3(S)-hydroxy-leukotriene B4 is a potent granulocyte chemotaxin in the guinea pig dermis

Inflammation Research -     

6-trans-leukotriene B4 is a neutrophil chemotaxin in the guinea pig dermis.

The products of the 5- and 12-lipoxygenase (5-LO, 12-LO) pathways of arachidonic acid metabolism are implicated as proinflammatory mediators in a number of disease states. 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] is present in large quantities in human psoriatic lesional skin and can be further metabolized by 5-LO to 5(S), 12(R)-dihydroxy-(6E,8Z,10E,14Z)-eicosatetraenoic acid (6-trans-LTB4). Furthermore, leukotriene B4 (LTB4) and the sulfidopeptide leukotrienes (LTC4, LTD4) can be transformed to 6-trans-LTB4. When injected into the guinea pig dermis, 6-trans-LTB4 (1.0, 10.0, 20.0 micrograms/intradermal site) caused a significant (P less than 0.02) infiltration of polymorphonuclear leukocytes (PMN) at 4 hr as assessed by histology and the levels of the PMN marker enzyme myeloperoxidase. 6-trans-LTB4 is a more potent PMN chemoattractant than 12(R)-HETE in the guinea pig dermis but is far less potent than LTB4. Pharmacological interdiction of leukotriene production or receptor binding should take into account the proinflammatory activity of 6-trans-LTB4.     

Inflammation of guinea pig dermis

Neutrophil (PMNL) infiltration is a prominent feature of human psoriasis. Psoriatic skin lesions contain abnormally high amounts of leukotriene B₄ (LTB₄), itself a potent PMNL chemoattractant both in vivo and in vitro. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-1-benzo-pyran-2-carboxylic acid}, an orally active LTB₄ receptor antagonist, was tested topically in models of skin inflammation induced by 200 nmol of the calcium ionophore A23187 or 200g phorbol-12-myristate-13-acetate (PMA) applied topically to the guinea pig ear as assessed by ear weight, levels of the PMNL marker enzyme myeloperoxidase (MPO), and histological examination (PMA model) at 4 and 18 h respectively. When coapplied topically with A23187 or PMA, SC-41930 significantly inhibited epidermal inflammation with ED₅₀ values of 0.6 and 4 mg, respectively. SC-41930 treatment also was associated with lowered dermal LTB₄ levels in both models. The PMA-induced skin inflammation model also was assessed histologically and revealed acanthosis, edema, PMNL infiltration, and rete ridge prominence as long as 96 h after a single application that was completely inhibited by SC-41930 topical coapplication. Furthermore, oral treatment (40 mg/kg) significantly reduced edema and inflammatory cell infiltration in both models. These models possess many of the characteristics of human psoriasis, and agents such as SC-41930 that demonstrate activity in these models may well have therapeutic utility in the treatment of human psoriasis.     

Leukotriene B4-induced neutrophil extracellular traps impede the clearance of Pneumocystis

Pneumocystis pneumonia (PCP) is a fungal pulmonary disease with high mortality in immunocompromised patients. Neutrophils are essential in defending against fungal infections; however, their role in PCP is controversial. Here we aim to investigate the effects of neutrophil extracellular traps (NETs) on Pneumocystis clearance and lung injury using a mouse model of PCP. Intriguingly, although neutrophils play a fundamental role in defending against fungal infections, NETs failed to eliminate Pneumocystis, but instead impaired the killing of Pneumocystis. Mechanically, Pneumocystis triggered Leukotriene B4 (LTB4)-dependent neutrophil swarming, leading to agglutinative NET formation. Blocking Leukotriene B4 with its receptor antagonist Etalocib significantly reduced the accumulation and NET release of neutrophils in vitro and in vivo, enhanced the killing ability of neutrophils against Pneumocystis, and alleviated lung injury in PCP mice. This study identifies the deleterious role of agglutinative NETs in Pneumocystis infection and reveals a new way to prevent NET formation, which provides new insights into the pathogenesis of PCP.     

Effect of a leukotriene B4 receptor antagonist on leukotriene B4-induced neutrophil chemotaxis in cavine dermis

Leukotriene B₄ (LTB₄) is a proinflammatory product of arachidonic acid metabolism that has been implicated as a mediator in a number of inflammatory diseases. When injected intradermally into the cavine, LTB₄ elicits a dose-dependent immigration (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)propoxy]-3,4-dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid, a potent LTB₄ receptor antagonist inhibited the chemotactic actions of LTB₄ when coadministered into the dermal site and when given intravenously or orally with ED₅₀ values of 200 ng, 0.5 mg/kg, and 0.6 mg/ kg respectively. This compound may well have application in disease states, such as inflammatory bowel disease and psoriasis, where LTB₄ is implicated as a proinflammatory mediator.     

