Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by
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免疫学杂志 2005年第2期
Abstract
cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14–22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3',5'-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B4 and TNF-, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H2O2 production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.
Introduction
Since its discovery, cAMP remains the archetypal "second messenger" responsible for directing cellular responses to extracellular signals. In the alveolar macrophage (AM),4 cAMP has largely inhibitory effects on a variety of components of cell activation, including phagocytosis (1), reactive oxygen intermediate (ROI) generation (2), and the production of inflammatory mediators such as TNF- (3). Modulation of macrophage activation is important for immunoregulation, yet the downstream mechanisms involved in these cAMP effects are incompletely defined. Classically, cAMP signaling involves the immediate activation of protein kinase A (PKA), which phosphorylates myriad downstream targets, such as the CREB. However, PKA-independent actions of cAMP have been recognized in various experimental systems. Recently, novel targets for cAMP signaling have been described that affect changes in cell function independently of PKA. These include cyclic nucleotide gated channels involved in the transduction of olfactory and visual signals and the guanine exchange proteins directly activated by cAMP (Epac-1 and -2) (4, 5).
Epac expression has been described in diverse cell types. A functional role for Epac-1 in integrin-mediated adhesion has been shown in nonmyeloid cell lines (6, 7), whereas Epac-2 appears to be important for pancreatic cell insulin granule exocytosis (8). In leukocytes, Epac-1 mRNA transcripts were detected in circulating human B cells, but not in peripheral blood T cells, monocytes, or neutrophils (9). Apart from this, no information about Epac-1 expression in primary myeloid cells, or its role in such cells, is available. We sought to examine the roles of Epac-1 and PKA in macrophage function. We focused on AMs, because these cells serve important functions as the resident immune effector cell in the distal lung. We now describe for the first time the presence of Epac-1 in primary phagocytic cells, and characterize the respective roles of Epac-1 vs PKA in the inhibitory effects of cAMP on various aspects of cell activation.
Materials and Methods
Animals and reagents
Wistar rats (Charles River Laboratories; 125–150 g, female) were 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. Myristoylated PKA inhibitory peptide 14-22 (PKI14–22) and Escherichia coli (055:B5) LPS were from Sigma-Aldrich. PGE2 was from Cayman Chemical. Calcium ionophore A23187, forskolin, and the PKA inhibitors H-89 and KT-5720 were from Calbiochem. cAMP analogs were from Biolog LSI. Experimental compounds showed no adverse effects on cell viability as determined by a trypan blue exclusion assay (not shown).
Cell isolation and culture
NR8383 rat AMs and RAW 264.7 murine macrophages (American Type Culture Collection) were maintained in RPMI 1640 containing 10% FBS and 1% antibiotics (complete medium) before use. Human AMs were obtained from healthy individuals by bronchoalveolar lavage as reported previously (10). Resident AMs from rats were obtained via ex vivo lung lavage as described (11) and were cultured in complete medium overnight before use, whereas cell lines were used on the day of subculturing.
Immunoblot analysis
Western blots were performed as previously described (12). Protein samples (90 μg for Epac-1, 50 μg for Epac-2) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with commercially available rabbit polyclonal Epac-1 (1:500; Upstate) or Epac-2 (1:300; Santa Cruz Biotechnology) Abs, followed by HRP-conjugated anti-rabbit secondary Abs and ECL Plus chemiluminescence detection reagents (Amersham Biosciences). Positive control cerebellar tissue lysate for Epac-2 came from Santa Cruz Biotechnology.
Phagocytosis and bacterial killing assays
Phagocytosis of either IgG-opsonized, FITC-labeled E. coli (Molecular Probes) or IgG-opsonized SRBC by rat AMs was assessed as previously described (1). The ability of Klebsiella pneumoniae to survive intracellularly following phagocytosis was assessed using a tetrazolium dye (MTT) reduction assay as described elsewhere (13). Preliminary experiments established that this assay provided similar results as did conventional CFU-based killing assays (not shown).
Assessment of AM H2O2 production
A solution containing 50 μM Amplex Red reagent (Molecular Probes), 10 U/ml HRP, and 3% rat immune serum-opsonized K. pneumoniae was prepared in PBS. AMs (5 x 105 cells/well) were pretreated for 30 min with compounds of interest as indicated in the figures before the addition of 0.1 ml of the above solution (50 bacteria per AM). Plates were incubated (37°C for 1 h), and the H2O2 concentration was determined according to the manufacturer’s instructions.
