CAFs shape myeloid-derived suppressor cells to promote stemness of intrahepatic cholangiocarcinoma through 5-lipoxygenase.

BACKGROUND AND AIMS: We previously demonstrated that cancer-associated fibroblasts (CAFs) promote tumor growth through recruitment of myeloid-derived suppressor cells (MDSCs). 5-lipoxygenase (5-LO) is highly expressed in myeloid cells and is critical for synthesizing leukotriene B4 (LTB4), which is involved in tumor progression by activating its receptor leukotriene B4 receptor type 2 (BLT2). In this study, we investigated whether and how CAFs regulate MDSC function to enhance cancer stemness, the driving force of the cancer aggressiveness and chemotherapy refractoriness, in highly desmoplastic intrahepatic cholangiocarcinoma (ICC). APPROACH AND RESULTS: RNA-sequencing analysis revealed enriched metabolic pathways but decreased inflammatory pathways in cancer MDSCs compared with blood MDSCs from patients with ICC. Co-injection of ICC patient-derived CAFs promoted cancer stemness in an orthotopic ICC model, which was blunted by MDSC depletion. Conditioned media (CM) from CAF-educated MDSCs drastically promoted tumorsphere formation efficiency and stemness marker gene expression in ICC cells. CAF-CM stimulation increased expression and activity of 5-LO in MDSCs, while 5-LO inhibitor impaired the stemness-enhancing capacity of MDSCs in vitro and in vivo. Furthermore, IL-6 and IL-33 primarily expressed by CAFs mediated hyperactivated 5-LO metabolism in MDSCs. We identified the LTB4-BLT2 axis as the critical downstream metabolite signaling of 5-LO in promoting cancer stemness, as treatment with LTB4 was elevated in CAF-educated MDSCs, or blockade of BLT2 (which was preferentially expressed in stem-like ICC cells) significantly reduced stemness-enhancing effects of CAF-educated MDSCs. Finally, BLT2 blockade augmented chemotherapeutic efficacy in ICC patient-derived xenograft models. CONCLUSIONS: Our study reveals a role for CAFs in orchestrating the optimal cancer stemness-enhancing microenvironment by educating MDSCs, and suggests the 5-LO/LTB4-BLT2 axis as promising therapeutic targets for ICC chemoresistance by targeting cancer stemness.

Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4.

[Figure: see text].

Isoflurane attenuates sepsis-associated lung injury.

Acute lung injury is one of major complications associated with sepsis, responsible for morbidity and mortality. Patients who suffer from acute lung injury often require respiratory support under sedations, and it would be important to know the role of sedatives in lung injury. We examined volatile anesthetic isoflurane, which is commonly used in surgical setting, but also used as an alternative sedative in intensive care settings in European countries and Canada. We found that isoflurane exposure attenuated neutrophil recruitment to the lungs in mice suffering from experimental polymicrobial abdominal sepsis. We found that isoflurane attenuated one of major neutrophil chemoattractants LTB4 mediated response via its receptor BLT1 in neutrophils. Furthermore, we have shown that isoflurane directly bound to BLT1 by a competition assay using newly developed labeled BLT1 antagonist, suggesting that isoflurane would be a BLT1 antagonist.

Leukotriene B4 and Its Receptor in Experimental Autoimmune Uveitis and in Human Retinal Tissues: Clinical Severity and LTB4 Dependence of Retinal Th17 Cells.

Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B4 (LTB4) and inhibiting complement component 5 (C5) activation. Nomacopan has been shown to provide therapeutic benefit in experimental autoimmune uveitis (EAU). Longer acting forms of nomacopan were more efficacious in mouse EAU models, and the long-acting variant that inhibited only LTB4 was at least as effective as the long-acting variant that inhibited both C5 and LTB4, preventing structural damage to the retina and a significantly reducing effector T helper 17 cells and inflammatory macrophages. Increased levels of LTB4 and C5a (produced upon C5 activation) were detected during disease progression. Activated retinal lymphocytes were shown to express LTB4 receptors (R) in vitro and in inflamed draining lymph nodes. Levels of LTB4R-expressing active/inflammatory retinal macrophages were also increased. Within the draining lymph node CD4+ T-cell population, 30% expressed LTB4R+ following activation in vitro, whereas retinal infiltrating cells expressed LTB4R and C5aR. Validation of expression of those receptors in human uveitis and healthy tissues suggests that infiltrating cells could be targeted by inhibitors of the LTB4-LTB4 receptor 1 (BLT1) pathway as a novel therapeutic approach. This study provides novel data on intraocular LTB4 and C5a in EAU, their associated receptor expression by retinal infiltrating cells in mouse and human tissues, and in attenuating EAU via the dual inhibitor nomacopan.

