The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 5454 -5461
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peres, C. M.
Right arrow Articles by Peters-Golden, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peres, C. M.
Right arrow Articles by Peters-Golden, M.

Specific Leukotriene Receptors Couple to Distinct G Proteins to Effect Stimulation of Alveolar Macrophage Host Defense Functions1

Camila M. Peres*,{ddagger}, David M. Aronoff{dagger}, Carlos H. Serezani*, Nicolas Flamand*, Lucia H. Faccioli{ddagger} and Marc Peters-Golden2,*

* Division of Pulmonary and Critical Care Medicine, and {dagger} Division of Infectious Diseases, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI 48109; and {ddagger} Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto São Paulo, Brazil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB4 and LTD4 also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both G{alpha}i and G{alpha}q proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB4 and LTD4. LTB4 but not LTD4 reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of Fc{gamma}R-mediated phagocytosis and bacterial killing by LTB4 was also PTX-sensitive, whereas that induced by LTD4 was not. LTD4 and LTB4 induced Ca2+ and intracellular inositol monophosphate accumulation, respectively, highlighting the role of G{alpha}q protein in mediating PTX-insensitive LTD4 enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific G{alpha}q/11 and G{alpha}i3 subunits, but not G{alpha}i2 or Gbeta{gamma}, in LTB4-enhanced phagocytosis. The selective importance of G{alpha}q/11 protein was also demonstrated in LTD4-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The lipid mediators leukotrienes (LT)3 are derived from the 5-lipoxygenase pathway of arachidonic acid metabolism. They are comprised of two specific classes, namely LTB4 and LTC4, LTD4, or LTE4; the latter are collectively known as cysteinyl LTs (cysLTs). LTB4 is a potent neutrophil chemoattractant (1), and the cysLTs are best known for their bronchoconstrictive properties, which account for the bioactivity long known as slow reacting substance of anaphylaxis (2). However, both LT classes are now recognized to enhance a variety of leukocyte functions, including adherence, phagocytosis (3), secretion of reactive oxygen intermediates and lysosomal hydrolases (4), as well as synthesis of chemokines (5).

LTB4 and cysLTs ligate two different types of heptahelical G protein-coupled receptors (GPCRs); these receptors are known as BLT type 1 (BLT1) and type 2 (BLT2) receptors (6, 7) and cysLT type 1 (cysLT1) and type 2 (cysLT2) receptors (8, 9). GPCRs are a large class of receptors that signal though the activation of small heterotrimeric G proteins (G{alpha}, Gbeta, and G{gamma}), as reviewed by Landry et al. (10). These G proteins are classified based on their {alpha} subunits into four families: G{alpha}s, which activates adenylate cyclase and Src tyrosine kinases; G{alpha}i, which classically inhibits adenylate cyclase but can also activate Src tyrosine kinases; G{alpha}q, which stimulates phospholipase Cbeta and Bruton’s tyrosine kinase; and G{alpha}12, which activates Bruton’s tyrosine kinase, GTPase-activating protein 1, and p115 Rho guanine exchange factor Lsc (10). To date, 27 G{alpha}, 5 Gbeta, and 14 G{gamma} subunits have been described, highlighting the complexity of this system (10). Further complicating matters is the fact that many GPCRs can activate more than one G protein family. For example, using recombinant cell system approaches, it was shown that all four LT receptors can couple to both G{alpha}i and G{alpha}q proteins, resulting in pertussis toxin (PTX)-sensitive reductions in cAMP and increase in intracellular Ca2+, respectively (8, 11). However, the relative role of specific G proteins in signaling from each of these receptors is poorly defined in primary cells.

The alveolar macrophage (AM) serves an important role in innate immune defense in the distal lung, which is essential to preserve pulmonary gas exchange (12). AMs both secrete and respond to both classes of LTs (3, 13, 14). Our laboratory has previously studied the effects of LTs on critical antimicrobial functions of primary AMs, such as the phagocytosis and intracellular killing of bacterial pathogens (3, 13, 14). Although LTB4 and cysLTs each activate antimicrobial functions, there are both similarities and differences in the intracellular signaling mechanisms they use to do so. For example, whereas both LTB4 and cysLTs are equally potent enhancers of AM phagocytosis of IgG-opsonized bacteria (3), only LTB4 does so by enhancing the phosphorylation of the tyrosine kinase Syk, a proximal effector in the Fc{gamma}R signaling pathway (15). Moreover, LTB4 has been shown to be more potent than cysLTs at stimulating NADPH oxidase activation and bacterial killing in AMs (14). These data indicate that although AMs express receptors for both LT classes, the downstream signal transduction and functional programs they activate are divergent. In this study, we focused on BLT1/cysLT1-elicited responses, as opposed to effects potentially mediated by the BLT2 or cysLT2 receptors. Prior studies demonstrated that 1) BLT1- and cysLT1-selective antagonists fully blocked the effects of LTB4 and cysLTs (respectively) on phagocytosis (3) and 2) that a dual cysLT1/cysLT2 antagonist showed no additional effects over a cysLT1-selective antagonist in preventing the cysLT-mediated enhancement of bacterial killing in AMs (14).

It is unknown whether these divergent responses reflect differential coupling to distinct classes of G proteins. In the present study, we sought to investigate the role of G{alpha}q vs G{alpha}i proteins in mediating the stimulation of AM phagocytosis and bacterial killing induced by LTB4 and cysLTs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Pathogen-free 125– to 150-g female Wistar rats were obtained from Charles River Breeding Laboratories and treated according to National Institutes of Health guidelines for the use of experimental animals with the approval of the University of Michigan Committee for the Use and Care of Animals.

Reagents

Lipofectin reagent, fura 2-acetoxymethyl ester and pluronic acid F-127 were obtained from Molecular Probes. PTX was from Calbiochem. LTB4 and LTD4 were purchased from Cayman Chemicals. Specific Abs against the following were purchased: phospho-Syk (Tyr525–526) and Syk (Cell Signaling Technology), beta-tubulin (Sigma-Aldrich), G{alpha}i1 (Chemicon International), G{alpha}i2 (D5 clone; Biomol), G{alpha}i3 (C-terminal 345–354), G{alpha}q/11 (internal 283–300), and Gbeta{gamma} (beta{gamma} complex) (Calbiochem). Ionomycin, o-phenylenediamine dihydrochloride, saponin, and peroxidase-labeled monoclonal anti-rabbit IgG were purchased from Sigma-Aldrich. The inositol monophosphate (IP1) ELISA kit was obtained from Cisbio-US. Required dilutions of all compounds were prepared immediately before use, and equivalent quantities of vehicle were added to the appropriate controls.