Leukotriene (LT)B4 and TNF- measurements
Rat AMs or NR8383 cells were cultured in 96-well plates (1 x 105 cells/well) in RPMI 1640. For LTB4 experiments, cultures were incubated for 15 min in the presence or absence of compounds of interest and then exposed to calcium ionophore A23187 (10 μM) for an additional 30 min to stimulate LTB4 production. LTB4 levels were quantified in culture supernatants by enzyme immunoassay (EIA; Assay Designs). Separately, cells were treated with compounds of interest for 60 min before the addition of LPS (100 ng/ml) in RPMI 1640 containing 1% FBS for 16 h. TNF- levels were quantified in supernatants by EIA (Assay Designs).
PKA activation assay
Rat AMs or NR-8383 cells (3 x 106 per well) were treated with vehicle, N6-benzoyladenosine-3',5'-cAMP (6-Bnz-cAMP) (2 mM), or 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP (8-pCPT-2'-O-Me-cAMP) (2 mM) for 15 min, and PKA activity was determined in whole-cell lysates using the SignaTECT PKA assay kit (Promega).
Statistical analysis
Data are represented as mean ± SE. Comparisons were performed with ANOVA followed by the Bonferroni test. Differences were considered significant if p < 0.05. Experiments were performed on 3 separate occasions.
Results and Discussion
cAMP-mediated inhibition of FcR-mediated phagocytosis in AMs is PKA independent
We previously found that PGE2 inhibited FcR-mediated phagocytosis by AMs through a cAMP-dependent mechanism (1). We sought to determine whether pharmacological inhibition of PKA could prevent the inhibitory effects of PGE2. As expected (Fig. 1A), pretreatment with PGE2 inhibited the ingestion of IgG-opsonized E. coli by rat AMs. However, preincubation of AMs with the standard PKA inhibitors H-89 and KT-5720 failed to prevent this inhibition. Because both H-89 and KT-5720 may inhibit kinases other than PKA, we used the highly specific, myristoylated peptide PKA inhibitor PKI14–22. As illustrated, PKI14–22 also failed to prevent inhibition of phagocytosis by PGE2. We verified these results using IgG-opsonized SRBC as an alternative phagocytic target (Fig. 1B). In addition, PKA inhibition did not prohibit the ability of forskolin, a direct activator of adenylate cyclase, from inhibiting FcR-mediated phagocytosis by AMs (Fig. 1A). Each of the three PKA inhibitors by itself had a small suppressive effect on phagocytosis (10% inhibition; data not shown), suggesting a requirement for PKA in FcR-mediated phagocytosis, which has also been observed in human neutrophils (14). These data suggest that an alternative, PKA-independent signaling pathway must be responsible for inhibiting phagocytosis.
FIGURE 1. cAMP-induced suppression of FcR-mediated phagocytosis by AMs is PKA independent. Rat AMs were pretreated with the PKA inhibitors H-89, KT-5720, or PKI14–22 (10 μM each), or vehicle followed by PGE2 (1 μM), forskolin (100 μM), or vehicle for 5 min, and then challenged with IgG-opsonized E. coli (A) or IgG-opsonized SRBC (B). Phagocytosis is expressed as percent inhibition compared with the control, to which no compounds were added. Results represent the mean (±SE) for three independent experiments performed in quadruplicate. *, p < 0.05 vs control.
Epac expression in macrophages
Because nothing is known about the presence or role(s) of Epac-1 or -2 in macrophages of any type, we assessed the presence of these isoforms in human and rat AMs. As demonstrated (Fig. 2A), only Epac-1 was expressed in these cells and its presence was also documented in the NR8383 rat AM cell line and RAW 264.7 macrophages. Epac-2 expression was documented in cerebellum, as expected (5), and to a minimal extent in RAW 264.7 cells, but not in AMs.