Effect of BRL-35135 on LTB4-induced guinea pig eosinophil chemotaxis

The effect of an atypical -adrenoceptor agonist, BRL-35135 on leukotriene B₄-induced-guinea pig eosinophil chemotaxis was studied. BRL-35135 and SC-41930 (leukotriene B₄-antagonist) inhibited the chemotaxis in a concentration-dependent manner (IC₅₀=9.0×10^–6 and 2.6×10^–7 M, respectively). However, isoproterenol, fenoterol and another atypical -agonist, BRL-37344 had no effects. The inhibitory effect of BRL-35135 was not affected by (±)-propranolol (10^–4 M). In contrast, the nonselective -adrenoceptor antagonist, (–)-alprenolol (10^–4 M) dextrally shifted the inhibitory curve of BRL-35135. The response to BRL-35135 was antagonized in a competitive manner by (–)-alprenolol, with the slope of the Schild plot close to unity, and a pA ₂ value of 5.62. These findings suggest that guinea pig eosinophils possess an atypical receptor, which differs from either ₁, ₂ or atypical -adrenoceptor on guinea pig ileum, and through which eosinophil chemotaxis can be modulated by BRL-35135.     

Leukotriene B4-induced permeability increase in postcapillary venules and its inhibition by three different antiinflammatory drugs

Leukotriene B₄ (LTB₄) is a potent chemotactic and chemokinetic substance that causes leukocyte accumulation and increases vascular permeability. We have used the hamster cheek pouch model to study the effect of some different antiinflammatory drugs on the LTB₄-induced microvascular permeability. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular leakage, which was measured as the number of venular leaky sites and as efflux of the vascular tracer. Topical application of LTB₄ (10^–8 M, 5 min) resulted in a reversible increase of the vascular permeability and could be repeated with 40-min intervals without any significant reduction in permeability response. The LTB₄-induced response could be inhibited by budesonide (a glucocorticoid) given locally, by iloprost (a prostacyclin analog) given intravenously and by a combined local and intravenous treatment with terbutaline (a ₂-receptor agonist) but not by local terbutaline treatment only.     

Leukotriene B4-induced hyperadhesiveness of endothelial cells for neutrophils: relation to CD54.

Leukotriene B4 (LTB4) induced an in vitro transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC), leading to a 2.2-fold increase in the binding of neutrophil granulocytes (PMN), which was less than that conferred by platelet activating factor (PAF) though more than thrombin did (3.4- or 2.0-fold increases, respectively). This study concerns the role of the adhesive molecules CD18 and CD54 for the LTB4- (as well as thrombin- and PAF-) induced endothelial hyperadhesiveness. The MoAbs 60.3 (to the CD18 molecule on PMN) and 84H10 (to one epitope of CD54 on the HUVEC) blocked the adherence of PMN to LTB4-treated HUVEC, whereas MoAb LB-2 (directed at another CD54 epitope) failed to do so. MoAb 84H10 blocked 43% of the thrombin-induced hyperadhesiveness, whereas the PAF response was unaffected. Thus, LTB4-induced HUVEC hyperadhesiveness may therefore be related to a specific domain on the CD54 (or on an antigenically related molecule) as well as being dependent on CD18, whereas the involvement of CD54 was much less or non-existent for the thrombin and PAF responses, respectively.     

Leukotriene B4 and inflammatory disease

Polymorphonuclear leukocytes (PMNL) are prominent at sites of acute inflammation. Their infiltration is stimulated under pathological conditions by a variety of agents which include bacteria, immune complexes and complement derived chemotactic peptides. Recently attention was focussed on the 5-lipoxygenase product leukotriene B₄ (LTB₄) which has been demonstrated to induce the key features associated with an acute inflammatory reaction. However, evidence supporting a pro-inflammatory role for LTB₄, and therefore the anti-inflammatory efficacy of 5-lipoxygenase inhibitors, is largely circumstantial. Moreover, there are concerns that other chemotactic factors, notably C₅a, may compensate for the absence of LTB₄. Here we challenge this view and, on the basis of recent experimental and clinical data suggest that LTB₄ does not simply duplicate the activity of C₅a. Instead we propose that their predominant site(s) of action differ in such a way that they may synergise in mediating PMNL recruitment.     

