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1.  Joint Tissues Amplify Inflammation and Alter Their Invasive Behavior via Leukotriene B4 in Experimental Inflammatory Arthritis 
Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB4 synthesis and to respond to LTB4 within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase–/– and leukotriene A4 (LTA4) hydrolase–/– mice, we demonstrate that FLSs generate sufficient levels of LTB4 production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs—which comprise the predominant lineage populating the synovial lining—are competent to metabolize exogenous LTA4 into LTB4 ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB4 3-fold without inducing the expression of LTA4 hydrolase protein. Moreover, LTB4 (acting via LTB4 receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB4 in joints, placing LTB4 regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.
PMCID: PMC3690310  PMID: 20876351
2.  Neutrophils in autoantibody-mediated arthritis: critical producers of FcRγ , the receptor for C5a, and LFA-1 
Arthritis and rheumatism  2010;62(3):753-764.
Neutrophils represent a prominent component of inflammatory joint effusions and are required for synovial inflammation in mouse models, but mechanisms are poorly understood. We developed a system to test the importance of production of specific factors by neutrophils in a mouse model of arthritis.
Neutrophil-deficient Gfi-1−/− mice were sub-lethally irradiated, then engrafted with donor bone-marrow cells (BMC), which resulted in production of mature neutrophils within two weeks. By reconstituting with BMC from mice lacking selected pro-inflammatory factors, mice specifically lacking these factors on neutrophils were generated. Arthritis was initiated by transfer of K/BxN serum to identify the role of defined neutrophil factors on arthritis incidence and severity.
Neutrophils lacking the signaling chain of stimulatory Fc receptors (FcRγ −/−) were unable to elicit arthritis, but neutrophils lacking Fcγ RIII still did so. Neutrophils lacking the chemotactic or adhesion receptors C5aR or CD11a/LFA-1 also failed to initiate arthritis but could enter joints in which inflammation had been initiated by wild-type neutrophils. Neutrophils unable to produce interleukin-1 α and β (IL-1αβ −/−) or leukotrienes (5-LO−/−) produced arthritis of intermediate severity. Inability of neutrophils to make tumor necrosis factor (TNF), or to express receptors for TNF or IL-1, had no effect on arthritis.
A novel transfer system was developed to identify neutrophil production of FcRγ , C5aR, and CD11a/LFA-1 as critical components of autoantibody-mediated arthritis. Neutrophil production of IL-1 and leukotriene B4 likely contributes to inflammation but is not essential. Molecular requirements for neutrophil influx into joints become more permissive after inflammation is initiated.
PMCID: PMC3057458  PMID: 20191628
arthritis; neutrophils; mouse model; inflammation; autoantibodies
3.  Role of PI3Kδ and PI3Kγ in inflammatory arthritis and tissue localization of neutrophils 
European journal of immunology  2008;38(5):1215-1224.
The p110δ isoform of class I phosphoinositide 3-kinase (PI3Ks) plays a major role in B cell receptor signaling, while its p110γ counterpart is thought to predominate in leukocyte chemotaxis. Consequently, emphasis has been placed on developing PI3Kγ selective inhibitors to treat disease states that result from inappropriate tissue accumulation of leukocytes. We now demonstrate that PI3Kδ blockade is effective in treating an autoimmune disorder in which neutrophil infiltration is required for tissue injury. Using the K/BxN serum transfer model of arthritis, in which neutrophils and leukotriene B4 (LTB4) participate, we show that genetic deletion or selective inhibition of PI3Kδ diminishes joint erosion to a level comparable to its PI3Kγ counterpart. Moreover, the induction and progression of joint destruction was profoundly reduced in the absence of both PI3K isoforms and correlated with a limited ability of neutrophils to migrate into tissue in response to LTB4. However, the dynamic interplay between these isoforms is not pervasive, as fMLP-induced neutrophil extravasation was primarily reliant on PI3Kγ. Our results not only demonstrate that blockade of PI3Kδ has potential therapeutic value in the treatment of chronic inflammatory conditions, but also provide evidence that dual inhibition of these lipid kinases may yield superior clinical results.
PMCID: PMC2972192  PMID: 18412166
Cell trafficking; neutrophils; kinases; autoimmunity
4.  A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis 
Neutrophil recruitment into tissue plays an important role in host defense and disease pathogenesis, including the inflammatory arthritides. A multitude of diverse chemoattractants have been implicated in neutrophil recruitment, suggesting that they have overlapping functions in mediating this critical biological response. However, here we demonstrate a unique, non-redundant role for the leukotriene B4 receptor BLT1 in mediating neutrophil recruitment into the joint in the K/BxN mouse model of inflammatory arthritis. We demonstrate that neutrophil expression of BLT1 was absolutely required for arthritis generation and chemokine production in this model, and that specific BLT1 inhibition reversed established disease. Adoptive transfer of wild-type (WT) neutrophils restored arthritis and chemokine production in BLT1−/− mice. Surprisingly, the primary effect of the transferred WT neutrophils into BLT1−/− mice was to promote the entry of endogenous BLT1−/− neutrophils into the joints of these mice. However, continued joint inflammation was dependent on the presence of WT neutrophils, indicating an ongoing specific requirement for BLT1-activated neutrophils in mediating BLT1−/− neutrophil recruitment by other chemoattractants. These experiments demonstrate that neutrophil BLT1 functions in a novel and essential non–cell-autonomous manner to enable the recruitment of additional neutrophils not expressing this receptor, thereby amplifying the inflammatory response in autoantibody-induced arthritis.