Isolation and culture of AMs

Resident AMs of >95% purity were obtained from rats via lavage (16) and resuspended in RPMI 1640 at 2 x 106 cells/ml. Cells adhered to tissue culture-treated slides or plates for 1 h (37°C, 5% CO2) followed by two washes with warm RPMI 1640. Cells were cultured overnight in RPMI 1640 containing 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B before use. The next day cells were washed twice with warm medium to remove nonadherent cells.

Fc{gamma}R-mediated phagocytosis assays

The phagocytosis of IgG-opsonized, nonviable, FITC-labeled Escherichia coli (Molecular Probes) was assessed as previously reported (17). Adherent cells were treated overnight with PTX (600 ng/ml) (37°C, 5% CO2) in serum-containing medium. In some experiments, cells were treated overnight with Lipofectin reagent (1% v/v), with or without isotype controls or specific Abs against different G proteins (1/500 dilution) in serum-free medium according to the manufacturer’s instructions and previous reports (18, 19). Some cells were transfected with both G{alpha}i2 and G{alpha}i3 blocking Abs together (each diluted 1/1000 to maintain the same ratio of total Ab to Lipofectin as when a single Ab was used). The next day, medium was removed and cells were replenished with fresh serum-free medium and allowed to stabilize for at least 1 h before the start of experiments. Drugs of interest were added 10 min before phagocytosis was initiated. Results are expressed as a percentage of the control, to which only vehicle was added.

Tetrazolium dye reduction assay of bacterial killing

The ability of AM to kill Klebsiella pneumoniae bacteria independent of their capacity for phagocytosis was quantified using a tetrazolium dye reduction assay as described elsewhere (14) and results are expressed as the percentage of survival of ingested bacteria.

Measurement of intracellular cAMP

Macrophages were cultured overnight in 6-well plates in RPMI 1640 plus 10% FBS at a concentration of 3 x 106 cells/well. Medium was then changed to serum-free medium and cells were incubated for 15 min with different concentrations of LTB4, LTD4, or vehicle control (DMSO). In some experiments, PTX (600 ng/ml) was added to the cells and incubated for various intervals before adding LTB4 or LTD4. Culture supernatants were aspirated and the cells were lysed by incubation for 20 min with 0.1 M HCl (22°C), followed by disruption using a cell scraper. Intracellular cAMP levels were determined by ELISA according to the manufacturer (Assay Designs).

Analysis of calcium mobilization

Freshly isolated AMs were centrifuged for 10 min at 750 x g, and then resuspended in HBSS containing 1.6 mM CaCl2. The warmed cell suspension (37°C, 5 x 106 cells/ml) was treated with 5 µM fura 2-acetoxymethyl ester in the presence of 30 µg/ml of pluronic acid F-127 for 1 h to enhance dye solubility and uptake (20). The suspension was washed twice with HBSS containing 1.6 mM CaCl2, and finally resuspended at a density of 5 x 106cells/ml and transferred into the magnetically stirred cuvette of a luminescence spectrometer (LS50B; PerkinElmer). Calcium mobilization was monitored using excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Data are presented as the ratio of fluorescence obtained at 340 and 380 (340/380) nm.

Measurement of intracellular IP1 accumulation

Macrophages were cultured overnight in 24-well plates in RPMI 1640 plus 10% FBS at a concentration of 2 x 106 cells/well. Medium was then changed to serum-free medium and cells were incubated for 1 h with 100 nM LTB4, LTD4, or vehicle control. Culture supernatants were aspirated and the cells were lysed by incubation for 30 min with 2.5% of lysis reagent (37°C). Intracellular IP1 levels were determined by ELISA according to the manufacturer.

Immunoprecipitation and Western blot analysis

Immunoprecipitation of Syk protein from AM lysates was performed as we have previously published (15). Western blotting was performed as previously described (21). Protein samples (40 µg) were resolved on 8% Tris-HCl polyacrylamide gels (containing 6 M urea for G{alpha} subunit Western blots) and subsequently transferred to a nitrocellulose membrane. Membranes were probed with primary Abs followed by HRP-conjugated anti-rabbit or anti-mouse secondary Abs and ECL Plus detection reagents (Amersham Biosciences).

Statistical analysis

Data are presented as the mean ± SEM and were analyzed with the Prism 4.0 statistical program (GraphPad Software). Comparisons among three or more experimental groups were performed with ANOVA followed by the Bonferroni correction. Differences were considered significant for values of p < 0.05. Experiments were performed on three or more separate occasions unless otherwise specified.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LTB4 but not LTD4 decreases intracellular cAMP in AMs

Both BLT1- and cysLT1-tranfected cells exhibit dual coupling to G{alpha}i and G{alpha}q proteins (7, 22, 23); however, few data are available in primary cells. To determine the relative contribution of G{alpha}i protein in BLT1 and cysLT1 signaling, our first approach was to investigate the levels of intracellular cAMP in rat AMs stimulated with LTB4 or LTD4 (Fig. 1). Rat AMs treated with LTB4 (15 min) showed a concentration-dependent decrease in intracellular cAMP. Decreases were observed at LTB4 concentrations as low as 100 pM and maximal inhibition (53.7 ± 7.4%) occurred at 1 nM (Fig. 1). We previously found that the maximal effects of LTB4 (and cysLTs) on Fc{gamma}R phagocytosis were also observed at 1 nM (3). LTD4 did not alter intracellular cAMP over the concentration range tested, causing a nonsignificant reduction only at 100 nM. These data suggest that LTB4, but not LTD4, activates a G{alpha}i-coupled receptor.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 1. Effect of LTB4 and LTD4 on intracellular cAMP concentration ([cAMP]i) in AM. AMs were incubated for 15 min with or without LTB4 or LTD4 at the indicated concentrations. Intracellular cAMP concentrations were determined as described in Materials and Methods. Data are expressed as relative units with the control value to which only vehicle was added = 1. Data are mean ± SEM for 3–10 separate experiments. *, p < 0.05 compared with control.