FIGURE 2. Expression of Epac proteins and their role in FcR-mediated phagocytosis. A, The presence of Epac-1 and -2 were determined by Western blotting using cell lysates from human and rat AMs, NR8383 cells, RAW 264.7 macrophages, and rat cerebellum. Representative results are shown. Bands are 115 kDa for Epac-1 and -2 proteins. B and C, Inhibition of FcR-mediated phagocytosis by cAMP analogs. B, Rat AMs were pretreated for 30 min with the dual PKA/Epac agonist (S)-p-8-(4-chloro-phenylthio)adenosine-3',5'-cAMP, the PKA specific agonist 6-Bnz-cAMP, or the Epac-1 specific agonist 8-pCPT-2'-O-Me-cAMP (2 mM each) or vehicle control, and then challenged with IgG-opsonized E. coli or SRBC. C, Rat AMs were pretreated for 30 min with 8-pCPT-2'-O-Me-cAMP or vehicle control, and then challenged with IgG-opsonized E. coli or SRBC. Phagocytosis is expressed as percent inhibition compared with the control, to which no compounds were added. Results show means ± SE for a representative experiment of three independent experiments performed in quadruplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control.
Selective Epac-1 activation inhibits FcR-mediated phagocytosis
Recently, phosphodiesterase-resistant cAMP analogs that are highly selective in their activation of either PKA or Epac have been developed (15). We used the best characterized of these compounds, the PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP (15). The specificity of these agents was confirmed by assessing the degree of PKA activation in both AMs and NR8383 cells. In these experiments, 8-pCPT-2'-O-Me-cAMP (2 mM) did not significantly alter PKA activity, whereas 6-Bnz-cAMP (2 mM) enhanced PKA activity in both AMs (2.67 ± 0.04-fold; p < 0.01; n = 3) and NR8383 cells (2.05 ± 0.04-fold; p < 0.01; n = 1) compared with untreated cells (data not shown). Such specificity of 8-pCPT-2'-O-Me-cAMP, even at millimolar concentrations, has been reported previously (16).
We investigated the effects of these cAMP analogs as well as the nonspecific (PKA and Epac) activator (S)-p-8-(4-chloro-phenylthio)adenosine-3',5'-cAMP on FcR-mediated phagocytosis. As shown (Fig. 2B), phagocytosis of IgG-opsonized targets was inhibited by the nonspecific PKA/Epac activator, whereas the PKA-specific activator 6-Bnz-cAMP failed to inhibit phagocytosis at concentrations as high as 2 mM. Mostnotably, the Epac activator suppressed phagocytosis to the same degree as the nonspecific cAMP analog, and this effect was dose dependent (Fig. 2C). These compounds clearly exerted distinctly different effects on phagocytosis at the indicated concentrations. The observed maximal degree of inhibition by PGE2 or 8-pCPT-2'-O-Me-cAMP differed between the two models of FcR-mediated phagocytosis used. We previously observed this difference for PGE2 (1), which likely reflects differences between the phagocytic targets used, different target-to-AM ratios, the source/type of opsonin used, and/or differences in assay sensitivity.
The mechanism by which Epac-1 activation inhibits phagocytosis remains unclear. Epac-1 activates the small GTPases Rap1 and Rap2 (4), and Rap1 was found to associate with late endocytic/phagocytic compartments of J774.A1 macrophage-like cells (17). In addition, Rap1 was shown to play a positive role in complement-mediated phagocytosis by the same cell line, through the functional activation of the macrophage integrin, M2 (18). However, Rap1 overexpression or inhibition in the J774.A1 cells did not affect FcR-mediated phagocytosis (18). Furthermore, although we have observed that PGE2, forskolin, and 8-pCPT-2'-O-Me-cAMP can induce Rap1 activation in primary rat AMs, we also found that Rap1 was activated by selective stimulation of PKA in these cells (data not shown). Thus, it seems unlikely that Rap-1 activation alone provides the basis for the distinct effects of Epac-1 on FcR phagocytosis in the AM. Whether Rap-independent pathways are involved in our model (such as the activation of the stress-activated c-Jun protein kinase cascade (19)) remains to be explored.
PKA activation suppresses inflammatory mediator synthesis by AMs
PGE2 and other cAMP-elevating compounds can alter the production of cytokines, chemokines, and lipid mediators by inflammatory cells (20, 21). As illustrated (Fig. 3A), PGE2 suppressed ionophore-stimulated LTB4 production by primary rat AMs, and this was mimicked by the PKA activator, whereas the Epac agonist had no effect. A similar profile was observed for LPS-stimulated TNF- production in both NR8383 cells and rat AMs (Fig. 3, B and C). These data implicate PKA, but not Epac-1, in the regulation of inflammatory mediator synthesis by AMs and further highlight the specificity of these cAMP analogs. Although previous studies have defined a role for PKA in mediating the suppressive effects of cAMP on LTB4 and TNF- production (20, 22), the present studies are, to our knowledge, the first to examine, and exclude, the potential involvement of Epac-1 in this setting.