PMCID: PMC2118298  PMID: 16567386
5.  Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis 
Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)–producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.
We utilized whole blood–derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C (U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.
Our data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release–activated channel–mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP+ cells capable of F-actin ring formation. These effects were dependent on Ca2+ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.
In conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca2+ signaling and the LTB4 signaling cascade.
PMCID: PMC4276054  PMID: 25443625
6.  Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis 
Immunity  2010;33(2):266-278.
A large and diverse array of chemoattractants control leukocyte trafficking, but how these apparently redundant signals collaborate in vivo is still largely unknown. We previously demonstrated an absolute requirement for the lipid chemoattractant leukotriene B4 (LTB4) and its receptor BLT1 for neutrophil recruitment into the joint in autoantibody-induced arthritis. We now demonstrate that BLT1 is required for neutrophils to deliver IL-1 into the joint to initiate arthritis. IL-1-expressing neutrophils amplify arthritis through the production of neutrophil-active chemokines from synovial tissue cells. CCR1 and CXCR2, two neutrophil chemokine receptors, operate non-redundantly to sequentially control the later phase of neutrophil recruitment into the joint and mediate all neutrophil chemokine activity in the model. Thus, we have uncovered a complex sequential relationship involving unique contributions from the lipid mediator LTB4, the cytokine IL-1, and CCR1 and CXCR2 chemokine ligands that are all absolutely required for effective neutrophil recruitment into the joint.
PMCID: PMC3155777  PMID: 20727790
7.  Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion 
Arthritis Research  1999;2(1):65-74.
Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.
Increased numbers of mast cells (MCs) are found in the synovial tissues and fluids of patients with rheumatoid arthritis (RA), and at sites of cartilage erosion. MC activation has been reported for a significant proportion of rheumatoid specimens. Because the MC contains potent mediators, including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines, its potential contributions to the processes of inflammation and matrix degradation have recently become evident.
Proinflammatory cytokines are important mediators of inflammation, immunity, proteolysis, cell recruitment and proliferation. Tumour necrosis factor (TNF) reportedly plays a pivotal role in the pathogenesis of RA, especially its ability to regulate interleukin (IL)-1β expression, this being important for the induction of prostanoid and matrix metalloproteinase production by synovial fibroblasts and chondrocytes. IL-15 has been assigned numerous biological effects and has been implicated as an important factor in TNF-α expression by monocyte/macrophages. Some in vitro studies have placed IL-15 upstream from TNF-α in the cytokine cascade, suggesting an interdependence between TNF, IL-1 and IL-15 for the promotion of proinflammatory cytokine expression in the rheumatoid joint.
To examine the in situ relationships of TNF-α, IL-1β and IL-15 in relation to MC activation in rheumatoid tissues by use of immunolocalization techniques; and to compare quantitatively the proinflammatory cytokine production by specific cell cultures and rheumatoid synovial explants with and without exposure to a MC secretagogue.
Materials and methods:
Samples of rheumatoid synovial tissue and cartilage–pannus junction were obtained from patients (n = 15) with classic late-stage RA. Tissue sections were immunostained for MC (tryptase) and the proinflammatory cytokines IL-1, TNF-α and IL-15. Rheumatoid synovial tissue explants were cultured in Dulbecco's modified Eagles medium (DMEM) containing either the MC secretagogue rabbit antihuman immunoglobulin (Ig)E, or control rabbit IgG. Primary rheumatoid synovial cell cultures, human articular chondrocytes, synovial fibroblasts and synovial macrophages were prepared as described in the full article. Conditioned culture media from these cultures were collected and assayed for IL-1β, TNF-α and IL-15 using enzyme-linked immunosorbent assay methodology.
Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Sites of MC activation were associated with localized oedema, and TNF-α, IL-1α and IL-1β production by a proportion of mononuclear inflammatory cells. By contrast, no evidence was found for IL-15 production in tissue sites containing either intact or activated MCs, and IL-15 expression, when observed, bore no relation to tissue sites where TNF-α and IL-1β were evident. The immunodetection of IL-15 was restricted to microfocal sites and was not typical of most junctional specimens, but was associated with a proportion of articular chondrocytes in a minority of junctional specimens.