 
AMs express G{alpha}i subunits 2 and 3 and G{alpha}q/11

Members of the G{alpha}i family include G{alpha}i1, G{alpha}i2, and G{alpha}i3 subunits and of the G{alpha}q family include G{alpha}q, G{alpha}11, G{alpha}14, G{alpha}15, and G{alpha}16 (10). Previous studies of G protein expression in AMs did not discriminate clearly among the three G{alpha}i subunits (24, 25, 26). To address that perspective, the expression of all three G{alpha}i subunits and G{alpha}q/11 in rat AM was examined by Western blot. Fig. 2A demonstrates the same membrane stripped and re-immunoblotted with Abs to the various G proteins. We observed bands at ~42 kDa corresponding to G{alpha}i2 and G{alpha}i3, and the characteristic doublet at 42 and 43 kDa representative of G{alpha}q and G{alpha}11, respectively. However, G{alpha}i1 expression was not detected by this method. As a positive control for the G{alpha}i1 Ab, we used rat brain and cerebellar cell lysates that are well known to express G{alpha}i1 protein (27). Unlike AMs, a band at the expected molecular mass for the G protein (~42 kDa) was observed in both cultured cell types (Fig. 2B).


Figure 2
View larger version (65K):
[in this window]
[in a new window]

  		FIGURE 2. Expression and PTX-induced ADP ribosylation of G{alpha}i subunits. A, Whole cell lysates from AMs were prepared and Western blot analysis for G protein subunits was performed; B, Lysates from rat brain and cerebellar cells (50 µg) were immunoblotted with anti-G{alpha}i1 Ab. C, AMs were incubated with PTX or heat-inactivated PTX (iPTX) (indicated with an asterisk) for different intervals. After cells were harvested, Western blot analysis was performed. One of three representative blots yielding similar results is shown.

 
We next used PTX in pharmacological studies of LT signaling in AMs. PTX is a classical inhibitor of G{alpha}i proteins that works by covalently ADP-ribosylating their {alpha} subunits, uncoupling them from their cognate membrane-bound receptors and thus preventing their activation (10). To evaluate the state of ADP ribosylation of G{alpha}i2 and G{alpha}i3 subunits in PTX-treated cells, we performed immunoblot analyses under conditions optimal for the separation of ribosylated subunits from their nonribosylated forms (28). This method allows the identification of a characteristic ~2-kDa upward shift in apparent moleular mass that occurs after ADP-ribosylation (28). As shown in Fig. 2C, ADP ribosylation was clearly seen in both G{alpha}i2 and G{alpha}i3 after 3 and 18 h incubation with active PTX (600 ng/ml), but not with heat-inactivated PTX. Slight ADP ribosylation of G{alpha}i3 was observed as early as 30 min. As all AM G{alpha}i subunits were completely ribosylated after overnight treatment, this time point and PTX dose were used in additional experiments.

PTX treatment prevents reduction of cAMP by LTB4

We next used PTX to explore the participation of G{alpha}i proteins in mediating the LTB4-induced decrease in intracellular cAMP release. AMs were pretreated with PTX overnight, followed by addition of LTB4 (1 nM) for 15 min. We found that PTX treatment completely abrogated the inhibitory effect of LTB4 on intracellular cAMP levels (Fig. 3A).


Figure 3
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 3. PTX treatment prevents reduction of cAMP by LTB4 and differentially modulates AM innate immune responses to LTB4 and LTD4. A, Cells were pretreated with PTX for 18 h, followed by LTB4 treatment at a final concentration of 1 nM for 15 min. Intracellular cAMP concentrations ([cAMP]i) were determined as shown in Materials and Methods. Data are expressed relative to the appropriate control value to which PTX was or was not added. Data are mean ± SEM from three separate experiments. B, AMs were preincubated with PTX for 18 h. The next day, cells were incubated with LTB4 (1 nM) or LTD4 (100 nM), or vehicle control for 10 min and then challenged with E. coli opsonized with specific rabbit polyclonal IgG. Phagocytic index (PI) was calculated as described (17 ) and expressed as a percentage of the control value in which no drugs were added. Mean ± SEM data are shown from four to seven separate experiments, each performed in sextuplet. C, Cells were pretreated for 18 h with PTX or vehicle and then incubated with 1 nM LTB4 for 3 min before the addition of IgG-SRBC (1:33 ratio), followed by incubation for 7 min at 37°C. Incubations were terminated by the addition of lysis buffer, and lysates were subjected to immunoprecipitation and immunoblotting. Immunoblots represent phosphorylated Syk detected with anti-phospho-Syk Ab (top) and the total amount of Syk protein evaluated with an anti-Syk Ab (bottom). The densitometry analysis represents mean ± SEM from independent experiments (right). D, AMs were preincubated with PTX and the next day were infected with 50:1 opsonized K. pneumoniae. Thirty min after infection, the cells were incubated with LTB4 (1 nM), LTD4 (100 nM), or vehicle. Microbicidal activity was assessed and expressed as the mean ± SEM percentage survival of ingested bacteria from four independent experiments, each performed in triplicate. *, p < 0.05; **, p < 0.001; ***, p < 0.001 only vs control; ##, p < 0.001 vs LTB4-treated cells.

 
PTX differentially modulates enhancement of Fc{gamma}R-mediated phagocytosis by LTB4 and LTD4

Our laboratory has demonstrated that Fc{gamma}R-mediated phagocytosis by AMs is enhanced by LTB4 and cysLTs (3, 29), though the exact mechanisms involved remain unclear. We hypothesized, based on our findings, that LTB4 stimulation of Fc{gamma}R-mediated phagocytosis would be at least partially PTX-sensitive, whereas the effects of LTD4 would not. Concentrations of 1 and 100 nM, respectively, of LTB4 and LTD4 were used because 1) those concentrations effectively enhanced both phagocytosis and bacterial killing in our previous studies (3, 14) and 2) they resulted in a maximal decrease (LTB4) and no effect (LTD4) in cAMP (Fig. 1). As expected, LTB4 and LTD4 increased the phagocytosis of IgG-E. coli in AMs by 50.0 ± 3.4% and 37.2 ± 8.5%, respectively (Fig. 3B). However, pretreatment of the cells with PTX completely abolished the ability of LTB4 to enhance Fc{gamma}R-mediated ingestion, but did not significantly attenuate LTD4 enhancement (Fig. 3B). These data suggest that stimulatory effects of BLT1 engagement on AM phagocytosis follow its ability to acutely decrease cAMP via G{alpha}i activation, whereas those of cysLT1 ligation are PTX-insensitive.

