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.
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.
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.
Cell trafficking; neutrophils; kinases; autoimmunity
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.
The purpose of this review is to summarize the role that murine models of arthritis are playing in the understanding of human rheumatoid arthritis and how leukotriene B4 (LTB4) is emerging as an important target in this field. Both the collagen-induced arthritis (CIA) model and the K/BxN serum transfer arthritis model have contributed to outline the potential mechanisms involved in inflammatory arthritis. Indeed, the CIA model has contributed to the development of effective anti-TNF and anti-IL-1β based treatments for RA that are currently in the clinic. Many recent studies in mouse models have suggested a critical role for LTB4 and its receptors in the development of inflammatory arthritis. Inhibitors of LTB4 biosynthesis as well as LTB4 receptors are protective in mouse models of RA and mice deficient in the LTB4 biosynthetic enzymes or LTB4 receptors are resistant to disease development suggesting several promising targets for RA in this pathway.
Rheumatoid Arthritis; Leukotriene B4 receptors; arthritis mouse models; FLAP
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.
arachidonic acid; neutrophil influx; knock-out; sex-related; chemotaxis
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.
Lipids; ICAM-1; LTB4 receptors; BLT1; BLT2
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.
arthritis; neutrophils; mouse model; inflammation; autoantibodies
Neuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary goal of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects.
Serum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU.
NMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux.
NMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors.
Leukotriene A4 Hydrolase (LTA4H) is a pro-inflammatory enzyme which generates the inflammatory mediator leukotriene B4 (LTB4). LTA4H also possesses aminopeptidase activity with unknown substrate and physiological significance. We identified the neutrophil chemoattractant, Pro-Gly-Pro (PGP), as this physiological substrate. PGP is a biomarker for chronic obstructive pulmonary disease (COPD), and is implicated in neutrophil persistence in the lung. In acute neutrophil driven inflammation, PGP was degraded by LTA4H, which facilitated the resolution of inflammation. In contrast, cigarette smoke, a major risk factor for the development of COPD, selectively inhibited LTA4H aminopeptidase activity, which led to the accumulation of PGP and neutrophils. These studies imply that therapeutic strategies that inhibit LTA4H to prevent LTB4 generation may not reduce neutrophil recruitment because of elevated PGP.
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.
Leukotriene B4 (LTB4), generated from arachidonic acid following lipoxygenase activity by a variety of inflammatory leucocytes, has been shown to be present in synovial fluid from patients with active rheumatoid arthritis. It does not persist as such, being converted to less active metabolites. The role of LTB4 as one of the natural mediators of inflammation is discussed.
Leukotriene B4 (LTB4) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane–spanning, G protein–coupled receptor, called BLTR (LTB4 receptor), has recently been identified as an LTB4 receptor. To determine if BLTR is the sole receptor mediating LTB4-induced leukocyte activation and to determine the role of LTB4 and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB4-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant–receptor pairs in vitro, LTB4 and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB4-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB4 in vivo.
receptors; leukotriene; chemotactic factors; inflammation mediators; knockout
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.
asthma; inflammation; monocytes; leukotrienes; receptors
cysteinyl-leukotrienes (LTC4, LTD4,
LTE4) are critical bronchoconstrictor and
eosinophilotactic mediators in asthma while LTB4 is a
potent neutrophil chemoattractant. Glucocorticosteroids are front line
anti-inflammatory treatment for asthma but the evidence that they
reduce leukotriene (LT) synthesis in vivo is poor.
randomised, double blind, placebo controlled, crossover trial
immunoassays were used to measure ex vivo synthesis of LTC4
and LTB4 by calcium ionophore stimulated blood leucocytes and bronchoalveolar lavage (BAL) cells of eight normal subjects and
eight patients with mild allergic asthma 4-6 hours after intravenous administration of a single 100 mg dose of methylprednisolone.
stimulated synthesis of LTC4 (but not LTB4) in
blood granulocytes tended to be higher in asthmatic subjects (mean 9.7 ng/106 cells) than in normal subjects
(4.2 ng/106 cells; p = 0.08) and intravenous
methylprednisolone reduced synthesis of LTC4 (but not
LTB4) to normal levels (2.9 ng/106 cells; 95%
CI for the reduction 1.0 to 12.5 ng/106 cells; p = 0.03).
In blood mononuclear cells methylprednisolone reduced LTC4
synthesis in asthmatic subjects from 1.26 to 0.79 ng/106
cells (95% CI for the reduction 0.26 to 0.79, p = 0.014) and tended to
reduce LTC4 synthesis in normal subjects from 1.51 to 0.86 ng/106 cells (p = 0.08). Methylprednisolone also
significantly reduced synthesis of LTB4 in mononuclear
cells from both subject groups (p = 0.014). It had no effect on LT
synthesis in BAL cells from either group nor on LT levels in BAL fluid.
methylprednisolone can reduce synthesis of leukotrienes in blood
granulocytes and mononuclear cells within six hours of a single
Neutrophils can be "primed" for an enhanced respiratory burst by lipopolysaccharide (LPS) in concentrations measurable in patients with septic shock. Leukotriene B4 (LTB4) is the primary eicosanoid product of neutrophils and is felt to be a mediator of host defense and inflammation. We investigated the in vitro effects of LPS on neutrophil production of LTB4 and the omega-oxidation metabolites of LTB4. Incubation of neutrophils with LPS in concentrations ranging from 0.01 to 100 ng/ml did not result in production of LTB4 or metabolites in the absence of a second stimulus. Priming neutrophils with LPS and then stimulating with opsonized zymosan, phorbol-myristate-acetate or a low concentration of the calcium ionophore A23187 resulted in enhanced production of LTB4. LPS priming of neutrophils occurred in a concentration dependent manner. LPS did not result in LTB4 production in response to the chemoattractant peptide FMLP. LPS priming of neutrophils had no effect on cytosolic calcium concentrations of resting or zymosan-stimulated cells. These results suggest that LPS might effect host defense and tissue injury by potentiating the effect of other stimulants on neutrophil production of LTB4. This LPS induced enhancement may represent an important pathogenetic pathway in patients with gram negative sepsis.
Lipid composition plays an important part in the structural and metabolic functions of cell membranes. In particular the production of inflammatory mediators such as prostaglandins and leukotrienes is dependent on polyunsaturated fatty acid precursors. Neutrophil leucocytes participate in inflammatory processes by their phagocytic and killing activities which can be monitored by measuring the photon emission (chemiluminescence). Chemiluminescence was measured in a luminol dependent system after stimulation by either particulate (zymosan) or soluble (phorbol myristate acetate) stimulus in a group of 10 patients with rheumatoid arthritis before and 21 and 45 days after treatment with a diet supplemented with eicosapentaenoic and docosahexaenoic acids. Ten patients with rheumatoid arthritis continuing their usual diet were used as control subjects. A progressive reduction of chemiluminescence stimulated by zymosan and phorbol myristate acetate was found in the patients treated with fish oil supplementation. This result correlated well with the reduction in erythrocyte sedimentation rate and an improvement of clinical parameters. The effects of fish oil derived lipids on neutrophil chemiluminescence are probably due to a change of the lipid composition of the cell membrane which is dependent on the esterification of eicosapentaenoic acid and docosahexaenoic acid in cellular membrane phospholipids. The modification of membrane lipid composition seems to interact in a non-specific way with the metabolic activation of neutrophils during phagocytosis.
Unmethylated CpG-oligodeoxynucleotides (ODNs) are generally thought of as potent adjuvants with considerable therapeutic potential to enhance immune responses against microbes and tumors. Surprisingly, certain so-called stimulatory CpG-ODNs strongly inhibited the effector phase of inflammatory arthritis in the K/BxN serum transfer system, either preventively or therapeutically. Also unexpected was that the inhibitory influence did not depend on the adaptive immune system cells mobilized in an immunostimulatory context. Instead, they relied on cells of the innate immune system, specifically on cross talk between CD8α+ dendritic cells and natural killer cells, resulting in suppression of neutrophil recruitment to the joint, orchestrated through interleukin-12 and interferon-γ. These findings highlight potential applications of CpG-ODNs and downstream molecules as antiinflammatory agents.
During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.
We previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: ±1 nM), similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the tick's ability to interfere with proper neutrophil function in inflammation.
These elements suggest that Ir-LBP is a “scavenger” of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.
The process of neutrophil adhesion to and migration through the microvascular endothelium, an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractants. Although both chemotactic peptides and lipid mediators enhance neutrophil adherence in vitro and in vivo, the mechanism(s) involved in the interaction between circulating neutrophils and microvascular endothelial cells is still not completely understood. In a microtiter well adherence assay, the chemotactic peptides, FMLP and C5a, and the lipid mediators, leukotriene B4 (LTB4) and platelet activating factor (PAF), enhanced human neutrophil adherence to cultured human microvascular endothelial cells as well as to human umbilical vein endothelial cells in a dose-dependent manner with a rapid time course. This stimulated adhesive interaction between neutrophils and cultured human endothelial cells was dependent on the expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control neutrophils. All four mediators enhanced expression of the glycoprotein family on the surface of normal neutrophils as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the glycoprotein common beta subunit. In addition, TS1/18 inhibited up to 100% the adherence of normal neutrophils to endothelial cells stimulated by maximal concentrations of FMLP, C5a, LTB4, or PAF. Moreover, HL-60 cells, human promyelocytic leukemia cells, neither increased glycoprotein surface expression nor adherence in response to stimulation. Thus, peptide and lipid mediators of the acute inflammatory response appear to enhance adherence of circulating neutrophils to the microvascular endothelium by a mechanism dependent on expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface.
Acetyl glyceryl ether phosphorylcholine (AGEPC) and leukotriene B4 (LTB4) induce concentration-dependent neutrophil aggregation. On a molar basis, LTB4 is approximately 10 to 100 times more potent than AGEPC. AGEPC-induced aggregation is attenuated by two inhibitors of arachidonate lipoxygenation, eicosatetraynoic acid and nordihydroguaiaretic acid, and to a lesser extent by the cyclooxygenase inhibitor, indomethacin. LTB4-induced aggregation is not readily reduced by the above inhibitors of arachidonic acid metabolism. Reverse phase high performance liquid chromatography, coupled with selective ion gas chromatography/mass spectrometry, shows that AGEPC stimulates neutrophils to synthesize sufficient LTB4 to account for the AGEPC response. In addition, the rate of LTB4 biosynthesis in response to AGEPC correlates well with the rate of AGEPC- and/or LTB4-induced neutrophils aggregation, and desensitization experiments indicate that AGEPC and LTB4 cross-desensitize. These data suggest that AGEPC-induced neutrophil aggregation may be mediated by LTB4.
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.
Neutrophils play a critical role in the clearance of bacteria from the lung and other organs by their capacity for phagocytosis and killing. Previously, we identified an important role for the leukotrienes in rat alveolar macrophage phagocytosis of Klebsiella pneumoniae. In this report, we explored the possibility that the leukotrienes play an important role in phagocytosis by neutrophils as well. Inhibition of endogenous leukotriene synthesis by 5-lipoxygenase knockout in mice or by pharmacologic means in human peripheral blood neutrophils attenuated phagocytosis of opsonized K. pneumoniae. Reduced phagocytosis was also observed in human neutrophils pretreated with a leukotriene B4 receptor but not a cysteinyl-leukotriene receptor antagonist. While leukotriene B4 reconstituted defective phagocytosis in leukotriene-deficient neutrophils and enhanced phagocytosis in neutrophils capable of leukotriene synthesis, leukotriene C4, leukotriene D4, 5-hydroperoxyeicosatetraenoic acid, and 5-oxo-eicosatetraenoic acid were ineffective. To determine the opsonin dependence of the leukotriene B4 augmentation of phagocytosis, we assessed the ability of leukotriene B4 to modulate neutrophil phagocytosis and the adherence of sheep erythrocytes opsonized with immunoglobulin G or the complement fragment C3bi. While leukotriene B4 augmented both Fc receptor- and complement receptor-mediated phagocytosis, increased adherence to leukotriene B4-treated neutrophils was limited to complement opsonized targets. In conclusion, we have identified a novel role for leukotriene B4 in the augmentation of neutrophil phagocytosis mediated by either the Fc or complement receptor.
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.
BACKGROUND: The movement of neutrophils into the colonic mucosa in ulcerative colitis is induced by chemokines including interleukin 8 (IL8) and leukotriene B4 (LTB4). AIMS: To compare the ability of mucosa from ulcerative colitis patients and controls to stimulate neutrophil movement, to define the contribution of LTB4 to this, and to define the relative biological importance of LTB4 and IL8. PATIENTS: Resected mucosa was obtained from seven control patients and 10 patients with ulcerative colitis. METHODS: Mucosal homogenate supernatants were used to stimulate isolated neutrophils and the effect assessed by the neutrophil shape change response. Responses were inhibited with either the LTB4 receptor antagonist SC41930- or neutralising anti-IL8 antibody. LTB4 was extracted and assayed by RIA. RESULTS: Homogenate supernatants from inflamed mucosa were more bioactive (median 1.2 mg/ml-1 induced 50% response) than those from uninflamed mucosa (4.25 mg/ml-1 induced 50% response; difference 2.8 mg/ml-1 (96.5% CI 0.5 to 6.1, p < 0.05). Maximal inhibition by SC41930 of the response was significantly greater in inflamed mucosa (54% median) than in uninflamed mucosa (34%). This inhibition correlated with the level of immunoreactive LTB4 (r = 0.78). Anti-IL8 reduced bioactivity of homogenate supernatants from inflamed mucosa (at 1:10 dilution) by 11.4% (IQR 1.2 to 51.8, p = 0.021) whereas SC41930 reduced it by 54.8% (35.6 to 77.5, p = 0.008). CONCLUSIONS: Inflamed colonic mucosa releases more neutrophil movement inducing bioactivity than uninflamed mucosa, and has greater LTB4 dependent activity. It yields both IL8 and LTB4 dependent activity but greater LTB4 dependent activity.