MC activation within synovial explant cultures was induced by the addition of polyclonal antibody to human IgE. MC activation significantly reduced the levels of TNF-α and IL1β released into the medium, this representing approximately 33% of control values. By contrast, MC activation had little effect on the levels of IL-15 released into the culture medium, the average value being very low in relation to the release of TNF-α and IL-1β . Thus, induced MC activation brings about changes in the amounts of released tryptase, TNF-α and IL-1β , but not of IL-15.
Four preparations of primary rheumatoid synovial cell cultures produced more IL-1β than TNF-α, with only modest values for IL-15 production, indicating that all three cytokines are produced and released as free ligands by these cultures. Of specific cell types that produced IL-15 in vitro, macrophages produced more than fibroblasts, which in turn produced more than chondrocytes. This demonstrates that all three cell types have the potential to produce IL-15 in situ.
The biological consequences of MC activation in vivo are extremely complex, and in all probability relate to the release of various combinations of soluble and granular factors, as well as to the expression of appropriate receptors by neighbouring cells. The subsequent synthesis and release of cytokines such as TNF-α and IL-1 may well follow at specific stages after activation, or may be an induced cytokine response by adjacent macrophagic or fibroblastic cells. However, because no IL-15 was detectable either in or around activated or intact MCs, and the induced MC activation explant study showed no change in IL-15 production, it seems unlikely that the expression of this cytokine is regulated by MCs. The immunohistochemistry (IHC) demonstration of IL-15 at sites of cartilage erosion, and especially by some chondrocytes of articular cartilage, showed no spatial relationship with either T cells or neutrophils, and suggests other functional properties in these locations. The lack of evidence for an in situ association of IL-15 with TNF and IL-1 does not support a role for IL-15 in a proinflammatory cytokine 'cascade', as proposed by other in vitro experiments. We believe that sufficient evidence is available, however, to suggest that MC activation makes a significant contribution to the pathophysiological processes of the rheumatoid lesion.
PMCID: PMC17805  PMID: 11219391
interleukin-15; interleukin-1β; mast cells; rheumatoid arthritis; tumour necrosis factor-α
8.  C5a receptor enables participation of mast cells in immune complex arthritis independent of Fc γ receptor modulation 
Arthritis and rheumatism  2010;62(11):3322-3333.
Mast cells are tissue-resident immune sentinels implicated in the pathogenesis of inflammatory joint disease. We hypothesized that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.
In vivo studies employed murine K/BxN arthritis, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR−/− and control mast cells were engrafted into mast cell-deficient W/Wv mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR−/− animals and in murine and human mast cell cultures.
Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR−/− mast cells adoptively transferred into W/Wv mice were incompetent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate expression of Fc γ receptors (FcγR) or otherwise alter activation threshold. In human mast cells, C5a promoted production of the neutrophil chemotaxin interleukin 8, and recruitment of neutrophils at 24h after serum administration was impaired in C5aR−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.
Stimulation via C5aR is required to unleash the pro-inflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism distinct from C5a-modulated FcγR expression.
PMCID: PMC2970731  PMID: 20662064
9.  Reduction in leukotriene B4 generation by bronchoalveolar lavage cells in asthma. 
Thorax  1995;50(1):67-73.
BACKGROUND--Leukotrienes are inflammatory mediators implicated in the pathogenesis of asthma. The capacity of inflammatory cells within the airways to generate leukotrienes may be altered in asthma. This hypothesis was tested using bronchoalveolar lavage (BAL) to sample cells within the airways from atopic asthmatic and normal subjects, and by measuring their capacity to generate leukotriene B4 (LTB4) and leukotriene C4 (LTC4) in response to A23187, a potent stimulus of leukotriene generation. METHODS--Bronchoalveolar lavage was performed in 12 mild asymptomatic atopic asthmatic patients and 12 normal subjects. Mixed BAL cell aliquots (approximately 80% alveolar macrophages) were incubated with 0-20 microM A23187 for 10 minutes and with 4 microM A23187 for 0-30 minutes, and leukotrienes were measured by radioimmunoassay and high performance liquid chromatography. RESULTS--Mixed BAL cells from asthmatic subjects generated less LTB4 than cells from normal subjects in dose response and time course experiments (area under the curve 81.5 (0.0-228.5) ng.min.10(-6) cells in asthmatic subjects and 197.9 (13.9-935.6) ng.min.10(-6) cells in normal subjects. There were no differences in LTC4 generation between BAL cells from asthmatic and normal subjects. CONCLUSIONS--Generation of LTB4 by BAL cells from atopic asthmatic subjects in response to A23187 was reduced. As the alveolar macrophage is the major source of LTB4 in BAL cells, these results probably reflect reduced generation of LTB4 by alveolar macrophages from asthmatic patients. This may be a consequence of monocyte migration into the lung, or altered alveolar macrophage function in asthma, or both.