Leukotriene B4 Receptor Type 2 Accelerates the Healing of Intestinal Lesions by Promoting Epithelial Cell Proliferation.

Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.

Macrophage LTB4 drives efficient phagocytosis of Borrelia burgdorferi via BLT1 or BLT2.

Unresolved experimental Lyme arthritis in C3H 5-lipoxygenase (5-LOX)-/- mice is associated with impaired macrophage phagocytosis of Borrelia burgdorferi In the present study, we further investigated the effects of the 5-LOX metabolite, leukotriene (LT)B4 on phagocytosis of B. burgdorferi Bone marrow-derived macrophages (BMDMs) from 5-LOX-/- mice were defective in the uptake and killing of B. burgdorferi from the earliest stages of spirochete internalization. BMDMs from mice deficient for the LTB4 high-affinity receptor (BLT1-/-) were also unable to efficiently phagocytose B. burgdorferi Addition of exogenous LTB4 augmented the phagocytic capability of BMDMs from both 5-LOX-/- and BLT1-/- mice, suggesting that the low-affinity LTB4 receptor, BLT2, might be involved. Blocking BLT2 activity with the specific antagonist, LY255283, inhibited phagocytosis in LTB4-stimulated BLT1-/- BMDMs, demonstrating a role for BLT2. However, the lack of a phagocytic defect in BLT2-/- BMDMs suggested that this was a compensatory effect. In contrast, 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid, a natural BLT2-specific high-affinity ligand, and resolvin E1, a BLT1 agonist, were both unable to boost phagocytosis in BMDMs from either 5-LOX-/- or BLT1-/- mice, suggesting a specific role for LTB4 in mediating phagocytosis in murine macrophages. This study demonstrates that LTB4 promotes macrophage phagocytosis of bacteria via BLT1, and that BLT2 can fulfill this role in the absence of BLT1.

Leukotriene B4 receptor-2 contributes to chemoresistance of SK-OV-3 ovarian cancer cells through activation of signal transducer and activator of transcription-3-linked cascade.

Inflammation and inflammatory mediators are intimately linked with chemoresistance through complex pathways in the tumor microenvironment. However, the mechanism by which inflammatory mediators (e.g., eicosanoids) contribute to chemoresistance remains elusive. In this study, we found that the low-affinity leukotriene B4 receptor-2 (BLT2) and its ligand leukotriene B4 were highly up-regulated in cisplatin-resistant SK-OV-3 ovarian cancer cells and play critical roles in mediating the chemoresistance through the activation of signal transducer and activator of transcription-3 (STAT-3) and the subsequent up-regulation of interleukin-6 (IL-6). BLT2 depletion with siRNA clearly abolished the chemoresistance to cisplatin in SK-OV-3 ovarian cancer cells and further increased cell sensitivity to cisplatin chemotherapy by down-regulating the 'STAT-3-IL-6' cascade. Enlarged tumor formation due to the cisplatin resistance of SK-OV-3 cells in cisplatin-treated athymic mice was also substantially reduced by co-treatment with the BLT2 inhibitor in vivo. Our study demonstrates that BLT2 is a novel contributor to cisplatin resistance in SK-OV-3 ovarian cancer cells and thus may be a potential therapeutic target for the treatment of cisplatin-resistant ovarian cancer.

The leukotriene B4-leukotriene B4 receptor axis promotes cisplatin-induced acute kidney injury by modulating neutrophil recruitment.

Cisplatin is an effective chemotherapeutic agent and widely used in treatment of various solid organ malignancies, including head and neck, ovarian, and testicular cancers. However, the induction of acute kidney injury (AKI) is one of its main side effects. Leukotriene B4 receptor 1 (BLT1) mediates the majority of physiological effects of leukotriene B4 (LTB4), a potent lipid chemoattractant generated at inflammation sites, but the role of the LTB4-BLT1 axis in cisplatin-induced AKI remains unknown. Here we found upregulated LTB4 synthesis and BLT1 expression in the kidney after cisplatin administration. Cisplatin was found to directly upregulate gene expression of leukotriene A4 hydrolase and stimulate LTB4 production in renal tubular epithelial cells. Reduced kidney structural/functional damage, inflammation, and apoptosis were observed in BLT1-/- mice, as well as in wild-type mice treated with the LTA4H inhibitor SC-57461A and the BLT1 antagonist U-75302. Neutrophils were likely the target of this pathway, as BLT1 absence induced a significant decrease in infiltrating neutrophils in the kidney. Adoptive transfer of neutrophils from wild-type mice restored kidney injury in BLT1-/- mice following cisplatin challenge. Thus, the LTB4-BLT1 axis contributes to cisplatin-induced AKI by mediating kidney recruitment of neutrophils, which induce inflammation and apoptosis in the kidney. Hence, the LTB4-BLT1 axis could be a potential therapeutic target in cisplatin-induced AKI.