LTB4-enhanced Syk activation is blocked by PTX

We previously showed in AMs that LTB4, but not other 5-lipoxygenase products including LTD4, increased phosphorylation of the protein tyrosine kinase Syk, a proximal effector in the Fc{gamma}R signaling pathway (15). We postulated that PTX would likewise suppress Syk phosphorylation following LTB4 treatment in AMs. As previously found at 100-fold higher concentrations (15), the addition of 1 nM LTB4 3 min before IgG-SRBC challenge augmented the degree of Syk phosphorylation evoked by Fc{gamma}R engagement (Fig. 3C). Indeed, PTX treatment attenuated the ability of LTB4 to enhance Syk phosphorylation (Fig. 3C) by ~38% as shown by band densitometry analysis (Fig. 3C, right). These data indicate that via BLT1, LTB4 promotes phagocytosis and Syk phosphorylation by activating G{alpha}i.

PTX differentially modulates enhancement of bacterial killing by LTB4 and LTD4

Along with phagocytosis, microbicidal activity is a critical step in the control of infection. We have demonstrated that endogenous and exogenous LTs enhance bacterial killing (14). We therefore determined the importance of G{alpha}i proteins in coupling BLT1 and cysLT1 to effects on killing of the clinically relevant Gram-negative pathogen K. pneumoniae. As previously demonstrated (14) and in Fig. 3D, both exogenous LTB4 (1 nM) and LTD4 (100 nM) increased AM microbicidal activity, reflected by a decrease in survival of ingested bacteria (~29% and 38%, respectively). Cell pretreatment with PTX abolished the LTB4 effects on bacterial killing (Fig. 3D), but not those of LTD4. These results indicate that although LTB4 and LTD4 are both capable of enhancing AM bactericidal activity, only the former does so via coupling of its cognate receptor to G{alpha}i.

LTB4 and LTD4 activate phospholipase C (PLC)beta

Given that LTD4 potentiation of Fc{gamma}R-mediated phagocytosis and microbicidal activity in AM was PTX-resistant (Fig. 3, B and D), and in light of the fact that the G{alpha}q pathway appears to be important in mediating cysLT receptor signaling in transfected cells (8, 11, 30), we examined G{alpha}q-mediated signaling by LTB4 and LTD4 in AM by evaluating Ca2+ mobilization and IP1 accumulation (IP1 is a stable product of inositol 1,4,5 triphosphate derived from PLCbeta activation). As shown in Fig. 4A, 300 nM LTD4 potently induced intracellular Ca2+ elevation. This response to LTD4 was totally blocked when cells were pretreated for 20 min with the cysLT1 antagonist, MK 571 (1 µM). Unlike LTD4, LTB4 was unable to mobilize intracellular Ca2+ to a detectable degree over a range of concentrations (1–300 nM) (Fig. 4B). This result was surprising because we have previously demonstrated that LTB4 increased intracellular Ca2+ concentration in murine dendritic cells (31) and in human neutrophils (32). We therefore used an additional approach for assessing G{alpha}q activation, namely, an ELISA to quantitate intracellular IP1 accumulation. Although LTB4 failed to induce Ca2+ mobilization, it (100 nM) did significantly increase accumulation of IP1 in a manner similar to LTD4 (Fig. 4C).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 4. LTD4 and LTB4 activate PLCbeta in AMs. For Ca2+ measurement, cells were loaded with fura 2-acetoxymethyl for 1 h, washed, and resuspended into a luminescence spectrometer. Calcium mobilization was monitored for ~30 s (stabilization), and then the cells were treated with LTD4 (A) and LTB4 (B), each at 300 nM. Calcium mobilization is expressed as a fluorescence ratio obtained from 340 and 380 (340/380) nm. Results are representative of four separate experiments. C, AMs were incubated for 1 h with 100 nM LTB4, LTD4, or vehicle control. Culture supernatants were aspirated and the cells were lysed by incubation for 30 min with 2.5% of lysis reagent. Intracellular IP1 levels were determinated by ELISA. Mean ± SEM from one representative of four independent experiments is shown. **, p < 0.01 vs control.

 
Ab blockade indicates that LTB4 responses are dependent on G{alpha}i3 and G{alpha}q subunits, whereas LTD4 responses are mediated by G{alpha}q

As an alternative approach to assess the roles of G{alpha}i and G{alpha}q in mediating the effects of LTs in AMs, and in an effort to discern roles of Gbeta{gamma} and specific G{alpha}i isoforms, phagocytosis studies were performed in cells pretreated overnight with Abs raised against rat G{alpha}i2, G{alpha}i3, G{alpha}q/11, and Gbeta{gamma} proteins or isotype controls (namely, IgG2b, the isotype control for anti-G{alpha}i2 or IgG, the isotype control for anti-G{alpha}i3 and anti-Gq). Abs were complexed with liposomes to facilitate delivery into the cytosol (18, 19). As expected, 1 nM LTB4 and 100 nM LTD4 significantly increased Fc{gamma}R phagocytosis compared with vehicle-treated cells (41 ± 4.8% and 94.6 ± 31%, respectively) (Fig. 5). Although G{alpha}i2 and G{alpha}i3 were expressed in AM, treatment of the cells with Abs against G{alpha}i3 completely abolished the ability of LTB4 to enhance phagocytosis, whereas anti-G{alpha}i2 Abs had no effect (Fig. 5A). When anti-G{alpha}i2 and anti-G{alpha}i3 were used together, phagocytic activity fell slightly below that of untreated cells. To confirm the lack of G{alpha}i2 involvement, we conducted additional experimental controls. We found that the responses to isotype control IgG2b and anti-G{alpha}i2 in the presence of LTB4 were similar, and that the responses to anti-G{alpha}i2 plus anti-G{alpha}i3 and isotype control IgG2b plus anti-G{alpha}i3, although slightly different, were not statistically significant, suggesting no role for G{alpha}i2 (Fig. 5A). AMs were also transfected with Lipofectin with G protein blocking Abs and/or the isotype control Abs in the absence of LT before assaying phagocytosis. No alterations in basal phagocytic capacities were observed with any of the blocking and isotype control Abs (data not shown), suggesting that this procedure does not cause a nonspecific depression of phagocytosis, instead suggesting that the effects of the blocking Abs are indeed specific to stimulation by LT. Consistent with the enhancement of LTB4- and LTD4-mediated IP1 and Ca2+, respectively, the blockade of G{alpha}q/11 signaling entirely abrogated the ability of LT to enhance Fc{gamma}R-mediated phagocytosis (Fig. 5). Conversely, we did not observe any effect of the G{alpha}i or Gbeta{gamma} Abs on LTD4 enhancement of phagocytosis (Fig. 5B). These data suggest that the effects of both BLT1 and cysLT1 ligation on phagocytosis required G{alpha}q/11 proteins, whereas the former also used the G{alpha}i3 protein.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Role of specific G protein subunits in LT-enhanced Fc{gamma}R-mediated phagocytosis. AMs were pretreated for 18 h with vehicle (1% v/v Lipofectin) ± subunit-specific blocking Abs or isotype controls (1:500) in serum-free medium. The medium was then replaced and cells were incubated with 1 nM LTB4 (A), 100 nM LTD4 (B), or vehicle control for 10 min and then challenged with E. coli opsonized with specific rabbit polyclonal IgG. Phagocytic index (PI) was calculated as described (17 ) and expressed as a percentage of the control value in which only vehicle was added. *, p < 0.05 vs control; #, p < 0.05; ##, p < 0.01 vs LTB4- and LTD4-treated cells. Mean ± SEM data are shown from two to three different experiments, each performed in sextuplet.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we extended our exploration of the mechanisms by which LTB4 and LTD4 modulate AM phagocytosis and bactericidal activity by focusing on the coupling of BLT1 and cysLT1 receptors to specific G proteins. Our results demonstrate that 1) LTB4, but not LTD4, decreases intracellular cAMP levels, a phenomenon prevented by PTX; 2) although Fc{gamma}R-mediated phagocytosis and microbicidal activity are enhanced by both LTs, only LTB4 does so in a PTX-sensitive manner; 3) both LTD4 and LTB4 elicit PLCbeta activation, as indicated by the ability of LTD4 to evoke detectable increases in intracellular Ca2+ and of both LTs to increase IP1; and 4) the blockade of specific G proteins indicates that whereas G{alpha}i3 and G{alpha}q proteins are used for LTB4-enhanced phagocytosis, only G{alpha}q is used by LTD4. A schematic representation of the role of G{alpha}i and G{alpha}q subunits in AM responses to LTs is depicted in Fig. 6.


