Published online before print August 28, 2003, doi: 10.1161/01.ATV.0000092941.77774.3C
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© 2003 American Heart Association, Inc.
Vascular Biology |
Mechanisms of Leukotriene B4–Triggered Monocyte Adhesion
From the Center for Immunology and Inflammatory Diseases (E.B.F., A.M.T., E.L., A.D.L., R.E.G.), the Cardiology Division (R.E.G.), and the Department of Radiation Oncology (L.M.), Massachusetts General Hospital, Charlestown, Mass; and the Wallenberg Neuroscience Center (A.P., C.O.), Lund University, Lund, Sweden.
Correspondence to Robert E. Gerszten, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital East-8307, 149 13th St, Charlestown, MA 02129. E-mail rgerszten{at}partners.org
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Abstract |
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Objective— Leukotriene B4 (LTB4) has been implicated in the trafficking of monocytes to inflammatory pathologic conditions, such as transplant rejection and atherosclerosis. The aim of this study was to determine the mechanisms by which LTB4 contributes to monocyte capture from the circulation.
Methods and Results— In in vitro and in vivo vascular models, the lipid chemoattractant LTB4 was an equipotent agonist of monocyte adhesion compared with the chemokine monocyte chemoattractant protein-1 (MCP-1). Adenoviral gene transfer of specific endothelial adhesion molecules and blocking monoclonal antibody studies demonstrated that LTB4 triggers both ß1- and ß2-integrin–dependent adhesion. Flow cytometry studies suggested that changes in integrin avidity or affinity, rather than alterations of integrin surface expression, were responsible for the chemoattractant-triggered arrest. Surprisingly, in contrast to the peptide chemokine MCP-1, LTB4 did not activate the phosphoinositide 3-kinase pathway, which is a functionally critical step in chemokine-triggered effector functions.
Conclusions— LTB4 is a potent trigger of monocyte adhesion under flow yet mediates its effects via pathways that appear to differ from peptide chemoattractants. A better understanding of the mechanisms of LTB4-induced monocyte trafficking might shed insight into disease pathogenesis and pinpoint critical steps for therapeutic intervention for multiple human inflammatory pathologic conditions.
Key Words: Please supply key words
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Introduction |
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Leukotriene B4 (LTB4), a product of the 5-lipoxygenase pathway of arachidonic acid metabolism, is associated with multiple inflammatory pathologic conditions, including human arthritis, asthma, allograft rejection, and atherosclerosis.1–4 Recently, investigators have begun to use receptor antagonists to address a causal link between LTB4 production and disease progression in animal models. Several of these studies have suggested a specific role for LTB4 in the contribution of monocytes to inflammatory pathologic conditions. For example, an LTB4 receptor antagonist markedly prolonged cardiac allograft survival in mice.3 Immunohistochemical analysis was most notable for decreased monocyte staining for the ß2-integrin CD11b, a potential marker of monocyte activation, suggesting that LTB4 contributes to monocyte effector functions during allograft rejection. Similarly, LTB4 has been more closely tied to atherogenesis, because plaques produce LTB44 and treatment of atherosclerosis-prone mice with an LTB4 receptor antagonist decreased both monocyte infiltration into lesions and lesion size.5 In the atherosclerosis studies, a pharmacologic receptor antagonist decreased ß2-integrin expression (CD11b) of circulating monocytes in a dose-dependent manner. This finding suggests that LTB4 might contribute to plaque development by enhancing monocyte recruitment secondary to LTB4-induced upregulation of ß2-integrin expression. Of note, however, knockout studies of both ß2 integrins and their endothelial counterligand, intercellular adhesion molecule-1 (ICAM-1), suggest a relatively minor role for this adhesion pathway in atherosclerotic lesion development.6,7 In contrast, the ß1-integrin–vascular cell adhesion molecule-1 (VCAM-1) pathway is critical for lesion development, because a murine model with altered ligand-binding sites of VCAM-1 has diminished lesion formation on a proatherogenic background.7 Thus, the effects of LTB4 on ß2-integrin expression on monocytes are unlikely to account for the demonstrated role of LTB4 in vascular lesion development.