PMCID: PMC473713  PMID: 7886653
10.  Cys-Leukotrienes Promote Fibrosis in a Mouse Model of Eosinophil-Mediated Respiratory Inflammation 
Leukotrienes (i.e., products of the 5-lipoxygenase pathway) are thought to be contributors to lung pathologies. Moreover, eosinophils have been linked with pulmonary leukotriene activities both as potential sources of these mediators and as responding effector cells. The objective of the present study was to define the role(s) of leukotrienes in the lung pathologies accompanying eosinophil-associated chronic respiratory inflammation. A transgenic mouse model of chronic T helper (Th) 2–driven inflammation expressing IL-5 from T cells and human eotaxin-2 locally in the lung (I5/hE2) was used to define potential in vivo relationships among eosinophils, leukotrienes, and chronic Th2-polarized pulmonary inflammation. Airway levels of cys-leukotrienes and leukotriene B4 (LTB4) are both significantly elevated in I5/hE2 mice. The eosinophil-mediated airway hyperresponsiveness (AHR) characteristic of these mice was abolished in the absence of leukotrienes (i.e., 5-lipoxygenase–deficient I5/hE2). More importantly, the loss of leukotrienes led to an unexpectedly significant decrease in collagen deposition (i.e., pulmonary fibrosis) that accompanied elevated levels of IL-4/-13 and TGF-β in the lungs of I5/hE2 mice. Further studies using mice deficient for the LTB4 receptor (BLT-1−/−/I5/hE2) and I5/hE2 animals administered a cys-leukotriene receptor antagonist (montelukast) demonstrated that the AHR and the enhanced pulmonary fibrosis characteristic of the I5/hE2 model were uniquely cys-leukotriene–mediated events. These data demonstrate that, similar to allergen challenge models of wild-type mice, cys-leukotrienes underlie AHR in this transgenic model of severe pulmonary Th2 inflammation. These data also suggest that an underappreciated link exists among eosinophils, cys-leukotriene–mediated events, and fibrotic remodeling associated with elevated levels of IL-4/-13 and TGF-β.
PMCID: PMC3931112  PMID: 23859654
5-lipoxygenase; asthma; eosinophils; montelukast; lung
11.  Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils. 
Journal of Clinical Investigation  1993;91(2):651-660.
Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.
PMCID: PMC288002  PMID: 8381824
12.  Cooperative and redundant signaling of leukotriene B4 and leukotriene D4 in human monocytes 
Allergy  2011;66(10):1304-1311.
Leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) are important immune mediators, often found concomitantly at sites of inflammation. Although, some of the leukotriene-mediated actions are distinctive (e.g. bronchial constriction for cysLTs), many activities such as leukocyte recruitment to tissues and amplification of inflammatory responses are shared by both classes of leukotrienes.
We used human monocytes to characterize leukotriene specific signaling, gene expression signatures and functions and to identify interactions between LTB4 and cysLTs induced pathways.
Responsiveness to leukotrienes was assessed using oligonucleotide microarrays, real-time PCR, calcium mobilization, kinase activation and chemotaxis assays.
Human monocytes were found to express mRNA for high- and low-affinity LTB4 receptors, BLT1 and BLT2, but signal predominantly through BLT1 in response to LTB4 stimulation as shown using selective agonists, inhibitors and gene knock-down experiments. LTB4 acting through BLT1 coupled to G protein α inhibitory subunit activated calcium signaling, p44/42 mitogen-activated protein kinase, gene expression and chemotaxis. Twenty-seven genes, including immediate-early genes, transcription factors, cytokines and membrane receptors were significantly upregulated by LTB4. LTB4 and LTD4 had similar effects on signaling, gene expression and chemotaxis indicating redundant cell activation pathways but co-stimulation with both lipid mediators was additive for many monocyte functions.
LTB4 and LTD4 display both redundant and cooperative effects on intracellular signaling, gene expression and chemotaxis in human monocytes. These findings suggest that therapies targeting either leukotriene alone may be less effective than approaches directed at both.
PMCID: PMC3170431  PMID: 21605126
asthma; inflammation; monocytes; leukotrienes; receptors
13.  Effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils. 
Journal of Clinical Investigation  1984;74(6):1922-1933.
Exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) have been compared with exogenous arachidonic acid for their capacity to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human neutrophils and for their suitability as parallel substrates in this pathway. The products from specific 14C- or 3H-labeled substrates were isolated by reverse phase high performance liquid chromatography (RP-HPLC) and were identified by elution of radiolabel at the retention times of the appropriate synthetic standards. Each product was also characterized by its ultraviolet (UV) absorption spectrum, and 7-hydroxy-DCHA was defined in addition by analysis of its mass spectrum. The metabolites, 5-hydroxyeicosatetraenoic acid, leukotriene B4 (LTB4), 6-trans-LTB4 diastereoisomers, 5-hydroxyeicosapentaenoic acid, 6-trans-leukotriene B5 diastereoisomers, leukotriene B5 (LTB5), and 7-hydroxy-DCHA were quantitated by integrated UV absorbance during resolution by RP-HPLC. LTB4 and LTB5 were also quantitated by radioimmunoassay of the eluate fractions, and leukotrienes C4 and C5 (LTC4 and LTC5, respectively) were quantitated by radioimmunoassay alone. None of the unlabeled exogenous fatty acids (5-40 micrograms/ml) altered the release of radioactivity from [14C]arachidonic acid-labeled, ionophore-activated neutrophils. The metabolism of 5 and 10 micrograms/ml of exogenous EPA by ionophore-activated, [14C]arachidonic acid-labeled neutrophils not only generated 5-hydroxyeicosapentaenoic acid, 6-trans-LTB5, LTB5, and LTC5, but also stimulated the formation of 5-hydroxyeicosatetraenoic acid, 6-trans-LTB4 diastereoisomers, and LTC4 from membrane-derived arachidonic acid. In contrast, LTB4 production was diminished throughout the EPA dose-response, beginning at 5 micrograms/ml EPA and reaching 50% suppression at 10 micrograms/ml and 84% suppression at 40 micrograms/ml. The selective decrease in extracellular LTB4 concentrations in the presence of EPA was not due to a change in the kinetic appearance of LTB4 or to an increase in conversion to its omega-oxidation metabolites. DCHA was metabolized to 7-hydroxy-DCHA, did not stimulate metabolism of membrane-derived arachidonic acid, did not appreciably inhibit LTB4 formation, and was not a substrate for leukotriene formation. Incremental doses of exogenous arachidonic acid resulted in increased production of 5-hydroxyeicosatetraenoic acid and 6-trans-LTB4 by ionophore-activated, [14C]arachidonic acid-labeled neutrophils without any change in LTB4 production. 5-hydroxyeicosapentaenoic acid and 7-hydroxy DCHA were inactive as chemotactic factors whereas 5-hydroxyeicosatetraenoic acid exhibited 2% of the potency of LBT4. Thus, exogenous DCHA does not appreciably interfere with the metabolism of membrane-derived arachidonic acid by ionophore-activated, [14C]arachidonic acid-labeled neutrophils and is converted only to a monohydroxy derivative. In contrast, exogenous EPA attenuates the generation of LTB4 and is converted to LTB5, which is a weak and partial agonist as compared with LTB4.
PMCID: PMC425378  PMID: 6096400
14.  5-Lipoxygenase-deficient mice infected with B. burgdorferi develop persistent arthritis 
The enzyme 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid into the leukotrienes, which are critical regulators of inflammation and inflammatory diseases, such as asthma and arthritis. Although leukotrienes are present in the synovial fluid of Lyme disease patients, their role in the development of Lyme arthritis has not been determined. In the current study, we used a murine model of Lyme arthritis to investigate the role 5-LO products might have in the development of this inflammatory disease. Following infection of Lyme arthritis-susceptible C3H/HeJ mice with B. burgdorferi, mRNA expression of 5-LO and 5-lipoxygenase-activating protein (FLAP) was induced in the joints, and the 5-LO product LTB4 was produced. Utilizing C3H 5-LO-deficient mice, we demonstrated that 5-LO activity was not necessary for the induction of Lyme arthritis, but that its deficiency resulted in earlier joint swelling and an inability to resolve arthritis as demonstrated by sustained arthritis pathology through day 60 post-infection. Although production of anti-Borrelia IgG was decreased in 5-LO-deficient mice, bacterial clearance from the joints was unaffected. Phagocytosis of B. burgdorferi and efferocytosis of apoptotic neutrophils was defective in macrophages from 5-LO-deficient mice, and uptake of opsonized spirochetes by neutrophils was reduced. These results demonstrate that products of the 5-LO metabolic pathway are not required for the development of disease in all models of arthritis, and that caution should be used when targeting 5-LO as therapy for inflammatory diseases.
PMCID: PMC3346295  PMID: 21270404
15.  Bacterial adherence and hemolysin production from Escherichia coli induces histamine and leukotriene release from various cells. 
Infection and Immunity  1985;50(1):271-278.