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 6. Schematic representation of the role of G{alpha}i and G{alpha}q in LT receptor-mediated responses. Ligation of cysLT1 by LTD4 exclusively results in activated G{alpha}q/11, which increases intracellular Ca2+ levels and activates protein kinase C (PKC). Binding of LTB4 to BLT1 results in activated G{alpha}i3, which leads to decreased intracellular cAMP levels and increased Fc{gamma}R-mediated Syk phosphorylation. This ligation also activates PLCbeta (via G{alpha}q/11), which increases intracellular inositol 1,4,5 triphosphate (IP3) accumulation, and contributes to coupling BLT1 signaling to enhance phagocytosis of IgG-coated target and bacterial clearance. The ability of both LTB4 and cysLTs to activate protein kinase C was reported previously by Mancuso et al. (13 ). AC, adenylate cyclase; DAG, diacylglycerol.

 
Previous studies using transfected cell lines have shown that BLT1 and cysLT1 are coupled to both G{alpha}q and G{alpha}i proteins (7, 8, 11, 22, 23), with the relative importance of these G proteins varying depending on the cell type and functions under investigation. For example, in BLT1-expressing RBL cells LTB4-induced chemotaxis, but not phosphatidylinositol hydrolysis and calcium mobilization, depended on G{alpha}i proteins (33). Hoshino et al. (34) demonstrated in human monocyte-like THP-1 cells that LTD4 signals through PTX-sensitive and PTX-insensitive pathways to mediate cellular activation. In human neutrophils, it has been shown that LTB4-induced calcium mobilization, chemotactic migration, and degranulation were PTX-sensitive, suggesting coupling to Gi protein (32). However, there is no information regarding the relative importance of particular G protein classes or subunits in LT receptor-mediated enhancement of phagocytosis and bacterial killing. In fact, we know of no such information about specific G protein subunit coupling to LT receptors in primary cells. In view of the importance of the AM in antimicrobial defense of the lung and as a target for the immunostimulatory actions of LTs, it is a relevant cell type in which to focus this investigation.

To address the participation of G{alpha}i protein in LTB4- and LTD4-mediated responses, we assessed the effects of each LT on intracellular cAMP levels. A clear distinction between the two was observed, as low concentrations of LTB4 but not LTD4 were able to reduce intracellular cAMP. LTB4 was very potent in this regard, as picomolar concentrations of LTB4 suppressed cAMP generation, and the effect was totally abolished by PTX, implicating a role for G{alpha}i signaling. These results are in agreement with other studies that have found that LTB4 inhibited forskolin-activated cAMP production by Chinese hamster ovary cells expressing BLT1 (7, 35). In contrast, LTB4 was found to increase cAMP in human neutrophils (36, 37). Such data highlight the underappreciated complexity of GPCR signaling in unique cell types. Unlike LTB4, cysLTs were reported not to alter cAMP levels in leukocytes (37) or in human airway smooth muscle cells (38), but to increase cAMP in a human epithelial cell line (39).

The suppressive effects of PGE2, a lipid mediator derived from the cyclooxygenase pathway, on AM phagocytosis and bacterial killing follow its ability to elevate cAMP via G{alpha}s-coupled receptors (17, 40). Considering that G{alpha}i protein activation serves as a negative regulator of G{alpha}s signaling (41), we hypothesized that changes in intracellular levels of cAMP might contribute to the opposing effects of LTs and PGE2 on AM innate immune responses.

Although both LTB4 and cysLTs enhance Fc{gamma}R-mediated phagocytosis and bacterial killing, we previously identified important differences in the molecular mechanisms triggered by BLT1 and cysLT1 activation that are responsible for these effects (14, 15). For example, LTB4, but not cysLTs, amplifies Fc{gamma}R-induced Syk activation through a Ca2+-dependent mechanism (15). We now demonstrate that BLT1 activation, but not cysLT1 activation, enhances Fc{gamma}R-mediated phagocytosis and Syk phosphorylation in a PTX-sensitive manner (Fig. 3, B and C), revealing a role for G{alpha}i proteins that was to our knowledge undocumented. Recently, Kuniyeda et al. (42) identified, in transfected cells, the intracellular region of BLT1 responsible for specific G{alpha}i-protein coupling.

