this inflammatory response.