We investigated the role of bacterial adherence and hemolysin production from Escherichia coli parent and genetically cloned strains as to their effects on histamine release from rat mast cells and leukotriene generation from human polymorphonuclear granulocytes. These mediators were involved in the induction of inflammatory disease processes and led, for example, to enhancement of vascular permeability, chemotaxis (leukotriene B4 [LTB4]), chemoaggregation, lysosomal enzyme release, and smooth muscle contraction, (LTC4, LTD4, and LTE4). Washed bacteria (E. coli K-12 MS+ Hly +/-; E. coli 536 MS+ MR +/-) as well as their culture supernatants were analyzed. Washed E. coli K-12 (Hly+), unlike Hly- strains, induced high amounts of histamine release from rat mast cells and chemotactic activity from human polymorphonuclear granulocytes. Significant leukotriene release was obtained with washed E. coli K-12 Hly+ strains and their bacterial culture supernatants. Leukotriene induction was dependent on the amount of hemolysin activity present in the supernatant. However, additional soluble factors should also be considered. The presence of hemolysin appeared to accelerate and enhance the rate of phagocytosis of bacteria by neutrophils. When E. coli 536 (MS+ MR +/- Hly +/-) strains were analyzed, the simultaneous presence of MR+ pili and hemolysin production led to an increase in histamine release as compared with MR- Hly+ strains. The genetically cloned MR+ Hly+ E. coli 536 strain induced higher amounts of leukotrienes as compared with the wild-type strain. Our data suggest a potent role for adhesins and hemolysin as virulence factors in inducing the release of inflammatory mediators.
PMCID: PMC262167  PMID: 2412960
16.  Human neutrophil FcγRIIA regulation by C5aR promotes inflammatory arthritis in mice 
Arthritis and Rheumatism  2011;63(2):467-478.
Rheumatoid arthritis culminates in joint destruction that in mouse models of disease, is supported by innate immune molecules including FcγRs and complement. However, the results may not predict outcomes in humans given the structural differences between murine and human activating FcγRs on neutrophils, a prominent component of joint exudates. In this study, we examined the role of the human neutrophil FcγRIIA in the development of arthritis and probed the underlying mechanism by which FcγRIIA initiated disease.
K/BxN serum transfer-induced arthritis was examined in mice that express FcγRIIA on neutrophils but lack their own activating FcγRs (γ-chain-deficient). The role of mast cells, complement (C3 and C5a) and CD18 integrins in FcγRIIA-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies respectively. Cross-talk between C5aR and FcγRIIA on neutrophils was evaluated in vitro.
Neutrophil FcγRIIA expression was sufficient to restore susceptibility to K/BxN serum induced neutrophil recruitment, synovitis and bone destruction in γ-chain-deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on CD18 integrin LFA-1 and C5aR. C5aR in addition significantly enhanced FcγRIIA mediated phagocytosis and oxidative burst in vitro.
Human and murine activating FcγRs on neutrophils are not functionally equivalent, and in humans may play a primary role in arthritis. Cross-talk between neutrophil C5aR and FcγRIIA is essential for disease thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.
PMCID: PMC3128197  PMID: 21280001
17.  Understanding the Mechanisms Controlling Leishmania amazonensis Infection In Vitro: The Role of LTB4 Derived From Human Neutrophils 
The Journal of Infectious Diseases  2014;210(4):656-666.
Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis–induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1).
PMCID: PMC4111911  PMID: 24634497
Leishmania amazonensis; LPG; human neutrophils; leukotriene B4; TLR2; NFkB
18.  Targeted Disruption of the Leukotriene B4Receptor in Mice Reveals Its Role in Inflammation and Platelet-Activating Factor–Induced Anaphylaxis 
Leukotrienes are derived from arachidonic acid and serve as mediators of inflammation and immediate hypersensitivity. Leukotriene B4 (LTB4) and leukotriene C4 (LTC4) act through G protein–coupled receptors LTB4 receptor (BLTR) and Cys-LTR, respectively. To investigate the physiological role of BLTR, we produced mice with a targeted disruption of the BLTR gene. Mice deficient for BLTR (BLTR−/−) developed normally and had no apparent hematopoietic abnormalities. Peritoneal neutrophils from BLTR−/− mice displayed normal responses to the inflammatory mediators C5a and platelet-activating factor (PAF) but did not respond to LTB4 for calcium mobilization or chemotaxis. Additionally, LTB4 elicited peritoneal neutrophil influx in control but not in BLTR−/− mice. Thus, BLTR is the sole receptor for LTB4-induced inflammation in mice. Neutrophil influx in a peritonitis model and acute ear inflammation in response to arachidonic acid was significantly reduced in BLTR−/− mice. In mice, intravenous administration of PAF induces immediate lethal anaphylaxis. Surprisingly, female BLTR−/− mice displayed selective survival (6 of 9; P = 0.002) relative to male (1 of 11) mice of PAF-induced anaphylaxis. These results demonstrate the role of BLTR in leukotriene-mediated acute inflammation and an unexpected sex-related involvement in PAF-induced anaphylaxis.