Previous work by other groups established that murine peritoneal macrophages express G{alpha}i1 and/or G{alpha}i2 as well as G{alpha}i3 (26), and guinea pig AMs express G{alpha}i1 and/or G{alpha}i2, but not G{alpha}i3 (24). However, these studies were limited by the use of reagents that could not discriminate between G{alpha}i1 and G{alpha}i2. Using more specific Abs, we demonstrated that only G{alpha}i2, not G{alpha}i1, is expressed in primary rat AMs, along with both G{alpha}i3 and G{alpha}q/11. Moreover, although both G{alpha}i2 and G{alpha}i3 were ribosylated by PTX treatment, we were able to determine the relative importance of individual G{alpha}i subunits in mediating coupling to BLT1 and cysLT1 by using Abs raised against rat G{alpha}i2, G{alpha}i3, G{alpha}q/11, or Gbeta{gamma}. To facilitate their entrance into the cells, Abs were complexed with liposomes, taking advantage of a novel intracellular delivery system that was developed in cell lines (18, 19). We believe this report is the first published using this functional knockdown approach in a primary cell. Other approaches have been used to block specific G proteins, including genetic deletion (43) and small interfering RNA technology (44). We chose to use the Lipofectin-based strategy for several reasons. First, primary AMs are difficult to transfect with small interfering RNA (45) and small interfering RNA has been shown to trigger antiviral responses that might significantly alter immune responses in vitro (46). Importantly, we did not find significant effects of liposome treatment on AM phagocytosis. Second, genetic knockdown can be complicated by compensatory alterations in expression of nontargeted G proteins. Lastly, liposomes are easy to use and the approach provides a facile method for protein blockade that is limited mostly by the availability of blocking Abs. The results of these studies (Fig. 5) indicated that LTB4/BLT1 effects were mediated by G{alpha}i3 and G{alpha}q, whereas LTD4/cysLT1 effects were mediated solely by G{alpha}q. Oh and Schnitzer (47) showed that G{alpha}q preferentially localizes in caveolae, while G{alpha}s and G{alpha}i localize in lipid rafts, suggesting compartmentation of GPCR signaling proteins in specific membrane microenvironments as a potential determinant of receptor-effector coupling.

The G{alpha}q/G{alpha}11/G{alpha}14/G{alpha}16 family is coupled to certain receptors and activates PLCbeta subtypes to mediate phosphatidylinositol hydrolysis, resulting in Ca2+ elevation (48). To address the activation of G{alpha}q/11, we first evaluated Ca2+ mobilization in response to LTB4 and LTD4. Our data show that LTD4 (300 nM) elevated Ca2+, whereas LTB4 (300 nM) did not. This result was unexpected because a rise in cytosolic Ca2+ concentration upon LTB4 ligation of BLT1 is widely recognized in neutrophils (49). These inconsistent data are also underscored by the requirement for intracellular Ca2+ in Syk activation by LTB4 during the ingestion of IgG-opsonized targets (15). In light of this requirement, we used an additional approach for assessing G{alpha}q activation, namely, inositol 1,4,5 triphosphate release in response to PLCbeta activation, as measured by an ELISA for intracellular IP1 production. Although LTB4 failed to induce Ca2+ mobilization, it did increase accumulation of IP1 in a manner similar to LTD4, suggesting that LTB4 indeed activates a G{alpha}q-coupled receptor. It is possible that the IP1 assay may be more sensitive than Ca2+ mobilization in our experimental system.

Although previous studies have established the role of specific G proteins in mediating signaling from LT receptors and other GPCRs in cell lines or transfected cells, our study explores this interaction as regards innate immune functions of rat primary macrophages. However, the relevance of our findings for other primary cell types is still uncertain and will require direct examination. Our data reveal a major divergence in the role of individual G protein subunits in mediating the stimulation of AM phagocytosis and microbicidal activity by LTB4 and LTD4. BLT1 uses both G{alpha}i3 and G{alpha}q proteins to activate these responses, whereas cysLT1 does so only via G{alpha}q. These findings emphasize the need to dissect signaling programs activated by specific GPCRs in relevant primary cells.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants HL078727 and HL058897 from the National Institutes of Health, by Grant RG-8909-N from the American Lung Association, and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil). N.F. is the recipient of a postdoctoral award from the Canadian Institutes of Health Research. Back

2 Address correspondence and reprint requests to Dr. Marc Peters-Golden, 6301 Medical Science Research Building III, Box 0642, University of Michigan Health System, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642. E-mail address: petersm{at}umich.edu Back

3 Abbreviations used in this paper: LT, leukotriene; cysLT, cysteinyl LT; cysLT1, cysLT type 1; BLT1, BLT type 1; AM, alveolar macrophage; GPCR, G protein-coupled receptor; PLC, phospholipase C; PTX, pertussis toxin; IP1, inositol monophosphate. Back