LTB4 binds to its high-affinity receptor BLT1, which is present on neutrophils, eosinophils, and monocytes.8 In the multistep paradigm of leukocyte recruitment, chemoattractants bind to their receptors on leukocytes, thereby converting rolling to firm adhesion via rapid integrin activation.9,10 Flow cytometric studies suggest that LTB4 can trigger the upregulation of overall levels of surface ß2-integrins on neutrophils (polymorphonuclear cells [PMNs]) and monocytes over the course of 10 minutes to several hours.11,12 Modulation of ß2-integrin expression over this time course, however, cannot account for rapid adhesive triggering. The impact of LTB4 on rapid changes in affinity (conformation) or avidity (multivalent binding or clustering) of ß2 or other integrins for their endothelial counterligands is poorly understood. In functional assays under static conditions, lipoxygenase products, including LTB4, increase leukocyte adhesion to endothelial monolayers. Indeed, these studies have suggested that lipoxygenase-triggered leukocyte adhesion might actually be due in large part to direct effects on human endothelial cells, although the relevant adhesive mechanisms have yet to be characterized.13–15
Thus, the mechanisms by which LTB4 contributes to monocyte activation and recruitment are not clearly defined. Here, we specifically addressed whether LTB4 can trigger the rapid changes in integrin affinity and/or avidity that promote monocyte capture under dynamic conditions as seen in the bloodstream, which has not previously been investigated. In light of recent atherosclerosis studies, we also explored the effects of LTB4 on monocyte activation of ß1-integrin–dependent pathways. Finally, in this study we studied signaling pathways triggered by LTB4 in human monocytes and compared these with cascades activated by peptide chemoattractants such as MCP-1. Dissecting the functionally relevant steps in which LTB4 promotes monocyte recruitment has important implications in the development of therapy for vascular diseases.
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Materials
RPMI-1640 medium, Dulbecco’s modified Eagle’s medium, and Dulbecco’s phosphate-buffered saline with or without Ca2+ and Mg2+ were purchased from BioWhittaker, Inc. Fetal bovine serum was obtained from Hyclone, Inc. Recombinant human MCP-1 and LTB4 were purchased from PeproTech, Inc, and Calbiochem, respectively. The specific BLT1 receptor antagonist CP-105,696 was a kind gift from Dr H.J. Showell, Pfizer, Inc, Groton, CT.12 The biochemical inhibitors LY294002, PD98059, SB203580, GF109203X, and BAPTA/AM were obtained from Alexis Biochemicals.
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics Human Cell Systems and cultured in endothelial cell growth medium according to the manufacturer’s instructions. For experimental use in the flow-plate apparatus, HUVECs (passages 2 to 5) were plated at confluence in 0.8-cm2 chambers on fibronectin-coated, plastic tissue-culture slides. HUVECs were infected for 48 hours with the indicated adenoviral vectors, as previously described.16
Recombinant Adenoviruses
The adenoviruses carrying the cDNA for the adhesion molecules E-selectin (AdE-sel), VCAM-1 (AdVCAM-1), and ICAM-1 (AdICAM-1) have been described previously.10,16 Large-scale production of adenovirus and determination of viral titers were performed as previously described.16 Wild-type adenovirus contamination was excluded by the absence of polymerase chain reaction–detectable E1 sequences.
Antibodies
Antibodies for signaling studies included phospho-Akt (Cell Signaling) and total Akt (Cell Signaling), phospho-Erk-1/2 (Cell Signaling), and total Erk-1/2 (Santa Cruz). Leukocyte integrin-blocking antibodies included HP 2.1 (to 
4ß1integrins, Serotec) and TS1/18 (to ß2-integrins, a kind gift from Dr F.W. Luscinskas, Boston, Mass). Antibodies to endothelial adhesion molecules16 included 7A9 and H4/18 (to human E-selectin), Hu5/3 (to human ICAM-1), E1/6 (which recognizes both human and rabbit VCAM-1), and IgG1 and IgG2A control monoclonal antibody (PharMingen). Antibodies to monocyte epitopes included CD11a and CD11b (both fluorescein isothiocyanate labeled), CD49d (allophycocyanin labeled), CD62L (phycoerythrin labeled), and isotype-matched control antibodies (PharMingen). The HUTS-4 antibody specific to the active conformation of ß1-integrins17 was obtained from Chemicon. The BLT1 receptor antibody (Clone 14F11) was obtained from Dr Christer Owman.18
Western Blotting
Freshly isolated human monocytes were resuspended in RPMI-1640/1% bovine serum albumin at 107 cells/mL and equilibrated at 37°C for 10 minutes. Monocytes (106 cells per condition) were left untreated or stimulated with LTB4 or MCP-1 at the indicated concentrations. At the indicated time points, whole-cell lysates were prepared as previously described.19 Precleared cell lysates were boiled for 5 minutes at 100°C in sodium dodecyl sulfate (SDS) sample buffer and resolved by SDS–polyacrylamide gel electrophoresis (PAGE; 10% gels). After transfer onto nitrocellulose membranes (Schleicher and Schuell), membranes were blocked and probed with the indicated antibodies according to the manufacturer’s instructions.
