Leukotriene B4 Augments and Restores FcγRs-dependent Phagocytosis in Macrophages*

Phagocytosis by macrophages is essential for host defense, i.e. preventing invasion of pathogens and foreign materials. Macrophages engulf immunoglobulin G (IgG)-opsonized particles through the action of the receptors for the Fc of IgG (FcγRs). Leukotriene B4 (LTB4) is a classical lipid chemoattractant derived from arachidonic acid. Leukotriene B4 receptor 1 (BLT1), a high affinity LTB4 receptor, is expressed in a variety of immune cells such as neutrophils, macrophages, and dendritic cells. Although LTB4 has been shown to enhance macrophage phagocytosis, few studies have investigated the intracellular mechanisms involved in this in detail. Furthermore, there have been no reports of the direct cross-talk between LTB4-BLT1 and IgG-FcγRs signaling. Here, we show that FcγRs-dependent phagocytosis was attenuated in BLT1-deficient macrophages as compared with wild-type (WT) cells. Moreover, cross-talk between LTB4-BLT1 and IgG-FcγRs signaling was identified at the level of phosphatidylinositol 3-OH kinase (PI3K) and Rac, downstream of Syk. In addition, the trimeric Gi protein (Gi) was found to be essential for BLT1-dependent phagocytosis. Surprisingly, we found that LTB4-BLT1 signaling restores phagocytosis in the absence of FcγRs signaling. These data indicate that LTB4-BLT1 signaling plays a pivotal role in macrophage phagocytosis and innate immunity.

Phagocytes such as macrophages prevent invasion of microorganisms and foreign materials by engulfment. Phagocytosis by macrophages is an important component of innate immunity. Efficient phagocytosis is achieved by opsonization of the targets by Ig and complement proteins. Fc␥Rs 3 on phagocytes play an essential role in recognition of opsonized materials (1,2). Binding of IgG-containing immune complexes to Fc␥Rs induces cross-linking of Fc␥Rs and subsequent phosphorylation of tyrosine residues in the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR common ␥-chain (FcR␥) by Src family kinases, such as Lyn and Hck. The phosphorylated ITAM then serves as docking sites for the Src homology 2 (SH2) domain of Syk kinase, subsequently triggering phosphorylation of Syk by Src family kinases (3). Activation of Syk triggers stimulation of the downstream effectors phospholipase C␥, PI3K, and GTPases of the Rho/Rac family. Activation of these downstream effectors results in various cellular events such as phagocytosis, cytokine/chemokine production, oxidative burst, and antibodydependent cellular cytotoxicity (4). However, the precise downstream pathways of Syk via Fc␥R are still unclear. For example, a variety of Syk substrates that are phosphorylated may account for the diverse cellular functions following Fc␥R ligation. Syk (5), PI3K (6), and the Rho family proteins (Rho (7) and Rac and Cdc42 (8 -10)) are all required for Fc␥Rs-dependent phagocytosis. LTB 4 is a classical lipid chemoattractant derived from arachidonic acid by the actions of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP), and LTA 4 hydrolase. LTB 4 attracts neutrophils and eosinophils by the binding to the LTB 4 -specific G-protein-coupled receptor, BLT1. BLT1, originally identified in our laboratory (11), is expressed in a variety of immune cells, including dendritic cells (12), differentiated T cells (13,14), and mast cells (15). Analysis of BLT1-deficient mice revealed the importance in macrophage biology, as atherogenesis was markedly attenuated in BLT1-deficient mice in an apolipoprotein E (apoE)-null background (16). However, although LTB 4 has been shown to activate macrophage phagocytosis, details of the intracellular mechanism of LTB 4 -dependent activation of phagocytosis remain to be elucidated. Recently, Serezani et al. (17) showed that Fc␥RI engagement results in tyrosine phosphorylation of BLT1 by Src and subsequent formation of a molecular complex of Fc␥RI and BLT1 within lipid rafts that drives phagocytic functions in rat alveolar macrophages. However, it remains to be determined whether there is direct cross-talk between LTB 4 -BLT1 and IgG-Fc␥Rs signaling pathways.