Leukotriene B4 in the immune system.

Leukotriene (LT) B4 is a biologically active molecule derived from arachidonic acid via the 5-lipoxygenase pathway. It mediates certain inflammatory and immunological reactions. The role of LTB4 in the immune system has been questioned since lymphocytes have been regarded to lack the enzymes involved in LTB4 formation. This review focuses on the recently described biosynthesis of LTB4 in B-lymphocytes and the effects of this compound on lymphocyte functions.     

A23187-induced pulmonary gas trapping and inflammation in the guinea pig

A brief A23187 aerosol exposure produced prolonged airway obstruction with granulocyte accumulation in conscious guinea pigs. Aminophylline, atropine, pyrilamine, salbutamol, SC-41930 (a leukotriene B₄ antagonist) and WEB 2086 (a platelet activating factor antogonist) were administered intravenously (i.v.) to evaluate their ability to prevent these changes. Inhaled salbutamol was also assessed. Aminophylline, atropine, and salbutamol (i.v. and aerosol) inhibited A23187-induced gas trapping (p<0.01). However, pyrilamine, SC-41930 and WEB 2086 did not influence this airway obstructive effect. Only atropine, inhaled salbutamol and SC-41930 inhibited the cell influx (p<0.01), while pyrilamine potentiated the inflammation (p<0.05). We conclude that A23187 produces a sustained bronchospasm and an intense granulocyte accumulation. The treatment agents tested differ considerably in their ability to alter A23187-induced airway obstruction and inflammation.     

Leukotriene B4 stimulation of macrophage cyclooxygenase metabolite synthesis

Inflammation Research -     

Inhibition of IKs in guinea pig cardiac myocytes and guinea pig IsK channels by the chromanol 293B

The chromanol derivative 293B was previously shown to inhibit a cAMP regulated K⁺ conductance in rat colon crypts. Subsequent studies on cloned K⁺ channels from the rat demonstrated that 293B blocks specifically I_sK channels expressed in Xenopus oocytes, but does not affect the delayed and inward rectifier Kv1.1 and Kir2.1, respectively. In the present study, the specificity of 293B for the cardiac K⁺ conductances I_Ks and I_Kr, and for the cloned guinea pig I_sK channel and the human HERG channel, which underly I_Ks and I_Kr, respectively, was analyzed. 293B inhibited both the slowly activating K⁺ conductance I_Ks in cardiac myocytes and guinea pig I_sK channels expressed in Xenopus oocytes with a similar IC₅₀ (2-6 μmol/1). In contrast, high concentrations of 293B had only a negligible effect on the more rapid activating I_Kr. Similarly, 293B exerted no effect on HERG channels expressed in Xenopus oocytes. In summary, 293B appears to be a rather specific inhibitor of I_Ks and the underlying I_sK channels.     

Leukotriene B4 augments neutrophil phagocytosis of Klebsiella pneumoniae

Neutrophils play a critical role in the clearance of bacteria from the lung and other organs by their capacity for phagocytosis and killing. Previously, we identified an important role for the leukotrienes in rat alveolar macrophage phagocytosis of Klebsiella pneumoniae. In this report, we explored the possibility that the leukotrienes play an important role in phagocytosis by neutrophils as well. Inhibition of endogenous leukotriene synthesis by 5-lipoxygenase knockout in mice or by pharmacologic means in human peripheral blood neutrophils attenuated phagocytosis of opsonized K. pneumoniae. Reduced phagocytosis was also observed in human neutrophils pretreated with a leukotriene B4 receptor but not a cysteinyl-leukotriene receptor antagonist. While leukotriene B4 reconstituted defective phagocytosis in leukotriene-deficient neutrophils and enhanced phagocytosis in neutrophils capable of leukotriene synthesis, leukotriene C4, leukotriene D4, 5-hydroperoxyeicosatetraenoic acid, and 5-oxo-eicosatetraenoic acid were ineffective. To determine the opsonin dependence of the leukotriene B4 augmentation of phagocytosis, we assessed the ability of leukotriene B4 to modulate neutrophil phagocytosis and the adherence of sheep erythrocytes opsonized with immunoglobulin G or the complement fragment C3bi. While leukotriene B4 augmented both Fc receptor- and complement receptor-mediated phagocytosis, increased adherence to leukotriene B4-treated neutrophils was limited to complement opsonized targets. In conclusion, we have identified a novel role for leukotriene B4 in the augmentation of neutrophil phagocytosis mediated by either the Fc or complement receptor.     