Received for publication July 25, 2007. Accepted for publication July 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Ford-Hutchinson, A. W., M. A. Bray, M. V. Doig, M. E. Shipley, M. J. Smith. 1980. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286: 264-265. [Medline]
  2. Lewis, R. A., K. F. Austen, R. J. Soberman. 1990. Leukotrienes and other products of the 5-lipoxygenase pathway: biochemistry and relation to pathobiology in human diseases. N. Engl. J. Med. 323: 645-655. [Medline]
  3. Mancuso, P., T. J. Standiford, T. Marshall, M. Peters-Golden. 1998. 5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae. Infect. Immun. 66: 5140-5146. [Abstract/Free Full Text]
  4. Serhan, C. N., A. Radin, J. E. Smolen, H. Korchak, B. Samuelsson, G. Weissmann. 1982. Leukotriene B4 is a complete secretagogue in human neutrophils: a kinetic analysis. Biochem. Biophys. Res. Commun. 107: 1006-1012. [Medline]
  5. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, R. M. Strieter, S. L. Kunkel. 1999. Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4. J. Immunol. 163: 6148-6154. [Abstract/Free Full Text]
  6. Yokomizo, T., K. Kato, K. Terawaki, T. Izumi, T. Shimizu. 2000. A second leukotriene B4 receptor, BLT2: a new therapeutic target in inflammation and immunological disorders. J. Exp. Med. 192: 421-432. [Abstract/Free Full Text]
  7. Yokomizo, T., T. Izumi, K. Chang, Y. Takuwa, T. Shimizu. 1997. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387: 620-624. [Medline]
  8. Lynch, K. R., G. P. O’Neill, Q. Liu, D. S. Im, N. Sawyer, K. M. Metters, N. Coulombe, M. Abramovitz, D. J. Figueroa, Z. Zeng, et al 1999. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399: 789-793. [Medline]
  9. Heise, C. E., B. F. O’Dowd, D. J. Figueroa, N. Sawyer, T. Nguyen, D. S. Im, R. Stocco, J. N. Bellefeuille, M. Abramovitz, R. Cheng, et al 2000. Characterization of the human cysteinyl leukotriene 2 receptor. J. Biol. Chem. 275: 30531-30536. [Abstract/Free Full Text]
  10. Landry, Y., N. Niederhoffer, E. Sick, J. P. Gies. 2006. Heptahelical and other G-protein-coupled receptors (GPCRs) signaling. Curr. Med. Chem. 13: 51-63. [Medline]
  11. Sarau, H. M., R. S. Ames, J. Chambers, C. Ellis, N. Elshourbagy, J. J. Foley, D. B. Schmidt, R. M. Muccitelli, O. Jenkins, P. R. Murdock, et al 1999. Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor. Mol. Pharmacol. 56: 657-663. [Abstract/Free Full Text]
  12. Gordon, S. B., R. C. Read. 2002. Macrophage defences against respiratory tract infections. Br. Med. Bull. 61: 45-61. [Abstract/Free Full Text]
  13. Mancuso, P., M. Peters-Golden. 2000. Modulation of alveolar macrophage phagocytosis by leukotrienes is Fc receptor-mediated and protein kinase C-dependent. Am. J. Respir. Cell. Mol. Biol. 23: 727-733. [Abstract/Free Full Text]
  14. Serezani, C. H., D. M. Aronoff, S. Jancar, P. Mancuso, M. Peters-Golden. 2005. Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase. Blood 106: 1067-1075. [Abstract/Free Full Text]
  15. Canetti, C., B. Hu, J. L. Curtis, M. Peters-Golden. 2003. Syk activation is a leukotriene B4-regulated event involved in macrophage phagocytosis of IgG-coated targets but not apoptotic cells. Blood 102: 1877-1883. [Abstract/Free Full Text]
  16. Peters-Golden, M., R. W. McNish, R. Hyzy, C. Shelly, G. B. Toews. 1990. Alterations in the pattern of arachidonate metabolism accompany rat macrophage differentiation in the lung. J. Immunol. 144: 263-270. [Abstract]
  17. Aronoff, D. M., C. Canetti, M. Peters-Golden. 2004. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP. J. Immunol. 173: 559-565. [Abstract/Free Full Text]
  18. Gomes, P., P. Soares-da-Silva. 2002. Role of cAMP-PKA-PLC signaling cascade on dopamine-induced PKC-mediated inhibition of renal Na+-K+-ATPase activity. Am. J. Physiol. 282: F1084-F1096.
  19. Gomes, P., P. Soares-da-Silva. 2002. Na+/H+ exchanger activity and dopamine D1-like receptor function in two opossum kidney cell clonal sublines. Cell. Physiol. Biochem. 12: 259-268. [Medline]
  20. Maruyama, I., T. Hasegawa, T. Yamamoto, K. Momose. 1989. Effects of pluronic F-127 on loading of fura 2/AM into single smooth muscle cells isolated from guinea pig taenia coli. J. Toxicol. Sci. 14: 153-163. [Medline]
  21. Kolodsick, J. E., M. Peters-Golden, J. Larios, G. B. Toews, V. J. Thannickal, B. B. Moore. 2003. Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation. Am. J. Respir. Cell. Mol. Biol. 29: 537-544. [Abstract/Free Full Text]
  22. Crooke, S. T., M. Mattern, H. M. Sarau, J. D. Winkler, J. Balcarek, A. Wong, C. F. Bennett. 1989. The signal transduction system of the leukotriene D4 receptor. Trends Pharmacol. Sci. 10: 103-107. [Medline]
  23. Pollock, K., J. Creba. 1990. Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate. Cell Signal. 2: 563-568. [Medline]
  24. Levistre, R., J. Masliah, G. Bereziat. 1993. Stimulatory and inhibitory guanine-nucleotide-binding regulatory protein involvement in stimulation of arachidonic-acid release by N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor from guinea-pig alveolar macrophages: differential receptor/G-protein interaction assessed by pertussis and cholera toxins. Eur. J. Biochem. 213: 295-303. [Medline]
  25. Shreeve, S. M., S. P. Sreedharan, M. P. Hacker, D. E. Gannon, M. J. Morgan. 2000. VIP activates GS and Gi3 in rat alveolar macrophages and Gs in HEK293 cells transfected with the human VPAC1 receptor. Biochem. Biophys. Res. Commun. 272: 922-928. [Medline]
  26. Garcia-Perganeda, A., J. M. Guerrero, M. Rafii-El-Idrissi, M. Paz Romero, D. Pozo, J. R. Calvo. 1999. Characterization of membrane melatonin receptor in mouse peritoneal macrophages: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein. J. Neuroimmunol. 95: 85-94. [Medline]
  27. Sternweis, P. C., J. D. Robishaw. 1984. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J. Biol. Chem. 259: 13806-13813. [Abstract/Free Full Text]
  28. Komatsu, M., A. M. McDermott, S. L. Gillison, G. W. Sharp. 1995. Time course of action of pertussis toxin to block the inhibition of stimulated insulin release by norepinephrine. Endocrinology 136: 1857-1863. [Abstract]
  29. Bailie, M. B., T. J. Standiford, L. L. Laichalk, M. J. Coffey, R. Strieter, M. Peters-Golden. 1996. Leukotriene-deficient mice manifest enhanced lethality from Klebsiella pneumoniae in association with decreased alveolar macrophage phagocytic and bactericidal activities. J. Immunol. 157: 5221-5224. [Abstract]
  30. Capra, V., S. Ravasi, M. R. Accomazzo, M. Parenti, G. E. Rovati. 2004. CysLT1 signal transduction in differentiated U937 cells involves the activation of the small GTP-binding protein Ras. Biochem. Pharmacol. 67: 1569-1577. [Medline]
  31. Canetti, C., D. M. Aronoff, M. Choe, N. Flamand, S. Wettlaufer, G. B. Toews, G. H. Chen, M. Peters-Golden. 2006. Differential regulation by leukotrienes and calcium of Fc{gamma} receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages. J. Leukocyte Biol. 79: 1234-1241. [Abstract/Free Full Text]
  32. Goldman, D. W., F. H. Chang, L. A. Gifford, E. J. Goetzl, H. R. Bourne. 1985. Pertussis toxin inhibition of chemotactic factor-induced calcium mobilization and function in human polymorphonuclear leukocytes. J. Exp. Med. 162: 145-156. [Abstract/Free Full Text]
  33. Haribabu, B., D. V. Zhelev, B. C. Pridgen, R. M. Richardson, H. Ali, R. Snyderman. 1999. Chemoattractant receptors activate distinct pathways for chemotaxis and secretion: role of G-protein usage. J. Biol. Chem. 274: 37087-37092. [Abstract/Free Full Text]
  34. Hoshino, M., T. Izumi, T. Shimizu. 1998. Leukotriene D4 activates mitogen-activated protein kinase through a protein kinase C{alpha}-Raf-1-dependent pathway in human monocytic leukemia THP-1 cells. J. Biol. Chem. 273: 4878-4882. [Abstract/Free Full Text]
  35. Martin, V., P. Ronde, D. Unett, A. Wong, T. L. Hoffman, A. L. Edinger, R. W. Doms, C. D. Funk. 1999. Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells. J. Biol. Chem. 274: 8597-8603. [Abstract/Free Full Text]
  36. Hopkins, N. K., A. H. Lin, R. R. Gorman. 1983. Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3',5'-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4. Biochim. Biophys. Acta 763: 276-283. [Medline]
  37. Claesson, H. E.. 1982. Leukotrienes A4 and B4 stimulate the formation of cyclic AMP in human leukocytes. FEBS Lett. 139: 305-308. [Medline]
  38. Rovati, G. E., M. Baroffio, S. Citro, L. Brichetto, S. Ravasi, M. Milanese, E. Crimi, V. Brusasco. 2006. Cysteinyl-leukotrienes in the regulation of beta2-adrenoceptor function: an in vitro model of asthma. Respir. Res. 7: 103[Medline]
  39. Gronroos, E., C. K. Thodeti, A. Sjolander. 1998. Leukotriene D4 induces a rapid increase in cAMP in the human epithelial cell line, Int 407: a potential role for this signal in the regulation of calcium influx through the plasma membrane. Cell. Calcium 24: 9-16. [Medline]
  40. Aronoff, D. M., C. Canetti, C. H. Serezani, M. Luo, M. Peters-Golden. 2005. Cutting edge: macrophage inhibition by cyclic AMP (cAMP): differential roles of protein kinase A and exchange protein directly activated by cAMP-1. J. Immunol. 174: 595-599. [Abstract/Free Full Text]
  41. Fimia, G. M., P. Sassone-Corsi. 2001. Cyclic AMP signalling. J. Cell. Sci. 114: 1971-1972. [Free Full Text]
  42. Kuniyeda, K., T. Okuno, K. Terawaki, M. Miyano, T. Yokomizo, T. Shimizu. 2006. Identification of the intracellular region of the leukotriene B4 receptor type-1 that specifically involves in Gi-activation. J. Biol. Chem. 282: 3998-4006. [Medline]
  43. Fan, H., D. L. Williams, B. Zingarelli, K. F. Breuel, G. Teti, G. E. Tempel, K. Spicher, G. Boulay, L. Birnbaumer, P. V. Halushka, J. A. Cook. 2006. Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by G{alpha}i proteins. Biochim. Biophys. Acta 1763: 1051-1058. [Medline]
  44. Zheng, B., C. Lavoie, T. D. Tang, P. Ma, T. Meerloo, A. Beas, M. G. Farquhar. 2004. Regulation of epidermal growth factor receptor degradation by heterotrimeric G{alpha}s protein. Mol. Biol. Cell. 15: 5538-5550. [Abstract/Free Full Text]
  45. Zhang, Z. H., X. Zeng, X. L. Hu, M. Y. Zhu, R. Hou, F. J. Shen, L. Zhang, M. J. Liao, X. P. Lv. 2005. Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli. Anim. Biotechnol. 16: 117-126. [Medline]
  46. Cullen, B. R.. 2006. Is RNA interference involved in intrinsic antiviral immunity in mammals?. Nat. Immunol. 7: 563-567. [Medline]
  47. Oh, P., J. E. Schnitzer. 2001. Segregation of heterotrimeric G proteins in cell surface microdomains: Gq binds caveolin to concentrate in caveolae, whereas Gi and Gs target lipid rafts by default. Mol. Biol. Cell. 12: 685-698. [Abstract/Free Full Text]
  48. Exton, J. H.. 1996. Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins. Annu. Rev. Pharmacol. Toxicol. 36: 481-509. [Medline]
  49. Murray, J., C. Ward, J. T. O’Flaherty, I. Dransfield, C. Haslett, E. R. Chilvers, A. G. Rossi. 2003. Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis. Br. J. Pharmacol. 139: 388-398. [Medline]