In this study, we investigated the function of BLT1 in macrophage phagocytosis using BLT1-deficient mice. BLT1 was necessary for Fc␥R-dependent macrophage phagocytosis. More importantly, we identified cross-talk between LTB 4 -BLT1 and IgG-Fc␥Rs signaling pathways at the level of PI3K and Rac, downstream of Syk. Finally, using FcR␥-deficient mice and retroviral gene transfer techniques, we showed that LTB 4 stimulation alone is able to induce phagocytosis in macrophages that are deficient in Fc␥Rs signaling.

EXPERIMENTAL PROCEDURES
Mice and BMM-BLT1-deficient mice (18) and FcR␥deficient mice (19) in a C57BL/6J background were previously described. All animal studies and procedures were approved by the Ethics Committees for Animal Experiments of Kyushu University. BMM were prepared from bone marrow suspensions from mouse femurs and tibias as described previously (20). Bone marrow cells were cultured in RPMI 1640 medium (WAKO, Osaka, Japan) containing 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin (Nacalai Tesque, Kyoto, Japan), and 30% L929 culture supernatants containing macrophage-colony stimulating factor (M-CSF) for 5 days and then used for the experiments (21).
Phagocytosis Assay-Zymosan-Alexa488 and an opsonizing reagent were purchased from Molecular Probes (Eugene, OR). Opsonization of zymosan-Alexa488 was performed according to the manufacturer's protocol. BMM were seeded on 35-mm glass bottom dishes and then cultured overnight. After incubation with opsonized-zymosan particles for 4 h at 37°C, BMM were washed with PBS, fixed with 4% paraformaldehyde/PBS, and then observed using a confocal laser scanning microscope (LSM510 META, Carl Zeiss, Germany). Where indicated, LTB 4 (final concentration of 100 nM) (Cayman Chemicals, Ann Arbor, MI) was added along with opsonized zymosan particles. For the inhibition assay, BMM were pretreated with the following reagents and times prior to incubation with zymosan particles: Flow Cytometry-BMM were incubated with 0.5 g/ml PE-anti-mCD64 antibody (BioLegend, San Diego), 0.5 g/ml PE-anti-mCD16/CD32 antibody (BD Biosciences), or 2.5 g/ml mIgG 2a -Alexa488 (eBioscience, San Diego) in PBS containing 2% FBS and 0.1% sodium azide. For the binding assay, BMM were pretreated with cytochalasin D (1 or 10 M, Calbiochem) for 30 min prior to incubation with opsonized zymosan particles or IgG beads. Cells were analyzed using a FACSCalibur with CellQuest (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR).
Rac Pulldown Assay-A cDNA encoding the Rac-binding domain of human PAK2 (PAK2-RBD; amino acids 74 -131) in pGEX-4T (23, 24) was introduced into Escherichia coli BL21 (DE3), and GST fusion protein (GST-PAK2-RBD) was expressed. The fusion protein was purified by glutathione-Sepharose 4B (GE Healthcare). For the in vitro pulldown assays, BMM cell lysates were mixed with GST-PAK2-RBD bound to glutathione-Sepharose 4B and allowed to incubate for 30 min at 4°C. After washing three times with lysis buffer, bound proteins were eluted with 10 mM reduced glutathione in 50 mM Tris, pH 8.0. The eluates were resolved by SDS-PAGE (12%), transferred to an Immobilon-P PVDF membrane, and then analyzed by immunoblotting using anti-Rac (BD Biosciences) and anti-GST (Nacalai Tesque) primary antibodies.
Retroviral Transfection-cDNAs encoding WT and mutant (YF) FcR␥ in pMX-IRES-rCD2 were transiently transfected into the packaging cell line, Phoenix (25,26). The cultured media were collected and used as viral suspensions. Bone marrow cells from FcR␥-deficient mice were cultured for 3 days with these viral suspensions and then differentiated into BMM as described above. FcR␥-expressing cells were isolated using MACS cell separators (Miltenyi Biotec, Germany) after incubation with biotinconjugated anti-rCD2 antibody (Cedarlane, Hornby, Canada) and streptavidin microbeads (Miltenyi Biotec). Viral suspensions for the expression of WT Rac and dominantnegative Rac (DN Rac, T17N) were prepared in a same way using pMXs-WT Rac-IRES-DsRed and pMXs-DN Rac-IRES-DsRed (kindly provided by Dr. Y. Fukui, Kyushu University) (27) and used for introduction into bone marrow cells from C57BL/6J mice.

BLT1 Is Required for Macrophage Phagocytosis via Fc␥Rs-
LTB 4 is a known potent chemoattractant for neutrophils, whereas the role of BLT1 in activating macrophages has not been clearly defined. To analyze the function of BLT1 in macrophages, we compared phagocytosis of opsonized zymosan in WT and BLT1-deficient BMM. After 4 h of incubation with opsonized zymosan, WT BMM phagocytosed 4.63 zymosan particles per M, and this increased to 8.37 upon stimulation of the cells with 100 nM LTB 4 (Fig.  1A, left panel). Inhibition of BLT1 using the BLT1 antago-nist CP105696 resulted in a decrease in phagocytosis (to 2.17) (Fig. 1A, left panel). BLT1-deficient BMM phagocytosed 1.33 opsonized zymosan particles per M, which was significantly lower than WT BMM (Fig. 1A, right panel). Neither stimulation of BLT1 by LTB 4 nor inhibition by CP105696 affected phagocytosis of opsonized zymosan in BLT1-deficient BMM.
Invading microorganisms are recognized not only by Fc␥Rs but also by pattern-recognition receptors, which recognize highly conserved invariant molecular patterns FIGURE 1. BLT1 is required for Fc␥R-dependent phagocytosis in macrophages. A and B, Phagocytosis of opsonized zymosan particles (A) and nonopsonized particles (B) in BMM from wild-type (WT) and BLT1-deficient (KO) mice is shown. Number of bound or phagocytosed zymosan particles per macrophage was counted under a confocal laser scanning microscopy. Data represent the means Ϯ S.E. *, p Ͻ 0.05; **, p Ͻ 0.001. C, engulfment of opsonized zymosan particles by BMM was observed using a confocal laser scanning microscope. Phagocytosed zymosan particles were observed as less bright spots (WT, Ϫ) than bright particles that represent zymosan particles bound on the cell surface. Differential interference contrast images are also presented to show similar numbers of the macrophages in each field. Representative photos are shown. such as lipopolysaccharide (LPS) and peptideglycan. Pattern-recognition receptors of zymosan have been reported to be Dectin-1, TLR2, and TLR6 (28). Phagocytosis of nonopsonized zymosan by WT BMM was much lower than that of opsonized particles and was comparable with phagocytosis by BLT1-deficient BMM (Fig. 1B). These results indicated that the effects of Dectin-1, TLR2, and TLR6 in the phagocytosis of opsonized zymosan by BMM were negligible and that the signaling pathways involved in the engulfment of opsonized particles was mediated mainly via Fc␥Rs. Because the phagocytosis of opsonized zymosan by macrophages requires Fc␥Rs (Fc␥RI, Fc␥RII, and Fc␥RIII), we measured cell surface expression of Fc␥Rs in WT and BLT1-deficient BMM. Flow cytometric analysis showed that the expression of Fc␥RI and Fc␥RII/III was similar between WT and BLT1-deficient BMM ( Fig. 2A). Furthermore, expression of functional Fc␥Rs examined by the binding of labeled IgG 1 , IgG 2b (data not shown), and IgG 2a was similar between WT and BLT1-deficient BMM (Fig.  2B). We also confirmed that LTB 4 did not up-regulate the expression of Fc␥Rs (Fig. 2, A and B). We also measured the binding of opsonized zymosan or IgG beads to BMM in the presence of cytochalasin D, which blocks internalization of bound particles by inhibiting actin polymerization. Similar levels of binding were observed between WT and BLT1-deficient BMM (Fig. 2C), which indicated that the attenuated phagocytosis observed in BLT1-deficient BMM was not due to the reduced cell surface expression of Fc␥Rs and binding of opsonized particles.