Ultrastructure of basophilic leukocytes and mast cells in normal and cutaneous basophil hypersensitivity-reacted guinea pig dermis.

Basophilic leukocytes and mast cells in guinea pig dermis in normal and cutaneous basophil hypersensitivity (CBH) reaction were examined by light and electron microscopy. Basophils were rare in the normal dermis and predominantly revealed in the CBH-reacted skin. Some infiltrating basophils of the reacted displayed an immediate attachment to mast cells. Cytoplasmic continuities were partially seen between them. They were most plentiful at 48 h after phytohemagglutinin injection and decreased in number thereafter. The basophils contained three types of granule. The vast majority of granules were Type I granules, basophil-specific granules. Type II granules were less frequently encountered and resembled a mast cell granule in the fine structure. Type III granules were scant and small-cored vesicles.     

Leukotriene B4 generation by human neutrophils following IgG-dependent stimulation

It has previously been shown that human neutrophils generate substantial quantities of LTB4 when stimulated with the calcium ionophore, A23187, or with unopsonized zymosan. We now report that normal human neutrophils produced substantial quantities of LTB4 (measured by radioimmunoassay and validated by RP-HPLC) when incubated with large non-phagocytosable IgG-coated beads (Sepharose 4B). LTB4 was identified in both the extra and intracellular compartments. The production of LTB4 was dependent upon the number of IgG-coated particles and the concentration of IgG bound to the beads. Release was maximal after a 15-30 min incubation time and was enhanced by prior activation of the neutrophils with the synthetic bacterial product f-met-leu-phe. Comparable LTB4 production was also observed when neutrophils were incubated with antigen (Aspergillus fumigatus)-coated beads sensitized with purified IgG obtained from the sera of patients with allergic bronchopulmonary aspergillosis. These results suggest a further mechanism by which neutrophils may be activated to produce inflammatory mediators in the tissues.     

Phospholipase A2-induced lung edema and its mechanism in isolated perfused guinea pig lung

Lung injury induced by phospholipase A₂ (PLA₂, 0.046 IU/ml perfusate) was studied in a continuous weighing system of isolated perfused guinea pig lungs. The results revealed that lung weight increased progressively during the 30-min perfusion of PLA₂. No change of pulmonary arterial pressure was observed in the same period. Albumin permeability-surface area product, lung index, lung water content, exudate from pleura, and angiotensin-converting-enzyme activity increased significantly at the end of 30 min PLA₂ perfusion.p-Bromophenacyl bromide, a PLA₂ inhibitor, may block the above changes nearly completely. The effects of inhibitors of cyclooxygenase (indomethacin, IM), lipoxygenase (diethylcarbamaxine, DE), and platelet-activating factor (SRI 63-441) on PLA₂-induced lung injury were also studied. We found: (1) PLA₂ may induce high permeability lung edema. The role of endothelial injury in the permeability change remains to be further investigated. (2) DE ameliorated lung injury significantly within 10 min of PLA₂ treatment but showed no effect after 15 min. IM ameliorated lung injury during the whole experimental period. SRI 63-441 had no effect. It is suggested that PLA₂ may damage lung by inducing products of cyclooxy genase and lipoxygenase besides its direct effect.Project supported by Natural Science Foundation of China, NO. 3880399.     

Lipopolysaccharide and granulocyte colony-stimulating factor delay neutrophil apoptosis and ingestion by guinea pig macrophages.

In previous studies, neutrophil-ingesting macrophages were clearly and easily observed in the peritoneal cavity of guinea pigs after intraperitoneal injection of thioglycolate medium, and phagocytosis of neutrophils by macrophages could be detected in in vitro cultures of peritoneal exudate cells. Using an in vitro system, we examined the effect of bacterial lipopolysaccharide and recombinant human granulocyte colony-stimulating factor on the apoptosis (programmed cell death) of neutrophils and their subsequent ingestion by macrophages. Lipopolysaccharide delayed karyopyknosis and apoptosis of neutrophils, as shown by endogenous endonuclease activity and a high proportion of trypan blue-excluding cells, and subsequent ingestion by autologous macrophages. Granulocyte colony-stimulating factor also delayed neutrophil karyopyknosis and ingestion by macrophages. When a thioglycolate medium was coinjected intraperitoneally with lipopolysaccharide into guinea pigs in the in vivo system, delays in neutrophil disappearance and ingestion by macrophages in the peritoneal cavity were also observed. We suggest that bacterial products and cytokines regulate neutrophil apoptosis and subsequent ingestion by macrophages at inflamed sites.