Inhibition of Leukotriene B4 Action Mitigates Intracerebral Hemorrhage-Associated Pathological Events in Mice.

Infiltration of neutrophils has been suggested to play an important role in the pathogenesis of intracerebral hemorrhage (ICH) for which effective therapeutic interventions remain unavailable. In the present study we focused on leukotriene B4 (LTB4) as a potent chemotactic factor for neutrophils in order to address its contribution to the pathologic events associated with ICH. ICH with hematoma expansion into the internal capsule that resulted in severe sensorimotor dysfunction was induced by injection of collagenase in mouse striatum. We found that LTB4 as well as mRNAs of 5-lipoxygenase (5-LOX) and 5-LOX-activating protein were increased in the brain after ICH. Daily treatment with a 5-LOX inhibitor zileuton (3 or 10 mg/kg, i.v.) prevented ICH-induced increase in LTB4, attenuated neutrophil infiltration into the hematoma, and ameliorated sensorimotor dysfunction. In addition, mice deficient in LTB4 receptor BLT1 exhibited a lower number of infiltrating neutrophils in the hematoma and lower levels of sensorimotor dysfunction after ICH than did wild-type mice. Similarly, daily treatment of mice with BLT antagonist ONO-4057 (30 or 100 mg/kg, by mouth) from 3 hours after induction of ICH inhibited neutrophil infiltration and ameliorated sensorimotor dysfunction. ONO-4057 also attenuated inflammatory responses of microglia/macrophages in the perihematoma region and axon injury in the internal capsule. These results identify LTB4 as a critical factor that plays a major role in the pathogenic events in ICH, and BLT1 is proposed as a promising target for ICH therapy.

Distinct cellular sources of hepoxilin A3 and leukotriene B4 are used to coordinate bacterial-induced neutrophil transepithelial migration.

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

Involvement of Leukotriene B4 Released from Keratinocytes in Itch-associated Response to Intradermal Interleukin-31 in Mice.

A recent study suggests that interleukin-31 (IL-31) exerts its effect via indirect mechanisms rather than through direct stimulation of cutaneous nerves. However, the underlying peripheral mechanisms of IL-31-induced itch in the skin remain unclear. Therefore, the present study investigated the peripheral mechanisms underlying IL-31-induced itch in mice. IL-31-induced itch-related response was inhibited by anti-allergic drugs (tranilast and azelastine), but not by an H1 histamine receptor antagonist (terfenadine). Furthermore, a 5-lipoxygenase inhibitor (zileuton), but not a cyclooxygenase inhibitor (indomethacin), and a leuko-triene B4 (LTB4) receptor antagonist (CMHVA) attenuated the action of IL-31. IL-31 receptor-immunoreactivity was observed in the epidermis and primary sensory neurones. IL-31 receptor mRNA was expressed in mouse keratinocytes and dorsal root ganglia neurones. IL-31 increased the production of LTB4 in mouse keratinocytes. These results suggest that IL-31 elicits itch not only through direct action on primary sensory neurones, but also by inducing LTB4 production in keratinocytes.

Systemic leukotriene B4 receptor antagonism lowers arterial blood pressure and improves autonomic function in the spontaneously hypertensive rat.

KEY POINTS: Evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4 ), a potent chemoattractant involved in the inflammatory response, but its mode of action is poorly understood. In the SHR, we observed an increase in T cells and macrophages in the brainstem; in addition, gene expression profiling data showed that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. When LTB4 receptor 1 (BLT1) receptors were blocked with CP-105,696, arterial pressure was reduced in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in systolic blood pressure (BP) indicators. These data provide new evidence for the role of LTB4 as an important neuro-immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension. ABSTRACT: Accumulating evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4 ), a potent chemoattractant involved in the inflammatory response. However, the mechanism for LTB4 -mediated inflammation in hypertension is poorly understood. Here we report in the SHR, increased brainstem infiltration of T cells and macrophages plus gene expression profiling data showing that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. Chronic blockade of the LTB4 receptor 1 (BLT1) receptor with CP-105,696, reduced arterial pressure in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in low and high frequency spectra of systolic blood pressure, and an increase in spontaneous baroreceptor reflex gain (sBRG). These data provide new evidence for the role of LTB4 as an important neuro-immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension.

Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function.

Imatinib, a tyrosine kinase inhibitor (TKI) has significantly improved clinical outcome for chronic myeloid leukemia (CML) patients. However, patients develop resistance when the disease progresses to the blast phase (BP) and the mechanisms are not well understood. Here we show that BCR-ABL activates BLT2 in hematopoietic stem/progenitor cells to promote leukemogenesis and this involves the p53 signaling pathway. Compared to normal bone marrow (NBM), the mRNA and protein levels of BLT2 are significantly increased in BP-CML CD34(+) stem/progenitor cells. This is correlated with increasing BCR-ABL expression. In contrast, knockdown of BCR-ABL or inhibition of its tyrosine kinase activity decreases Blt2 protein level. BLT2 inhibition induces apoptosis, inhibits proliferation, colony formation and self-renewal capacity of CD34(+) cells from TKI-resistant BP-CML patients. Importantly, the inhibitory effects of BCR-ABL TKI on CML stem/progenitor cells are further enhanced upon combination with BLT2 inhibition. We further show that BLT2 activation selectively suppresses p53 but not Wnt or BMP-mediated luciferase activity and transcription. Our results demonstrate that BLT2 is a novel pathway activated by BCR-ABL and critically involved in the resistance of BP-CML CD34(+) stem/progenitors to TKIs treatment. Our findings suggest that BLT2 and p53 can serve as therapeutic targets for CML treatment.

Ras promotes transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition via a leukotriene B4 receptor-2-linked cascade in mammary epithelial cells.

Inflammation and inflammatory mediators are inextricably linked with epithelial-mesenchymal transition (EMT) through complex pathways in the tumor microenvironment. However, the mechanism by which inflammatory mediators, such as the lipid inflammatory mediators, eicosanoids, contribute to EMT is largely unknown. In the present study we observed that BLT2, leukotriene B4 receptor-2, is markedly up-regulated by oncogenic Ras and promotes EMT in response to transforming growth factor-ß (TGF-ß) in mammary epithelial cells. Blockade of BLT2 by the BLT2 inhibitor LY255283 or by siRNA reduced EMT induced by Ras in the presence of TGF-ß. In addition, stimulation of BLT2 by the addition of a BLT2 ligand, such as leukotriene B4, restored EMT in the presence of TGF-ß in human immortalized mammary epithelial MCF-10A cells. We further searched BLT2 downstream components and identified reactive oxygen species and nuclear factor κB as critical components that contribute to EMT. Taken together, these results demonstrate for the first time that a BLT2-linked inflammatory pathway contributes to EMT. This provides valuable insight into the mechanism of EMT in mammary epithelial cells. In addition, considering the implications of EMT with the stemness of cancer cells, our finding may contribute to a better understanding of tumor progression.

Leukotriene B4-leukotriene B4 receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8⁺ T cells.

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1ß. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1ß. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis.

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors.

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.

Leukotriene B4 Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells.

BACKGROUND/AIMS: Arachidonic acid (AA) and its metabolites are important endogenous lipid messengers. In this study, we test the effect of Leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of AA, on L-type calcium channels in A7r5 rat aortic vascular smooth muscle cells. METHODS: L-type calcium channel currents were recorded by a patch-clamp technique. The mRNA expression of CaV1.2 was determined by Real-time RT-PCR. The protein expression of CaV1.2 and p38 activity was determined by Western blot analysis. RESULTS: LTB4 inhibits L-type channel currents in A7r5 cells in a dose-and time- dependent manner. LTB4 reduced the mRNA/protein expression of CaV1.2 channels in A7r5 cells. BLT1 receptor antagonist LY29311 abrogated the inhibitory effect of LTB4, while BLT2 receptor antagonist LY255283 had no effect. 5Z-7-oxozeaenol and SB203580, which block TAK1 and p38 kinase respectively, abrogated the LTB4 inhibitory effect on L-type calcium channels. LTB4 increased p38 activity in A7r5 cells. Blockage of Src, PI3K, JNK and NF-x03BA;B kinase had no effects on LTB4 inhibition of L-type calcium channel currents in A7r5 cells. CONCLUSION: We conclude that LTB4 inhibits L-type calcium channels through BLT1-TAk1-p38 signaling pathway. The LTB4 inhibitory effect on L-type calcium channels may be involved in its pathological processes such as atherosclerosis.