This article has been cited by other articles:


Home page
 BloodHome page
C. H. Serezani, D. M. Aronoff, R. G. Sitrin, and M. Peters-Golden
Fc{gamma}RI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions
Blood, October 8, 2009; 114(15): 3316 - 3324.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. P. Lee, C. H. Serezani, A. I. Medeiros, M. N. Ballinger, and M. Peters-Golden
Crosstalk between Prostaglandin E2 and Leukotriene B4 Regulates Phagocytosis in Alveolar Macrophages via Combinatorial Effects on Cyclic AMP
J. Immunol., January 1, 2009; 182(1): 530 - 537.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. I. Medeiros, A. Sa-Nunes, W. M. Turato, A. Secatto, F. G. Frantz, C. A. Sorgi, C. H. Serezani, G. S. Deepe Jr., and L. H. Faccioli
Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells
J. Immunol., December 15, 2008; 181(12): 8544 - 8551.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Chung, C. H. Serezani, S. K. Huang, J. N. H. Stern, D. B. Keskin, R. Jagirdar, T. G. Brock, D. M. Aronoff, and M. Peters-Golden
Rap1 Activation Is Required for Fc{gamma} Receptor-Dependent Phagocytosis
J. Immunol., October 15, 2008; 181(8): 5501 - 5509.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Canetti, C. H. Serezani, R. G. Atrasz, E. S. White, D. M. Aronoff, and M. Peters-Golden
Activation of Phosphatase and Tensin Homolog on Chromosome 10 Mediates the Inhibition of Fc{gamma}R Phagocytosis by Prostaglandin E2 in Alveolar Macrophages
J. Immunol., December 15, 2007; 179(12): 8350 - 8356.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peres, C. M.
Right arrow Articles by Peters-Golden, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peres, C. M.
Right arrow Articles by Peters-Golden, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS