Leukotriene B4 Is a Major Determinant of Leukocyte Recruitment During Otitis Media

BackgroundPathogens of otitis media (OM) induce inflammatory responses in the middle ear (ME), characterized by mucosal hyperplasia, leukocyte infiltration, and inflammatory mediators, including arachidonic acid metabolites. We studied the role of the eicosanoid leukotriene B4 (LTB4) in OM.MethodsExpression of LTB4-related genes was evaluated by gene array and single-cell RNA-Seq in MEs infected with nontypeable Haemophilus influenzae (NTHi). An inhibitor of LTB4 receptor 1 (i.e. U75302) was also used to block LTB4 responses.ResultsME expression of LTB4-related genes was observed by gene arrays and scRNA-Seq. However, not all genes involved in LTB4 generation occurred in any one specific cell type. Moreover, LTB4 receptor inhibition significantly reduced mucosal hyperplasia and virtually eliminated leukocyte infiltration.ConclusionsME expression of LTB4-related genes suggest a functional role in OM disease. The fact that LTB4-generation is spread across different cell types is consistent with a transcellular pathway of eicosanoid biosynthesis involving cell-to-cell signaling as well as transfer of biosynthetic intermediates between cells. The dramatic reduction in ME leukocyte infiltration caused by U75302 indicates that LTB4 plays a major role in ME inflammatory cell recruitment, acting via the LTB4R1 receptor. Given that there are many other chemotactic factors that occur in the ME during OM, the ability of LTB4 to activate leukocytes and stimulate their extravasation may explain the effects of inhibition. Reduction in mucosal hyperplasia due to U75302 administration may be secondary to the reduction in leukocytes since LTB4R1 is not expressed by mucosal epithelial or stromal cells. The results suggest that LTB4 receptor antagonists could be useful in treating OM.

Leukotriene receptors (version 2020.3) in the IUPHAR/BPS Guide to Pharmacology Database

The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [34, 37]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [196] and the vanilloid TRPV1 ligand-gated nonselective cation channel [217]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [37]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [96, 243, 272], GPR17 [57] and GPR99 [168].

Leukotriene receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [31, 34]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [189] and the vanilloid TRPV1 ligand-gated nonselective cation channel [210]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [34]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [91, 236, 265], GPR17 [53] and GPR99 [161].

The leukotriene receptors as therapeutic targets of inflammatory diseases

Leukotrienes (LTs) are inflammatory mediators derived from arachidonic acid. LTs include the di-hydroxy acid LT (LTB4) and the cysteinyl LTs (CysLTs; LTC4, LTD4 and LTE4), all of which are involved in both acute and chronic inflammation. We and other groups identified a high-affinity LTB4 receptor, BLT1; the LTC4 and LTD4 receptors, CysLT1 and CysLT2; and the LTE4 receptor, GPR99. Pharmacological studies have shown that BLT1 signaling stimulates degranulation, chemotaxis and phagocytosis of neutrophils, whereas CysLT1 and CysLT2 signaling induces airway inflammation by increasing vascular permeability and the contraction of bronchial smooth muscle. Recently, we and other groups suggested that the LTB4–BLT1 axis and the cysteinyl LTs–CysLT1/2 axis are involved in chronic inflammatory diseases including asthma, atopic dermatitis, psoriasis, atherosclerosis, arthritis, obesity, cancer and age-related macular degeneration using animal models for disease and gene knockout mice. This review describes the classical and novel functions of LTs and their receptors in several inflammatory diseases and discusses the potential clinical applications of antagonists for LT receptors and inhibitors of LT biosynthesis.

Elevation of Plasma Leukotriene B4 on G-CSF-Induced Hematopoietic Stem Cells Mobilization In Normal Individuals

Abstract 4437 Background: Previously, we had reported that mobilization kinetics of CD34+ cells association of CD44 and CD31 expression during continuous intravenous administration of G-CSF in normal donors(Stem Cells 18:281-286, 2000, BMT 36:1027-1032, 2005). Meanwhile, a number of studies have reported the mechanisms of G-CSF-induced HSCs mobilization, but the underlying mechanisms are not clear yet. Some chemokines, macrophage inflammatory protein-1α (MIP-1α/CCL3), stem cell-derived factor 1α (SDF-1α/CCL12), interleukin 8 (IL-8/CXCL8), and GROβ/CXCL2, can mobilize HSCs, and we also reported that LTB4, which has considerable functional overlap with the chemokine family of chemoattractant peptides, had mobilized HSCs in murine model. In this study, we have investigated whether G-CSF may affect plasma LTB4 level during HSCs mobilization and G-CSF-induced HSCs mobilization may be modulated by LTB4. Method: To investigate that G-CSF may modulate plasma LTB4 level, G-CSF (Filgrastim, Kirin Brewery Co. Tokyo, Japan; 10 microgram/kg/day) was administered subcutaneously into 4 healthy donors for 5 days and then, apheresis were performed on day 4 and 5, and were collected for their serum plasma at base line, 24hours, 48hours and 72hours after 1st dose of G-CSF. LTB4 concentration was measured by enzyme-linked immunosorbent assay (Parameter™ LTB4 Assay; R&D Systems, Mineapolis, MN, USA). Meanwhile, to evaluate the effect of LTB4 inhibition on G-CSF-induced HSCs mobilization, LTB4APA and U75302, which are LTB4 receptor antagonists were given to C57BL/6 mice followed by G-CSF 5μ g or LTB4 1μ g administration intravenously 2 hours later. 24 hours after the G-CSF injection or 4 hours after LTB4 injection, peripheral blood samples were obtained and analyzed for HSCs mobilization by flow cytometry using Sca-1, CD45R(B220), CD116, Gr-1 and TER119. Results: Plasma LTB4 levels of healthy donors demonstrated increases in 24hrs after G-CSF administration; 415.3±112.1 pg/ml before treatment of G-CSF and 706.4±154.7 pg/ml 24hours after 1st dose of G-CSF (p=0.005), and then plasma LTB4 levels were decreased continuously after peak level on 24hours. Meanwhile, when LTB4 receptor antagonists were given, the number of HSCs were decreased in the G-CSF mobilized mice; 4.69×103 cells/ml blood before treatment of G-CSF, 80.08×103 cells/ml blood after treatment G-CSF without LTB4 receptor antagonist, 9.24×103 cells/ml blood after treatment of G-CSF with LTB4APA 1μ g respectively(p<0.05, compared with the data of G-CSF alone), 22.86×103 cells/ml blood after treatment of G-CSF with U75302 1μ g (p<0.05, compared with the data of G-CSF alone). The blocking effects on mobilization of HSCs by LTB4 receptor antagonists were also demonstrated in the LTB4 mobilized mice (data not shown). Conclusions: We observed that G-CSF increases plasma LTB4 levels during HSCs mobilization in healthy donors, and that LTB4 inhibition by LTB4 receptor antagonists in murine model downregulate the G-CSF-induced HSCs mobilization. These indicate that LTB4 may be involved in the downstream pathway of G-CSF-induced HSCs mobilization. Through the our results, we hypothesize that G-CSF increase LTB4 level in plasma during HSCs mobilization, and LTB4 receptor activation by increased LTB4 level in plasma may contribute HSCs mobilization in vivo. Currently, we are investigating the cellular and molecular mechanism(s) of potential role of LTB4 during G-CSF induced peripheral blood progenitor cell mobilization. Disclosures: No relevant conflicts of interest to declare.