LTB4 is a signal-relay molecule during neutrophil chemotaxis

Neutrophil recruitment to inflammation sites purportedly depends on sequential waves of chemoattractants. Current models propose that leukotriene B(4) (LTB(4)), a secondary chemoattractant secreted by neutrophils in response to primary chemoattractants such as formyl peptides, is important in initiating the inflammation process. In this study we demonstrate that LTB(4) plays a central role in neutrophil activation and migration to formyl peptides. We show that LTB(4) production dramatically amplifies formyl peptide-mediated neutrophil polarization and chemotaxis by regulating specific signaling pathways acting upstream of actin polymerization and MyoII phosphorylation. Importantly, by analyzing the migration of neutrophils isolated from wild-type mice and mice lacking the formyl peptide receptor 1, we demonstrate that LTB(4) acts as a signal to relay information from cell to cell over long distances. Together, our findings imply that LTB(4) is a signal-relay molecule that exquisitely regulates neutrophil chemotaxis to formyl peptides, which are produced at the core of inflammation sites.     

Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4

We investigated the involvement of monocyte chemoattractant protein (MCP)-1 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that CLP induced a dramatic increase in MCP-1 production in the peritoneum, followed by an increase in the recruitment of leukocytes. MCP-1 blockade with anti-MCP-1 antiserum significantly decreased the survival rate following CLP, which was accompanied by an enhanced recovery of viable bacteria from the peritoneum. This was likely due to the reduction in the recruitment and activation of both macrophages and neutrophils. To understand the mechanisms whereby MCP-1 may influence neutrophil infiltration, levels of chemokines known to attract neutrophils were monitored, which showed that peritoneal levels of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1alpha were not altered with anti-MCP-1 Abs. However, anti-MCP-1 Abs reduced the peritoneal levels of leukotriene B4 (LTB4) by 59%. The i.p. injection of MCP-1 into normal mice resulted in elevated levels of LTB4 in the peritoneum. In vitro, MCP-1 stimulated the production of LTB4 from peritoneal macrophages, in a dose-dependent manner. A specific LTB4 receptor antagonist (CP-105, 696) inhibited CLP-induced recruitment of both neutrophils and macrophages, which was accompanied by a reduced level of MCP-1 in the peritoneum. Finally, administration of CP-105,696 was extremely detrimental to the survival of mice following CLP. These experiments demonstrate that endogenous MCP-1 serves as an indirect mediator to attract neutrophils via the production of LTB4, and suggest the cross-talk can occur between MCP-1 and the lipid mediator LTB4 during septic peritonitis.     

IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4

IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease severity and reduced articular inflammation and joint destruction. Furthermore, MK-886-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Finally, we showed that IL-18-activated human peripheral blood neutrophils produced significant amounts of LTB(4) that were effectively blocked by the MK. Together, these findings provide a novel mechanism whereby IL-18 can promote inflammatory diseases.     

Temporin A and related frog antimicrobial peptides use formyl peptide receptor-like 1 as a receptor to chemoattract phagocytes

Many mammalian antimicrobial peptides (AMPs) have multiple effects on antimicrobial immunity. We found that temporin A (TA), a representative frog-derived AMP, induced the migration of human monocytes, neutrophils, and macrophages with a bell-shaped response curve in a pertussis toxin-sensitive manner, activated p44/42 MAPK, and stimulated Ca(2+) flux in monocytes, suggesting that TA is capable of chemoattracting phagocytic leukocytes by the use of a G(ialpha) protein-coupled receptor. TA-induced Ca(2+) flux in monocytes was cross-desensitized by an agonistic ligand MMK-1 specific for formyl peptide receptor-like 1 (FPRL1) and vice versa, suggesting that TA uses FPRL1 as a receptor. This conclusion was confirmed by data showing that TA selectively stimulated chemotaxis of HEK 293 cells transfected with human FPRL1 or its mouse ortholog, murine formyl peptide receptor 2. In addition, TA elicited the infiltration of neutrophils and monocytes into the injection site of mice, indicating that TA is also functionally chemotactic in vivo. Examination of two additional temporins revealed that Rana-6 was also able to attract human phagocytes using FPRL1, but temporin 1P selectively induced the migration of neutrophils using a distinct receptor. Comparison of the chemotactic and antimicrobial activities of several synthetic analogues suggested that these activities are likely to rely on different structural characteristics. Overall, the results demonstrate that certain frog-derived temporins have the capacity to chemoattract phagocytes by the use of human FPRL1 (or its orthologs in other species), providing the first evidence suggesting the potential participation of certain amphibian antimicrobial peptides in host antimicrobial immunity.     

Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37

The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.     

Cathelicidin-related antimicrobial peptide is required for effective lung mucosal immunity in Gram-negative bacterial pneumonia

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, cathelicidin-related antimicrobial peptide (CRAMP), to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp(-/-)) demonstrate impaired lung bacterial clearance, increased bacterial dissemination, and reduced survival in response to intratracheal K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 h in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.     

Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis

Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.     

Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells

The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation.     

Detergent solubilization of human neutrophil leukotriene B4 receptors

Specific leukotriene B4 (LTB4) receptors in human neutrophils were solubilized by treatment of "receptor fraction" membranes with the zwitterionic detergent (3-[(3-cholamidopropyl)-dimethylammonio]1-propane sulfonate (CHAPS). The soluble receptors were assayed by polyethylene glycol (PEG) precipitation coupled with Millipore filtration. The solubilized receptors retained all of the characteristics of the receptor sites in intact neutrophils. The binding of LTB4 was rapid, reversible and stereospecific. Mathematical modeling analysis revealed biphasic binding of [3H] LTB4 indicating two classes of binding sites. The high affinity binding site had a dissociation constant of 1.93 nM and Bmax of 281 fmoles/mg protein; the low affinity binding site had a dissociation constant of 78.92 nM and Bmax of 2522 fmoles/mg protein. Competitive binding experiments with structural analogs of LTB4 demonstrate that the interaction between LTB4 and its binding site is stereospecific and correlates with the relative biological activity of the analogs. These data suggest that it may be possible to purify the LTB4 receptor from human neutrophil membranes.     

Transepithelial migration of neutrophils in response to leukotriene B4 is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade

The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B(4) (LTB(4)) is known to be a principal chemoattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB(4) induces transepithelial migration of neutrophils. We found that LTB(4) induces concentration-dependent transmigration of DMSO-differentiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB(4) receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB(4)-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB(4) signaling to transepithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB(4) led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB(4) was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB(4) receptor antagonist.     

BLT1 and BLT2: the leukotriene B(4) receptors

Two receptors for leukotriene B(4) (LTB(4)) have been molecularly identified: BLT1 and BLT2. Both receptors are G protein-coupled seven transmembrane domain receptors, whose genes are located in very close proximity to each other in the human and mouse genomes. The two receptors differ in their affinity and specificity for LTB(4): BLT1 is a high-affinity receptor specific for LTB(4), whereas BLT2 is a low-affinity receptor that also binds other eicosanoids. The two receptors also differ in their pattern of expression with BLT1 being expressed primarily in leukocytes, whereas BLT2 is expressed more ubiquitously. By mediating the activities of LTB(4), these receptors participate both in host immune responses and in the pathogenesis of inflammatory diseases. Reduced disease severity in animal inflammatory models seen with LTB(4) receptor antagonists and in mice with targeted deletion of BLT1 have revealed important roles for LTB(4) and its receptors in regulating pathologic inflammation.     

Antiviral effects of a transgenic RNA-dependent RNA polymerase

Transgenic expression of the RNA-dependent RNA polymerase 3D(pol) inhibited infection of Theiler's murine encephalitis virus (TMEV), a picornavirus from which it was derived. Here, we infected 3D(pol) transgenic mice with another picornavirus, as well as an alphaherpesvirus and a rhabdovirus. 3D(pol) transgenic FVB mice had significantly lower viral loads and survived longer after infection with all three types of viruses than nontransgenic FVB mice. Viral inhibition among three different types of virus by transgenic 3D(pol) suggests that the mechanism of action is not the direct interference with picornaviral 3D(pol) but instead may be the changing of host cells to an antiviral state before or after viral infection occurs, as basal interferon levels were higher in 3D(pol) transgenic mice before infection. Further study of this mechanism may open new possibilities for future antiviral therapy.     