Dual role of leukotriene B4 receptor type 1 in experimental sepsis.

BACKGROUND: The controversial results from different studies suggested that leukocyte recruitment mediated by leukotriene B4 (LTB4) and its receptor might improve pathogen clearance, but might also aggravate organ injury during sepsis. The present study was performed to compare the effect of BLT1 ligand LTB4 and its antagonist U-75302 on the development of sepsis. METHODS: Sepsis in mice was induced by cecal ligation and puncture (CLP). The mice were allocated into sham group, CLP group, U-75302 group, and LTB4 group. In the latter three groups, CLP mice were treated by intraperitoneal saline, U-75302, and LTB4, respectively. Their effect on the progression of sepsis were compared by histopathologic tests, level of systemic cytokines, counts of immune cells and bacterial clearance, and survival rate. RESULTS: The histopathologic tests showed that U-75302 attenuated lung injury, whereas LTB4 aggravated liver injury. LTB4 increased the plasma levels of interleukin-6, tumor necrosis factor-α, and U-75302 increased the level of plasma interleukin-10. LTB4 increased whereas U-75302 reduced the neutrophil numbers in the peritoneal lavage fluid. LTB4 also increased the number of peritoneal and splenic CD4(+) and CD8(+) T cells. Bacterial clearance in blood and peritoneal lavage fluid was significantly enhanced in the LTB4 group. Both U-75302 and LTB4 did not change the survival rate significantly compared with vehicle, but mortality in the LTB4 group was significantly higher than in the U-75302 group. Dose response analyses were also performed to compare the effect of U-75302 and LTB4 at different doses. Different doses of both agents did not influence the survival rate of CLP mice. CONCLUSIONS: U-75302 attenuates sepsis-induced organ injury, whereas LTB4 increases the leukocyte recruitment toward infection site, but LTB4 showed a more lethal effect than U-75302 during polymicrobial sepsis.

The leukotriene B4 receptor (BLT) antagonist BIIL284 decreases atherosclerosis in ApoE-/- mice.

Leukotriene B4 (LTB4) induces proinflammatory signaling through BLT receptors expressed in atherosclerotic lesions. Either genetic or pharmacological targeting of the high affinity LTB4 receptor, BLT1, reduces atherosclerosis in different mouse models. The low affinity BLT2 receptor for LTB4 may transduce additional pro-atherogenic signaling, but combined BLT1 and BLT2 receptor antagonism has not previously been explored in atherosclerosis. The aim of the present study was to unravel the effects of the BLT receptor antagonist BIIL284 in apolipoprotein E deficient mice in terms of atherosclerotic lesion size and composition, as well as on arterial matrixmetalloproteinase (MMP) activity and plasma cytokines. Oral administration of BIIL284 (0.3-3mg/kg) dose-dependently decreased atherosclerotic lesion size after 12 weeks. In addition, significantly smaller aortic lesions were observed in mice treated with BIIL284 (3mg/kg) for 24 weeks. The reduced atherosclerosis was associated with less lesion smooth muscle cells, less arterial MMP activities and lower plasma levels of TNF-α and IL-6. Taken together, these results suggest a therapeutic value of BLT receptor antagonism in atherosclerosis.

Activation of the leukotriene B4 receptor 2-reactive oxygen species (BLT2-ROS) cascade following detachment confers anoikis resistance in prostate cancer cells.

The majority of prostate cancer-related deaths are associated with advanced and metastatic malignancies. Although anoikis resistance has been recognized as one of the hallmarks of metastatic prostate malignancies, the molecular events that cause anoikis resistance are poorly understood. In this study, we found that the detachment of PC-3 prostate cancer cells caused a time-dependent increase in the expression level of the leukotriene B4 receptor-2 (BLT2) and that BLT2 played a critical role in establishing anoikis resistance in these cells. Blocking BLT2 with the pharmacological inhibitor LY255283 or with RNAi knockdown clearly abolished anoikis resistance and resulted in severe apoptotic death. Additionally, we demonstrated that the activation of NADPH oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) were downstream of BLT2 signaling and led to the activation of NF-κB, thus establishing anoikis resistance during cell detachment. Furthermore, we observed that the ectopic expression of BLT2 in normal prostate PWR-1E cells rendered the cells resistant to anoikis and apparently diminished apoptotic cell death following detachment. Taken together, our results suggest that BLT2-NOX-ROS-NF-κB cascade induction during detachment confers a novel mechanism of anoikis resistance in prostate cancer cells and potentially contributes to prostate cancer progression.