Monocyte Isolation
Human monocytes were purified from healthy donors by Ficoll-Hypaque density gradient centrifugation at 15°C (LSM, Organon Teknika) followed by negative-selection magnetic bead purification (Miltenyi Biotech), as previously described.19 Monocyte preparations were >92% pure as determined by flow cytometry.
Flow Cytometry
Monocytes were resuspended in RPMI-1640/1% fetal bovine serum at 5 to 10x107 cells/mL and equilibrated at 37°C for 10 minutes. Cells (0.5 to 1x106 cells per condition) were either left unstimulated or treated with LTB4 or MCP-1 (100 nmol/L for each) for 1 or 10 minutes. Reactions were stopped by addition of ice-cold 1x phosphate-buffered saline without Ca2+ and Mg2+/0.2% NaN3/2% fetal bovine serum and incubation for 
5 minutes on ice. Monocytes were washed once with fluorescence-activated cell sorting (FACS) buffer (1x phosphate-buffered saline without Ca2+ and Mg2+/0.2% NaN3/1% fetal bovine serum) and blocked for 10 minutes in the same buffer supplemented with 10% human serum. After an incubation of 20 minutes on ice with the indicated fluorescently tagged primary antibodies, cells were washed twice in FACS buffer and fixed in 1x phosphate-buffered saline without Ca2+ and Mg2+/1% paraformaldehyde. For FACS analysis of whole blood, 200-µL aliquots were labeled with the indicated antibodies as described earlier, followed by a 20-minute incubation with FACS lysis solution according to the manufacturer’s instructions (Becton Dickinson). For analysis of ß1-integrin activation with the HUTS-4 antibody, monocytes were either left unstimulated or treated for 1 minute at 37°C in whole blood with either manganese (1 mmol/L), LTB4 (100 nmol/L), or MCP-1 (100 nmol/L). Isotype-matched, fluorescently labeled, nonbinding antibodies were included as controls. Samples were then analyzed with a Becton-Dickinson FACS set to detect fluorescence, forward scatter, and side scatter.
Calcium Flux Studies in Monocytes
Purified human monocytes (6x106) were loaded with 15 µg fura-2 AM (Molecular Probes) in the dark for 30 minutes at 37°C in RPMI-1640/1% fetal bovine serum and washed twice in a buffer containing 145 mmol/L NaCl, 4 mmol/L KCl, 1 mmol/L NaHPO4, 0.8 mmol/L MgCl2, 18 mmol/L CaCl2, 25 mmol/L HEPES, and 22 mmol/L glucose. The data are presented as the relative ratio of fluorescence at 340 and 380 nm after stimulation of monocytes with the indicated agonist (100 nmol/L).10,20
In Vitro Adhesion Assays Under Flow Conditions
Monocyte-EC interactions were analyzed in a commercially available, parallel-plate, laminar-flow chamber (Immunetics) as previously described.19,21 For blocking antibody studies, monocytes were incubated for 10 minutes at 4°C with the indicated antibody (10 µg/mL) and then diluted with perfusion medium to 5x105 cells/mL. For inhibitor studies, monocytes were pretreated with CP-105,696, PD98059, SB203580, GF109203X, or BAPTA/AM (all 10 µmol/L for 10 minutes at 4°C) or LY 294002 (50 µmol/L for 30 minutes at 16°C). When indicated, LTB4 (1 to 100 nmol/L) was added to the flow of incoming monocytes (room temperature). Monocyte adhesion (>3 seconds) was quantified for each coverslip on at least 5 randomly chosen high-power fields 1 minute before and 1 minute after addition of the chemokine. The cells were perfused at an estimated shear stress of 2.0 dyne/cm2 (flow rate of 0.78 mL/min). The entire period of perfusion was recorded on videotape.