PMCID: PMC2193219  PMID: 10934231
arachidonic acid; neutrophil influx; knock-out; sex-related; chemotaxis
19.  Deficiency of CXCR2, but not other chemokine receptors, attenuates a murine model of autoantibody-mediated arthritis 
Arthritis and rheumatism  2010;62(7):1921-1932.
Chemokines coordinate leukocyte trafficking in homeostasis and during immune responses. Prior studies of their role in arthritis have employed animal models with both an initial adaptive immune response and an inflammatory effector phase. This study focused on chemokines and their receptors in the effector phase of arthritis using the K/BxN serum-transfer model.
A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle, synovial fluid and peripheral blood. Upregulation of chemokines was confirmed by quantitative RT-PCR. The functional relevance of chemokine induction was assessed by transferring serum into mice deficient in CCR1–CCR7, CCR9, CXCR2, CXCR3, CXCR5, CX3CR1, CCL2 or CCL3. Further mechanistic analysis of CXCR2 involved treatment of arthritic mice with a CXCR2 antagonist, bone-marrow transfers with CXCR2+/− and CXCR2−/− donors and recipients, flow cytometry of synovial cells, and competition experiments measuring enrichment of CXCR2-expressing neutrophils in arthritic joints of mice with mixed CXCR2+/+ and CXCR2−/− bone-marrow.
Gene-expression profiling revealed upregulation of the CXCR2 ligands CXCL1, CXCL2 and CXCL5 in the joint in parallel with disease activity. CXCR2−/− mice had attenuated disease relative to CXCR2+/− littermates, as did mice receiving the CXCR2 inhibitor, while deficiency of other chemokine receptors did not affect arthritis severity. CXCR2 was required only on hematopoietic cells and was widely expressed on synovial neutrophils. CXCR2-expressing neutrophils were preferentially recruited to arthritic joints in the presence of CXCR2-deficient neutrophils.
CXCR2 (but not other chemokine receptors) is critical for the development of autoantibody-mediated arthritis, exhibiting a cell-autonomous role in neutrophil recruitment to inflamed joints.
PMCID: PMC2994550  PMID: 20506316
20.  Monocyte Chemoattractant Protein 1 Regulates Pulmonary Host Defense via Neutrophil Recruitment during Escherichia coli Infection▿ 
Infection and Immunity  2011;79(7):2567-2577.
Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1−/− mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B4 (LTB4), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1−/− mice after infection. E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1−/− mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1−/− mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB4.
PMCID: PMC3191985  PMID: 21518788
21.  Comparison of four hemolysin-producing organisms (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) for release of inflammatory mediators from various cells. 
Journal of Clinical Microbiology  1988;26(3):544-551.
We investigated the role of various hemolysin-producing strains (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) in induction of inflammatory mediators, e.g., histamine release from rat mast cells as well as the chemiluminescence response and the release of lipoxygenase transformation products from human polymorphonuclear neutrophils. Our data show that the hemolysin-positive bacteria as well as the hemolysin-positive culture supernatants were active in inducing the chemiluminescence response, leukotriene (LTB4 and LTC4) release from human granulocytes, and histamine release from rat mast cells. The degree of leukotriene release was dependent on the hemolysin type and on the expression of hemolysin activity. The E. coli alpha-hemolysin and the aerolysin-producing A. hydrophila were the most potent stimuli whether washed bacteria or bacterial supernatant was used. Bacteria expressing the S. marcescens hemolysin and the listeriolysin were only poor inducers of leukotriene generation. In contrast to leukotriene generation, all hemolysin-positive strains induced nearly the same histamine release in a dose-dependent manner. Our data suggest a potent role for various hemolysins as virulence factors in inducing the release of inflammatory mediators.
PMCID: PMC266329  PMID: 2451679
22.  Intrapulmonary Administration of Leukotriene B4 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 B4 (LTB4), reactive oxygen species (ROS), and reactive nitrogen species (RNS) production is unclear, as is the contribution of these factors to host immunity. Here, we investigated: 1) the role of CXCL1 in LTB4, NADPH oxidase, and iNOS expression in lungs and neutrophils; and 2) whether LTB4 after infection reverses innate immune defects in CXCL1-/- mice via regulation of NADPH oxidase and iNOS. Our results demonstrate reduced neutrophil influx, attenuated LTB4 levels, and decreased ROS and iNOS production in the lungs of CXCL1-/- mice following K. pneumoniae infection. Using neutrophil depletion and repletion, we found that neutrophils are the predominant source of pulmonary LTB4 following infection. To treat immune defects in CXCL1-/- mice, we intrapulmonary administered LTB4. Post-infection LTB4 treatment reversed immune defects in CXCL1-/- mice and improved survival, neutrophil recruitment, cytokine/chemokine expression, NF-κB/MAPK activation, and ROS/RNS production. LTB4 also enhanced MPO, H2O2, 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 LTB4 could be used to correct defects in neutrophil recruitment and function in individuals lacking or expressing malfunctional CXCL1.