Leukotriene B4 protects latently infected mice against murine cytomegalovirus reactivation following allogeneic transplantation

Human CMV is often associated with transplant rejection and opportunistic infections such as pneumonia in immunosuppressed patients. Current anti-CMV therapies, although effective, show relatively high toxicity, which seriously limits their long-term use. In this study, we provide evidence that leukotriene B(4) (LTB(4)) plays an important role in the fight against murine CMV (MCMV) infection in vivo. Intravenous administration of 50 and 500 ng/kg/day of LTB(4) to mice infected with a lethal dose of MCMV significantly increases their survival (50 and 70%, respectively), compared with the placebo-treated group (10% of survival). In mice infected with a sublethal dose of MCMV and treated daily with 50 ng/kg/day of LTB(4), the salivary gland viral loads were found to be reduced by 66% compared with the control group. Furthermore, using an allogeneic bone marrow transplantation mouse model, the frequency of MCMV reactivation from latently infected mice was much lower (38%) in LTB(4) (500 ng/kg)-treated mice than in the placebo-treated group (78%). Finally, in experiments using 5-lipoxygenase-deficient mice, MCMV viral loads in salivary glands were found to be higher in animals unable to produce leukotrienes than in the control groups, supporting a role of endogenous 5-lipoxygenase products, possibly LTB(4), in host defense against CMV infection.     

Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB(4) signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6(-/-) mice. In vitro, GRK6(-/-) PMN showed increased chemokinetic and chemotactic responses to LTB(4). GRK6(-/-) PMN respond to LTB(4) with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB(4) receptor desensitization in the absence of GRK6. However, pre-exposure to LTB(4) renders both GRK6(-/-) as well as wild-type PMN refractory to restimulation with LTB(4), indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.     

Human neutrophil defensins increase neutrophil uptake of influenza A virus and bacteria and modify virus-induced respiratory burst responses

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.     

Intrapulmonary administration of leukotriene B(4) augments neutrophil accumulation and responses in the lung to Klebsiella infection in CXCL1 knockout mice

In prior studies, we demonstrated that 1) CXCL1/KC is essential for NF-κB and MAPK activation and expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine in Klebsiella-infected lungs, and 2) CXCL1 derived from hematopoietic and resident cells contributes to host immunity against Klebsiella. However, the role of CXCL1 in mediating neutrophil leukotriene B(4) (LTB(4)), reactive oxygen species (ROS), and reactive nitrogen species (RNS) production is unclear, as is the contribution of these factors to host immunity. In this study, we investigated 1) the role of CXCL1 in LTB(4), NADPH oxidase, and inducible NO synthase (iNOS) expression in lungs and neutrophils, and 2) whether LTB(4) postinfection reverses innate immune defects in CXCL1(-/-) mice via regulation of NADPH oxidase and iNOS. Our results demonstrate reduced neutrophil influx, attenuated LTB(4) levels, and decreased ROS and iNOS production in the lungs of CXCL1(-/-) mice after Klebsiella pneumoniae infection. Using neutrophil depletion and repletion, we found that neutrophils are the predominant source of pulmonary LTB(4) after infection. To treat immune defects in CXCL1(-/-) mice, we intrapulmonarily administered LTB(4). Postinfection, LTB(4) treatment reversed immune defects in CXCL1(-/-) mice and improved survival, neutrophil recruitment, cytokine/chemokine expression, NF-κB/MAPK activation, and ROS/RNS production. LTB(4) also enhanced myeloperoxidase, H(2)O(2,) RNS production, and bacterial killing in K. pneumoniae-infected CXCL1(-/-) neutrophils. These novel results uncover important roles for CXCL1 in generating ROS and RNS in neutrophils and in regulating host immunity against K. pneumoniae infection. Our findings suggest that LTB(4) could be used to correct defects in neutrophil recruitment and function in individuals lacking or expressing malfunctional CXCL1.     

Inhibition of leukotriene biosynthesis abrogates the host control of Mycobacterium tuberculosis

Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B(4) and the anti-inflammatory substance lipoxin A(4) by approximately 50%, increased 60-day mortality from 14% to approximately 57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by approximately 15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) - presumably leukotrienes - positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB(4)) rather than a suppressor (LXA(4)) of antimicrobial defense, suggesting the major effect of leukotrienes.     

Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein

We produced human leukotriene B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.     

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.