Rat Mesentery Adhesion Studies
Intravital microscopy of rat mesentery vasculature was performed similar to previously described methods.22,23 In brief, male Sprague-Dawley rats (400 to 500 g) were anesthetized with a ketamine/xylazine solution. After a midline abdominal incision, the mesentery was exteriorized, placed on a Plexiglas stage, kept moist with a warm saline solution, and maintained at 37°C. A fluorescence microscope (Zeiss Axioplan) was used to visualize the mesenteric microcirculation. The images were projected by a high-resolution video camera (Hamamatsu C2400, intensified CCD) onto a high-resolution video monitor (Sony). Images were recorded on a video recorder (Panasonic SVHS). The rats were allowed to stabilize for 30 minutes after surgery, and areas adjacent to 10- to 50-µm-diameter postcapillary venules were chosen for observation. For each experiment, 2x106 calcein-labeled human monocytes (untreated or pretreated with LTB4 [10 nmol/L at 37°C for 5 minutes]) were superfused through a microcannula (KZ1106, Kent Scientific) into the mesenteric artery. We counted the number of adherent monocytes in at least 30 random 10x fields within 5 minutes of introduction via the cannula, as determined offline by review of the videotape. A leukocyte was judged to be adherent when it had remained stationary for >30 seconds.
Statistical Analysis
Data are expressed as the mean±SD. Statistical comparison of means was performed by a 2-tailed, unpaired, Student’s t test. The null hypothesis was rejected at P<0.05.
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Results |
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As shown in Figure 1, 78% of purified monocytes expressed BLT1, as determined by a recently generated anti-BLT1 monoclonal antibody.18 We examined the effects of LTB4 on monocyte adhesion in a parallel-plate, laminar-flow model. We used adenoviral vectors that allowed us to manipulate specific EC-leukocyte interactions and studied the impact of chemokines on integrin-activated arrest. The adenoviral constructs confer expression of the specific adhesion molecule of interest without affecting other adhesion molecules or perturbing the underlying biology of the ECs.10,16,19 We transfected monolayers with adenoviral constructs for either VCAM-1 or ICAM-1 to assess the relative contribution of ß1- and ß2-integrin–dependent firm adhesion, respectively. To enhance initial monocyte tethering, monolayers were also cotransduced with an adenoviral vector carrying the cDNA for human E-selectin (AdE-sel).10 As shown in Figure 2A, addition of LTB4, at physiologically relevant concentrations markedly augmented monocyte adhesion, comparable to the augmentation seen by the chemokine MCP-1.10 Interestingly, LTB4 markedly enhanced both VCAM-1– and ICAM-1–dependent monocyte firm arrest, suggesting rapid activation of both ß1- and ß2-integrins. To assess whether the increased adhesion conferred by LTB4 was due to effects on the monocytes, ECs, or both, we pretreated endothelial monolayers with LTB4, vigorously washed them, and then performed flow assays. In contrast to prior studies performed in static assays, we saw no enhancement of monocyte arrest under physiological flow conditions (Figure 2A). However, isolated monocytes that were pretreated with LTB4 and then washed showed marked adhesion in the chamber (Figure 2A), suggesting that LTB4 was exerting its proadhesive effect directly on the leukocytes in this system. Pretreatment of monocytes with the specific BLT1 antagonist CP-105,6965,12 inhibited LTB4-triggered arrest of monocytes (Figure 2B), demonstrating that this effect was mediated by BLT1 on monocytes.
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To verify the specificity of the adhesive mechanisms that contribute to monocyte arrest in vitro, we used monoclonal antibodies to block the function of ß1- and ß2-integrins (Figure 2C). To block ß1-integrin–dependent interactions in these experiments, we used an antibody to 4-integrins, which associate with the ß1-subunit.10,16 On monolayers transduced with VCAM-1, LTB4-induced firm arrest was blocked by a monoclonal antibody to 4ß1-integrins, whereas a monoclonal antibody to ß2-integrins failed to abrogate interactions. Conversely, on monolayers transduced with ICAM-1, the monoclonal antibody to ß2-integrins significantly decreased firm adhesion, whereas the monoclonal antibody to 4ß1-integrins had no effect.
The in vitro flow chamber allows one to dissect the contribution of specific adhesion pathways in monocyte recruitment. However, no in vitro system can mimic exactly the complex biology present in the vasculature in vivo. Thus, to validate the physiologic relevance of our findings, we also performed in vivo experiments in the rat mesentery. As shown in Figure 3, LTB4-activated monocytes that were infused into the rat mesentery showed enhanced adhesion in this system as well.
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Prior studies have suggested that LTB4 might enhance recruitment of leukocytes into inflamed tissues by upregulating overall levels of leukocyte surface integrins, specifically ß2-integrin family members.11,12 In parallel with our functional assays, we performed flow cytometry analysis of human monocytes after stimulation with LTB4 or MCP-1 to assess whether increases in leukocyte integrin surface expression might be playing a role in our system. We saw no changes in the levels of ß1- and ß2-integrins on isolated human monocytes at the time point (1 minute) when chemoattractants increased adhesion in our system, nor up to 10 minutes (Figure 4A). However, we could reproducibly demonstrate rapid changes in ß1-integrin activation after agonist exposure, as assessed by the increased binding of an available ß1-integrin activation epitope–specific antibody (Figure 4B). Thus, chemoattractant-triggered changes in leukocyte integrin avidity or affinity were responsible for our functional findings in the adhesion assays under physiological flow.
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Finally, we investigated the signaling pathways by which LTB4 might confer its functional effects. The phosphoinositide 3-kinase (PI 3-K) family has been implicated as a functionally critical pathway in chemokine receptor signaling.19,24–26 Chemokines activate PI 3-K isoforms at nanomolar concentrations. Inhibition of the PI 3-K axis, either biochemically or by genetic manipulation, decreases chemokine-induced chemotaxis and adhesion of leukocyte subsets, including monocytes. As previously described, the chemokine MCP-1 activated PI 3-K, as assessed by Western blotting for the phosphorylated form of the obligate downstream target, Akt19 (Figure 5). Surprisingly, we did not see any evidence of PI 3-K pathway activation triggered by LTB4, although calcium signaling and extracellular signal–regulated kinase activation were robustly activated by both agonists. Functional adhesion assays performed in parallel with the signaling studies showed no effect of the specific PI 3-K inhibitor LY294002 on LTB4-triggered monocyte arrest. In contrast, LY294002 inhibited monocyte firm adhesion triggered by MCP-1, as previously described19 (Figure 6). We used inhibitors of the mitogen-activated protein kinase pathway, PD98059 (mitogen-activated protein kinase/extracellular-signal-related kinase kinase inhibitor) and SB203580 (p38 mitogen-activated protein kinase inhibitor), as well as an inhibitor of protein kinase C (GF109203X), because these pathways have been implicated in chemoattractant signaling by other investigators.27,28 We also inhibited calcium signaling with BAPTA-AM, an intracellular calcium chelator. However, in our system, these agents did not inhibit firm arrest triggered by either LTB4 or MCP-1 (data not shown).
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Discussion |
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Although recent studies of LTB4 have underscored its importance in multiple inflammatory models, the mechanisms by which LTB4 enhances leukocyte recruitment remain incompletely defined. Here we show that LTB4 is sufficient to trigger monocyte firm arrest under physiologic flow in vitro and in vivo. Studies with adenoviral vectors and blocking monoclonal antibodies showed that not only ICAM-1/ß2-integrin–dependent adhesion but also VCAM-1/ß1-integrin–dependent adhesion is augmented by LTB4. Furthermore, flow cytometry studies suggest that the adhesion triggered by LTB4 is not dependent on increases in overall levels of monocyte surface integrins but rather due to effects on leukocyte integrin affinity and/or avidity for endothelial ligands. Finally, both signaling studies and functional assays show that in contrast to the classic peptide chemokine MCP-1, LTB4 appears to trigger effector functions in purified human monocytes in a PI 3-K–independent manner.
The data presented here are consistent with a growing body of evidence implicating LTB4 in leukocyte recruitment. Prior studies of isolated human neutrophils and monocytes have suggested that LTB4 can modulate overall levels of ß2-integrins for 10 minutes to several hours.11,12 Our data therefore extend prior work, because we used functional assays that mimicked the dynamic conditions in the bloodstream. This system is extremely sensitive to rapid changes in integrin activation that precede changes in levels of overall integrin surface expression. Thus, in addition to modulation of integrin surface levels, LTB4 enhances monocyte integrin activation, a more rapidly occurring step relevant to monocyte accumulation.
Recent studies suggest that specific chemoattractants confer specific effector functions in leukocytes.10,29,30 For example, certain chemokines appear more efficiently coupled to adhesion pathways, whereas others couple more effectively to pathways specific for chemotaxis. It is believed that this type of specialization of chemokines and chemokine receptors has been honed for "the opulence of host protection."30 Rather than a system in which chemoattractant receptors subserve multiple functions, the efficiency of inflammatory cell infiltration might be increased by a "division of labor" between receptors, with each subserving specific, nonredundant functions. Thus, it was not entirely anticipated that LTB4 would trigger monocyte adhesion under physiologic flow. Understanding the specific steps in which LTB4 enhances monocyte recruitment has important implications in defining the therapeutic windows for intervention in vascular disease.
We also demonstrate previously unappreciated effects of LTB4 on monocyte ß1-integrin–dependent adhesion. This finding is particularly noteworthy with respect to atherosclerosis, wherein ß1-integrin–mediated monocyte recruitment appears to be most critical to lesion development. Adhesion studies performed with explanted carotid arteries from atherosclerosis-prone mice have shown a critical role for 4ß1-integrins but not ß2-integrins on monocyte recruitment.29 Furthermore, knockout studies have shown surprisingly modest effects of loss of the ICAM-1/ß2-integrin pathway, in marked contrast to the VCAM-1/ß1-integrin pathway, in atherosclerotic lesion development.6,7,31 Thus, LTB4-triggered rapid changes of monocyte ß1-integrins, as we have demonstrated, might be the critical step in monocyte recruitment induced by the LTB4 produced by atherosclerotic plaques.4 The importance of ß1-integrins/VCAM-1 in recruitment to atherosclerotic lesions might also explain why PMNs are rarely observed in lesions, because PMNs express BLT1 but have low levels of the VCAM-1 counterligand 4ß1-integrin.29 Reduction of atherosclerotic lesion size by an LTB4 receptor antagonist has been postulated to result from inhibition of LTB4-induced upregulation of monocyte ß2-integrin expression. Although we hypothesize that the LTB4 effect on ß1-integrin activation is more relevant for monocyte capture into lesions, LTB4 effects on ß2-integrins might participate in more chronic aspects of lesion development. Furthermore, LTB4-triggered rapid affinity or avidity changes of monocyte ß2-integrins might be critical for initial monocyte capture in other inflammatory pathologic conditions.
The intracellular signaling events that underlie LTB4-triggered functional phenomena are incompletely understood. One group has reported that LTB4-dependent lysosomal enzyme release in rat basophilic leukemia (RBL) cells transfected with BLT1 is resistant to the PI 3-K inhibitor wortmannin,32 although others have reported contradictory findings in a similar system.33,34 Studies have also demonstrated that wortmannin is able to inhibit LTB4-triggered chemotaxis of RBL cells,32 although at concentrations (100 nmol/L) that might invoke nonspecific inhibition of other pathways.35 In freshly isolated human monocytes, as opposed to cultured cell lines, it is surprising that LTB4-triggered activation does not appear to involve the PI 3-K pathway, as demonstrated by both our signaling and functional assays. This is in contrast to peptide chemokines such as MCP-1, whose activation of PI 3-K is clearly necessary for both chemotaxis and adhesion in human monocytes. We believe that the critical difference between our work and prior studies is our use of primary human monocytes. We have previously published significant differences in PI 3-K signaling between primary human monocytes and other cell types.19 Other investigators have also suggested important limitations of leukemic cell lines to study PI 3-K–dependent pathways, including abnormal localization of PI 3-K targets such as Akt and high basal levels of PI 3-K activation due to the lack of key counterregulatory pathways, such as PTEN and SHIP.36 The spectrum of signaling pathways utilized by LTB4 in distinct cell types and the functionally relevant pathways by which LTB4 triggers adhesion specifically in monocytes, remain the subjects of future investigation.
Unanswered and also of obvious interest is whether the signaling pathways activated by MCP-1 and LTB4 converge. If the signaling pathways were completely nonredundant, one might expect that coadministration of the 2 ligands would synergistically trigger adhesion. Interestingly, in preliminary studies in our laboratory, we did not observe synergy in terms of adhesion triggered by coadministration of MCP-1 and LTB4 across a range of concentrations. In addition, pretreatment of monocytes with the specific BLT1 antagonist CP-105,696 did not impair MCP-1–triggered adhesion, thus suggesting that MCP-1–triggered responses are not downstream of BLT1 in this functional context. We anticipate that future studies with unbiased protein display technologies, specifically phosphoproteomics, will shed insight into the differential signaling mediated by lipid versus peptide chemoattractants. Finally, our data suggest that emerging therapeutic interventions aimed specifically at inhibiting the PI 3-K axis might have variable effects, depending on the local chemoattractant environment in a given pathologic process.
In contrast to prior experiments performed under static conditions, we did not observe augmented adhesion after direct activation of ECs with LTB4, followed by perfusion with monocytes. The presence of shear and differences between leukocyte subsets and endothelial preparations might have contributed to the findings in the present study. A more thorough characterization of BLT1 and BLTR2 receptor density and function in ECs in vitro and in vivo also merits future evaluation.