PMCID: PMC3311767  PMID: 22379035
23.  5-Lipoxygenase Reaction Products Modulate Alveolar Macrophage Phagocytosis of Klebsiella pneumoniae 
Infection and Immunity  1998;66(11):5140-5146.
The leukotrienes are potent lipid mediators of inflammation formed by the 5-lipoxygenase-catalyzed oxidation of arachidonic acid. Although the effects of leukotrienes on neutrophil chemotaxis and activation have been established, their role in modulating innate host defense mechanisms is poorly understood. In a previous study (M. Bailie, T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden, J. Immunol. 157:5221–5224, 1996), we used 5-lipoxygenase knockout mice to establish a critical role for endogenous leukotrienes in pulmonary clearance and alveolar macrophage phagocytosis of Klebsiella pneumoniae. In the present study, we investigated the role of specific endogenous leukotrienes in phagocytosis of K. pneumoniae and explored the possibility that exogenous leukotrienes could restore phagocytosis in alveolar macrophages with endogenous leukotriene synthesis inhibition and enhance this process in leukotriene-competent cells. Rat alveolar macrophages produced leukotriene B4 (LTB4), LTC4, and 5-hydoxyeicosatetraenoic acid (5-HETE) during the process of phagocytosis, and the inhibition of endogenous leukotriene synthesis with zileuton and MK-886 dramatically attenuated phagocytosis. We also observed a reduction in phagocytosis when we treated alveolar macrophages with antagonists to the plasma membrane receptors for either LTB4, cysteinyl-leukotrienes, or both. In leukotriene-competent cells, LTC4 augmented phagocytosis to the greatest extent, followed by 5-HETE and LTB4. These 5-lipoxygenase reaction products demonstrated similar relative abilities to reconstitute phagocytosis in zileuton-treated rat alveolar macrophages and in alveolar macrophages from 5-lipoxygenase knockout mice. We conclude that endogenous synthesis of all major 5-lipoxygenase reaction products plays an essential role in phagocytosis. The restorative and pharmacologic effects of LTC4, LTB4, and 5-HETE may provide a basis for their exogenous administration as an adjunctive treatment for patients with gram-negative bacterial pneumonia.
PMCID: PMC108641  PMID: 9784515
24.  Neutrophil activation by anti-proteinase 3 antibodies in Wegener's granulomatosis: role of exogenous arachidonic acid and leukotriene B4 generation 
The Journal of Experimental Medicine  1996;184(4):1567-1572.
Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA- positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody- related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha- priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5- hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4- related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha- priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5- lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.
PMCID: PMC2192817  PMID: 8879231
25.  Leukotriene B4 and its Metabolites Prime the Neutrophil Oxidase and Induce Pro-Inflammatory Activation of Human Pulmonary Microvascular Endothelial Cells 
Shock (Augusta, Ga.)  2011;35(3):240-244.
Leukotrienes are pro-inflammatory lipid mediators, derived from arachidonic acid via 5-lipoxygenase (5-LO). Leukotriene B4 (LTB4) is an effective neutrophil (PMN) chemoattractant, as well as being a major product of PMN priming. LTB4 is rapidly metabolized into products that are thought to be inactive, and little is known about the effects of LTB4 on the pulmonary endothelium. We hypothesize that LTB4 and its metabolites are effective PMN priming agents and cause pro-inflammatory activation of pulmonary endothelial cells.
Isolated PMNs were primed (5 min, 37°C) with serial concentrations 10−11–10−5M of LTB4 and its metabolites: 6-trans-LTB4, 20-OH-LTB4, and 20-COOH-LTB4 and then activated with fMLP. Primary human pulmonary microvascular endothelial cells (HMVECs) were incubated with these lipids (6 hrs, 37°C, 5% CO2) and intercellular adhesion molecule-1 (ICAM-1) was measured by flow cytometry. PMN adhesion was measured by myeloperoxidase assays and to ensure that these reactions were specific to the LTB4 receptors BLT1 and BLT2 were antagonized with CP105,696 (BLT1) or silenced with siRNA (BLT1 and BLT2).
LTB4 and its metabolites primed PMNs over a wide range of concentrations depending upon the specific metabolite. In addition, at high concentrations these lipids also caused increases in the surface expression of ICAM-1 on HMVECs and induced HMVEC-mediated adhesion of PMNs. Silencing of BLT2 abrogated HMVEC activation and blockade of BLT1 inhibited the observed PMN priming activity. We conclude that LTB4 and its ω-oxidation and non-enzymatic metabolites prime PMNs over a range of concentrations and activate HMVECs. These data have expanded the repertoire of causative agents in ALI and post-injury multiple organ failure.
PMCID: PMC3056395  PMID: 20926984
Lipids; ICAM-1; LTB4 receptors; BLT1; BLT2

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