LTB 4 Potentiates Fc␥R-dependent Rac Activation-To determine whether the cross-talk between the LTB 4 -BLT1 and IgG-Fc␥R signaling pathways resulted in enhanced phagocytosis in macrophages, we examined the activation of Syk induced by incubation with IgG beads and LTB 4 . Syk phosphorylation is one of the initial events induced by Fc␥R cross-linking by opsonized particles (5). Syk was phosphorylated following the addition of IgG beads and LTB 4 in a time-dependent manner, and there was no difference between WT and BLT1-deficient BMM (Fig. 3A). Thus, reduced phagocytosis in BLT1-deficient BMM was most likely not due to alternation in Syk phosphorylation. Rac is intimately involved in macrophage phagocytosis and functions downstream of G i -coupled G-protein-coupled receptors, including BLT1 (29). LTB 4 activated Rac in WT BMM but not in BLT1-deficient BMM (Fig. 3B). The addition of IgG beads alone induced the activation of Rac in WT BMM, and there was a synergistic increase in Rac activation upon co-treatment with LTB 4 (Fig. 3C, left  panel). In contrast, IgG beads-dependent activation of Rac was greatly attenuated in BLT1-deficient BMM, and there was no synergistic activation by LTB 4 (Fig. 3C, right panel). Furthermore, treatment of WT BMM with the BLT1 antagonist CP105696 attenuated Rac activation by IgG beads, and this effect was not apparent in BLT1-deficient BMM (Fig. 3C). These data suggested that LTB 4 was either included in the culture media or produced by macrophages during phagocytosis and activated BLT1 in macrophages. Quantification of LTB 4 in the media during phagocytosis by ELISA and LC-MS/MS (30) revealed trace amounts of LTB 4 15 min after the addition of IgG beads that were below the limit of detection (5 pg/ml) (data not shown). In addition, Rac activation induced by LTB 4 was inhibited by an inhibitor of G i protein, PTX (Fig. 3D). This result indicates that BLT1dependent Rac activation is mediated by G i . Fig. 1, LTB 4 enhanced phagocytosis in WT BMM. To investigate the signaling molecules engaged in phagocytosis that were affected by BLT1 deficiency, we examined the effects of various signal transduction inhibitors on the binding and phagocytosis of opsonized zymosan in WT macrophages. Src family kinase inhibitor (PP2), Syk inhibitor (Piceatannol), PI3K inhibitor (LY294002), and Rac inhibitor (NSC23766) all prevented phagocytosis in the absence of LTB 4 (Fig. 4A, Ϫ). PTX had a partial effect, and the G q inhibitor (YM-254890) had no effect on phagocytosis (Fig. 4A). Under conditions of 100 nM LTB 4 stimulation, inhibition of Src family kinases and Syk did not inhibit phagocytosis of opsonized zymosan (Fig.  4A, ϩ). Thus, LTB 4 abrogated the inhibitory effects of PP2 and Piceatannol. In contrast, in the presence of LTB 4 , LY294002, NSC23766, and PTX were all effective in inhibiting phagocytosis. These data suggested that there is cross-talk between the LTB 4 -BLT1 and IgG-Fc␥Rs signaling pathways at a specific step downstream of Syk. To verify the function of Rac in phagocytosis, we retrovirally introduced WT and dominant-negative (DN) form (T17N) of Rac into WT macrophages, and we then examined the binding and phagocytosis of opsonized zymosan in the presence or absence of LTB 4 . Infection of WT Rac greatly increased phagocytosis in the absence of LTB 4 , and this was further augmented by LTB 4 (Fig. 4B, WT Rac). In cells that expressed DN Rac, phagocytosis was greatly reduced and was not activated by LTB 4 (Fig.  4B, DN Rac).

LTB 4 Enhances Macrophage Phagocytosis-As shown in
LTB 4 Signaling Is Sufficient to Induce Macrophage Phagocytosis in the Absence of Fc␥Rs Signaling-FcR␥ is important for phagocytosis not only in cell activation but also in the efficient assembly and cell surface expression of Fc␥RI and Fc␥RIII, which capture opsonized particles (4). To determine whether LTB 4 -BLT1 signaling was sufficient to induce macrophage phagocytosis in the absence of Fc␥Rs signaling, we investigated phagocytosis in FcR␥-deficient macrophages retrovirally transfected with WT FcR␥ or a mutant form of FcR␥ (YF) in which two tyrosine residues in the ITAM were replaced by phenylalanine. The expression levels of retrovirally transfected WT and YF FcR␥ are shown in supplemental Fig. 1. The YF mutant is deficient in transducing the signals induced by FcR␥ in mast cells and basophils, but surface expression of FcRs remains intact (25,26). Phagocytosis in cells expressing WT FcR␥ was readily apparent and was further enhanced by LTB 4 (Fig.  5A, WT), similar to WT macrophages (Fig. 4A, Ϫ). Phagocytosis in cells expressing FcR␥ YF was reduced as expected, and interestingly, LTB 4 stimulation restored the levels of phagocytosis in FcR␥ YF-infected cells (Fig. 5A,  YF). To verify these observed effects of LTB 4 on phagocytosis in FcR␥ YF cells, we treated cells with LY294002, NSC23766, and PTX. All of the inhibitors suppressed LTB 4 -dependent phagocytosis of opsonized zymosan (Fig.  5B). Taken together, these data indicated that LTB 4 -BLT1 signaling is sufficient to induce phagocytosis when opsonized particles bound to Fc␥Rs.

DISCUSSION
In the host defense mechanism against pathogens, phagocytosis by macrophages plays an essential role. Macrophages engulf IgG-opsonized particles via recognition and  DECEMBER 24, 2010 • VOLUME 285 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 41117 binding by Fc␥Rs. FcR␥ is particularly important for phagocytosis; FcR␥ is a common subunit of Fc␥Rs and is required for the efficient assembly and cell surface expression of Fc␥Rs. FcR␥-deficient mice are more susceptible to infection due to defects in the phagocytosis of IgG-coated particles (31). The intracellular signaling pathways involved in Fc␥Rs-dependent phagocytosis have been studied in detail. Binding of opsonized particles to Fc␥Rs and subsequent cross-linking of the receptors induces ITAM phosphorylation of FcR␥ by Src family kinases. Syk is then recruited to the phosphorylated ITAM and activated by Src family kinases. The downstream events of Syk activation trigger stimulation of PI3K and the Rac GTPase, resulting in phagocytosis (4).

Cross-talk of Fc␥Rs and BLT1 Signaling in Phagocytosis
In addition to Fc␥Rs, certain lipid-derived mediators are known to modulate phagocytosis. Among them, the classical lipid mediator LTB 4 is a potent activator of phagocytes (32). Although LTB 4 has been shown to activate phagocytosis in macrophages, the molecular details of the underlying intracellular mechanism are lacking. Furthermore, there have been no reports of the direct cross-talk between LTB 4 -BLT1 and IgG-Fc␥Rs signaling pathways. In this FIGURE 3. LTB 4 potentiates Rac activation during Fc␥R-dependent phagocytosis. A, effect of BLT1 deficiency on Syk phosphorylation in BMM stimulated with IgG beads and LTB 4 . BMM from WT and BLT1-deficient mice were incubated with IgG beads and 100 nM LTB 4 for the indicated periods of time. Cell extracts were separated by SDS-PAGE and immunoblotted with the indicated antibodies. B and C, effect of BLT1 deficiency on Rac activation. BMM from WT and BLT1-deficient mice were stimulated with IgG beads in the presence or absence of LTB 4 (B and C) and BLT1 antagonist CP105696 (C). Activated Rac (GTP-Rac) was pulled down with GST-PAK followed by immunoblotting. TCL, total cell lysates. GTP-Rac/Rac ratio was normalized to 1 for the WT (unstimulated) (C and D). Data represents the means Ϯ S.D. (n ϭ 3). ##, p Ͻ 0.05 versus #. D, effect of PTX on BLT1-dependent Rac activation in WT BMM. Cells were stimulated with LTB 4 in the presence or absence of PTX. Rac pulldown assay was performed as described above. Data represent the meansϮ S. D. (n ϭ 3). *, p Ͻ 0.05; **, p Ͻ 0.01. study, analysis of the role of BLT1 on Fc␥Rs-dependent phagocytosis using BLT1-deficient macrophages showed that LTB 4 -BLT1 signaling plays a pivotal role in the engulfment of opsonized particles by macrophages. Flow cytometric analysis showed that the attenuated phagocytosis in BLT1-deficient macrophages was not due to the reduced expression of Fc␥Rs on the cell surface or impaired binding of opsonized particles. These results suggested that there may be cross-talk between LTB 4 -BLT1 and IgG-Fc␥Rs signaling pathways. Immunoblotting and inhibition assays showed that LTB 4 -BLT1 signaling intersects with IgG-Fc␥Rs signaling at the level of PI3K and Rac, downstream of Syk. Based on these results, we explored whether LTB 4 -BLT1 signaling was sufficient to restore phagocytosis in the absence of Fc␥Rs signaling. Because FcR␥-deficient macrophages lack surface expression of Fc␥Rs, we adopted a relatively complex experiment in which WT or mutated (YF) FcR␥ was retrovirally introduced into FcR␥-deficient macrophages. FcR␥ YF lacks key tyrosine residues in the ITAM and does not transduce FcRs signals; however, in FcR␥ YF-expressing cells, expression of the receptors for the Fc of IgE (Fc⑀Rs) on the cell surface is intact (25). Thus, the FcR␥ YF mutant does not affect the expression of FcRs. This method enabled us to analyze the roles of LTB 4 -BLT1 signaling in phagocytosis in the absence of Fc␥Rs-dependent intracellular signaling. Phagocytosis in FcR␥ YF-reconstituted macrophages was reduced, as expected. Interestingly LTB 4 stimulation restored this defect in phagocytosis. Based on these results, we propose a model in which cross-talk between IgG-Fc␥Rs and LTB 4 -BLT1 signaling regulates phagocytosis in macrophages (Fig. 6). The data presented here support a novel mechanism whereby G-protein-coupled receptor stimulation restores macrophage phagocytosis in the absence of Fc␥Rs signaling in the following manner. LTB 4 -BLT1 signaling induces both Rac activation through G i protein and PI3K activation, which in turn enhance phagocytosis synergistically with Fc␥R signaling.
One remaining question from this study is the source of LTB 4 during phagocytosis. Macrophages as well as neutrophils express all of the enzymes required for LTB 4 biosynthesis (cytosolic phospholipase A 2 , 5-lipoxygenase, FLAP, and LTA 4 hydrolase) and produce LTB 4 in response to increased intracellular calcium (33). We attempted to quantify LTB 4 production in macrophages during phagocytosis by ELISA and LC-MS/MS techniques (30), but we were only able to detect trace amounts of LTB 4 that were below the detection limit of the assays (5 pg/ml). Although there are no reports on LTB 4 production in response to Fc␥Rs signaling, it is reasonable to consider that phospholipase C␥-dependent calcium mobilization leads to LTB 4 production upon cross-linking of Fc␥Rs by immune complexes. Macrophages are known to express CYP4F18, an LTB 4oxidizing cytochrome P450 enzyme (34). Thus, LTB 4 produced during phagocytosis might be oxidized rapidly by this enzyme under our experimental conditions. Recently, transcellular LTB 4 production is intensively studied. In this case, LTA 4 predominantly produced in phagocytic cells is transcellularly transferred to a variety of cells that express LTA 4 hydrolase, where it is converted to a large amount of LTB 4 (35). Macrophages are adherent cells, and it is rea-sonable to consider that more LTB 4 is produced upon phagocytosis in vivo. LTB 4 production is increased at the site of inflammation (36 -43). In this study, we stimulated macrophages with LTB 4 at a concentration of 100 nM. It has been reported that LTB 4 in excess of 100 nM is produced in human neutrophils by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and formyl-methionylleucyl-phenylalanine (44) and that LTB 4 levels reach 50 nM following stimulation of human monocytes with the calcium ionophore A23187 (45) or LPS (46). Thus, it is possible that macrophages are exposed to LTB 4 above the level in vivo. When macrophages infiltrate into inflammatory sites, they are activated by lipid mediators, including LTB 4 . Activated macrophages engulf pathogens and release cytokines and chemokines to recruit neutrophils. LTB 4 produced by recruited neutrophils results in further activation of macrophages. By this positive feedback loop, macrophage phagocytosis is further accelerated and plays an important role in the clearance of pathogens. The results of several recent studies suggested the importance of BLT1 in atherosclerosis. BLT1 deficiency resulted in reduced atherogenesis in an apoE-deficient background (16), and this effect was explained by reduced level of production of monocyte chemoattractant protein-1 (MCP-1) in BLT1deficient macrophages (47). Future studies investigating the possible role of LTB 4 -BLT1 signaling, especially Rac activation, in MCP-1 production in macrophages would be of particular interest.  Syk activation triggers stimulation of PI3K and a GTPase Rac, resulting in an acceleration of phagocytosis. PI3K and Rac also play a pivotal role in phagocytosis downstream of BLT1. LTB 4 -BLT1 signaling induces both Rac activation through G i protein and PI3K activation, and these activations lead to an augmentation of phagocytosis.