In summary, these results indicate that LTB4 can trigger rapid changes in the avidity of both ß1- and ß2-integrins for their counterligands, thus enhancing monocyte capture under physiologic flow conditions. The intracellular signaling pathways appear to differ from the relevant pathways triggered by peptide chemokines such as MCP-1. Identification of the molecular mechanisms of monocyte recruitment by LTB4 could provide future targets for therapeutic intervention in a host of important human inflammatory pathologic conditions.
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Acknowledgments |
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The authors gratefully acknowledge support from the NIH to S.S., A.T., A.D.L., and R.E.G. E.F. is a Feodor Lynen Research Fellow of the Alexander von Humboldt Foundation. The authors wish to thank Sabina Islam, Seddon Thomas, and Christoph Hess for assistance with the flow cytometry studies. We thank Anthony Rosenzweig, Jennifer Allport, and Thomas Force for thoughtful discussions.
Received June 18, 2003; accepted July 31, 2003.
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References |
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1. Griffiths RJ, Pettipher ER, Koch K, Farrell CA, Breslow R, Conklyn MJ, Smith MA, Hackman BC, Wimberly DJ, Milici AJ, et al. Leukotriene B4 plays a critical role in the progression of collagen-induced arthritis. Proc Natl Acad Sci U S A. 1995; 92: 517–521.
2. Turner CR, Breslow R, Conklyn MJ, Andresen CJ, Patterson DK, Lopez-Anaya A, Owens B, Lee P, Watson JW, Showell HJ. In vitro and in vivo effects of leukotriene B4 antagonism in a primate model of asthma. J Clin Invest. 1996; 97: 381–387.[Medline] [Order article via Infotrieve]
3. Weringer EJ, Perry BD, Sawyer PS, Gilman SC, Showell HJ. Antagonizing leukotriene B4 receptors delays cardiac allograft rejection in mice. Transplantation. 1999; 67: 808–815.[CrossRef][Medline] [Order article via Infotrieve]
4. De Caterina R, Mazzone A, Giannessi D, Sicari R, Pelosi W, Lazzerini G, Azzara A, Forder R, Carey F, Caruso D, et al. Leukotriene B4 production in human atherosclerotic plaques. Biomed Biochim Acta. 1988; 47: S182–S185.[Medline] [Order article via Infotrieve]
5. Aiello RJ, Bourassa PA, Lindsey S, Weng W, Freeman A, Showell HJ. Leukotriene B4 receptor antagonism reduces monocytic foam cells in mice. Arterioscler Thromb Vasc Biol. 2002; 22: 443–449.
6. Kubo N, Boisvert WA, Ballantyne CM, Curtiss LK. Leukocyte CD11b expression is not essential for the development of atherosclerosis in mice. J Lipid Res. 2000; 41: 1060–1066.
7. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos JC, Connelly PW, Milstone DS. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001; 107: 1255–1262.[Medline] [Order article via Infotrieve]
8. Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature. 1997; 387: 620–624.[CrossRef][Medline] [Order article via Infotrieve]
9. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994; 76: 301–314.[CrossRef][Medline] [Order article via Infotrieve]
10. Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone MA Jr, Luster AD, Luscinskas FW, Rosenzweig A. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature. 1999; 398: 718–723.[CrossRef][Medline] [Order article via Infotrieve]
11. Vaddi K, Newton RC. Regulation of monocyte integrin expression by ß-family chemokines. J Immunol. 1994; 153: 4721–4732.[Abstract]
12. Showell HJ, Conklyn MJ, Alpert R, Hingorani GP, Wright KF, Smith MA, Stam E, Salter ED, Scampoli DN, Meltzer S, Reiter LA, Koch K, Piscopio AD, Cortina SR, Lopez-Anaya A, Pettipher ER, Milici AJ, Griffiths RJ. The preclinical pharmacological profile of the potent and selective leukotriene B4 antagonist CP-195543. J Pharmacol Exp Ther. 1998; 285: 946–954.
13. Patricia MK, Kim JA, Harper CM, Shih PT, Berliner JA, Natarajan R, Nadler JL, Hedrick CC. Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells. Arterioscler Thromb Vasc Biol. 1999; 19: 2615–2622.
14. Palmblad J, Lerner R, Larsson SH. Signal transduction mechanisms for leukotriene B4 induced hyperadhesiveness of endothelial cells for neutrophils. J Immunol. 1994; 152: 262–269.[Abstract]
15. Hoover RL, Karnovsky MJ, Austen KF, Corey EJ, Lewis RA. Leukotriene B4 action on endothelium mediates augmented neutrophil/endothelial adhesion. Proc Natl Acad Sci U S A. 1984; 81: 2191–2193.
16. Gerszten R, Luscinskas F, Ding H, Dichek D, Stoolman L, Gimbrone M Jr, Rosenzweig A. Adhesion of memory lymphocytes to VCAM-1-transduced human vascular endothelial cells under simulated physiological flow conditions in vitro. Circ Res. 1996; 79: 1205–1215.
17. Luque A, Gomez M, Puzon W, Takada Y, Sanchez-Madrid F, Cabanas C. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (9355–425) of the common ß1 chain. J Biol Chem. 1996; 271: 11067–11075.
18. Pettersson A, Boketoft A, Sabirsh A, Nilsson NE, Kotarsky K, Olde B, Owman C. First-generation monoclonal antibodies identifying the human leukotriene B4 receptor-1. Biochem Biophys Res Commun. 2000; 279: 520–525.[CrossRef][Medline] [Order article via Infotrieve]
19. Gerszten RE, Friedrich EB, Matsui T, Hung RR, Li L, Force T, Rosenzweig A. Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions. J Biol Chem. 2001; 276: 26846–26851.
20. Wheeler ME, Luscinskas FW, Bevilacqua MP, Gimbrone MA Jr. Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion. J Clin Invest. 1988; 82: 1211–1218.
21. Friedrich EB, Sinha S, Li L, Dedhar S, Force T, Rosenzweig A, Gerszten RE. Role of integrin-linked kinase in leukocyte recruitment. J Biol Chem. 2002; 277: 16371–16375.
22. Scalia R, Gefen J, Petasis NA, Serhan CN, Lefer AM. Lipoxin A4 stable analogs inhibit leukocyte rolling and adherence in the rat mesenteric microvasculature: role of P-selectin. Proc Natl Acad Sci U S A. 1997; 94: 9967–9972.
23. Jain RK, Munn LL, Fukumura D. Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer. 2002; 2: 266–276.[CrossRef][Medline] [Order article via Infotrieve]
24. Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, Sozzani S, Mantovani A, Altruda F, Wymann MP. Central role for G protein-coupled phosphoinositide 3-kinase-
in inflammation. Science. 2000; 287: 1049–1053.
25. Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzki I, Joza N, Mak TW, Ohashi PS, Suzuki A, Penninger JM. Function of PI3K- in thymocyte development, T cell activation, and neutrophil migration. Science. 2000; 287: 1040–1046.
26. Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D. Roles of PLC-ß2 and -ß3 and PI3K- in chemoattractant-mediated signal transduction. Science. 2000; 287: 1046–1049.
27. Heit B, Tavener S, Raharjo E, Kubes P. An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J Cell Biol. 2002; 159: 91–102.
28. Carnevale KA, Cathcart MK. Protein kinase C-ß is required for human monocyte chemotaxis to monocyte chemoattractant protein 1. J Biol Chem. 2003; 278: 25317–25322.
29. Huo Y, Weber C, Forlow SB, Sperandio M, Thatte J, Mack M, Jung S, Littman DR, Ley K. The chemokine KC, but not monocyte chemoattractant protein-1, triggers monocyte arrest on early atherosclerotic endothelium. J Clin Invest. 2001; 108: 1307–1314.[CrossRef][Medline] [Order article via Infotrieve]
30. Rollins BJ. Chemokines and atherosclerosis: what Adam Smith has to say about vascular disease. J Clin Invest. 2001; 108: 1269–1271.[CrossRef][Medline] [Order article via Infotrieve]
31. Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, Beaudet AL. P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med. 2000; 191: 189–194.
32. Haribabu B, Zhelev DV, Pridgen BC, Richardson RM, Ali H, Snyderman R. Chemoattractant receptors activate distinct pathways for chemotaxis and secretion: role of G-protein usage. J Biol Chem. 1999; 274: 37087–37092.
33. Yokomizo T, Izumi T, Shimizu T. Leukotriene B4: metabolism and signal transduction. Arch Biochem Biophys. 2001; 385: 231–241.[CrossRef][Medline] [Order article via Infotrieve]
34. Ito N, Yokomizo T, Sasaki T, Kurosu H, Penninger J, Kanaho Y, Katada T, Hanaoka K, Shimizu T. Requirement of phosphatidylinositol 3-kinase activation and calcium influx for leukotriene B4-induced enzyme release. J Biol Chem. 2002; 277: 44898–44904.
35. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994; 269: 5241–5248.
36. Astoul E, Edmunds C, Cantrell DA, Ward SG. PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models. Trends Immunol. 2001; 22: 490–496.[CrossRef][Medline] [Order article via Infotrieve]
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