FIGURE 3. Regulation of AM production of LTB4 and TNF- by PKA. A, Rat AMs were pretreated for 15 min with PGE2 (1 μM), forskolin (100 μM), 8-pCPT-2'-O-Me-cAMP (2 mM), or 6-Bnz-cAMP (2 mM) followed by ionophore A23187 (10 μM) for 30 min. LTB4 levels were quantified by EIA and expressed as a percentage of the control to which no compounds were added. Shown are the mean (±SE) for three experiments performed in quintuplicate. B and C, NR8383 cells (B) or rat AMs (C) were cultured as described and then pretreated for 15 min with PGE2 (1 μM), 8-pCPT-2'-O-Me-cAMP, or 6-Bnz-cAMP at the concentrations noted. Cells were then incubated for 16 h at 37°C, and TNF- levels were quantified in the culture supernatants by EIA. Shown are the mean (±SE) for a representative of three independent experiments. *, p < 0.05; **, p < 0.01 vs control.
Epac-1 and PKA are both involved in the suppression of AM microbicidal activity
Elevations in cAMP suppressed the microbicidal activity of THP-1 monocytes infected with Brucella in a PKA-dependent manner (23). However, the influence of Epac-1 activation on bacterial killing was not assessed and remains unknown. Furthermore, the ability of cAMP to regulate the bactericidal activity of AMs has not been determined. Thus, we assessed the effect of cAMP elevation and selective PKA or Epac-1 activation on the ability of rat AMs to kill immune serum-opsonized K. pneumoniae, which are ingested largely via FcR-mediated pathways (24). We previously found that cAMP elevation impaired the phagocytosis of opsonized K. pneumoniae (1). We now demonstrate that PGE2 also inhibited the ability of AMs to kill successfully ingested bacteria compared with untreated cells (Fig. 4A). Interestingly, both 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP inhibited AM microbicidal activity to the same degree as PGE2. The specific mechanisms whereby cAMP negatively regulates bacterial killing are unclear. The production of ROIs (such as H2O2) by NADPH oxidase represents a key bactericidal mechanism of the macrophage, and AM killing of K. pneumoniae depends on NADPH oxidase activity (our unpublished data). In addition, Dent et al. (2) showed that cAMP inhibited zymosan-stimulated H2O2 production by human AMs. As shown in Fig. 4B, infection with immune serum-opsonized K. pneumoniae provoked a 15-fold increase in AM H2O2 production. PGE2, 8-pCPT-2'-O-Me-cAMP, and 6-Bnz-cAMP each suppressed AM H2O2 production 30–40% compared with untreated, infected cells. Although effects on AM bactericidal activity and H2O2 production were parallel, it is uncertain whether cAMP-induced reduction in ROI production alone accounts for the impairment of bacterial killing. Nonetheless, these results implicate both Epac-1 and PKA activation in the suppression of AM microbicidal function and ROI generation.
FIGURE 4. Activation of PKA or Epac-1 suppresses AM microbicidal activity and H2O2 generation. A, Following infection and phagocytosis of immune serum-opsonized K. pneumoniae (multiplicity of infection, 50:1) for 30 min (37°C), rat AMs were either treated with PGE2 (100 nM), 8-pCPT-2'-O-Me-cAMP (2 mM), 6-Bnz-cAMP (2 mM), or vehicle for 90 min at 37°C (to allow bacterial killing), or were placed at 4°C (phagocytosis control) (13 ). The survival of ingested bacteria is expressed relative to the 4°C control (dashed line). As indicated, untreated cells killed 30% of phagocytosed bacteria. Data are the mean (±SE) of a representative of three experiments performed in quadruplicate. *, p < 0.05 vs control. B, AMs were pretreated with PGE2 (100 nM), 8-pCPT-2'-O-Me-cAMP (2 mM), 6-Bnz-cAMP (2 mM), or vehicle for 30 min followed by infection with opsonized K. pneumoniae. H2O2 production was assessed in culture supernatants. *, p < 0.05 vs uninfected cells; #, p < 0.05 vs infected but untreated AMs.
Conclusion
We demonstrate for the first time the presence of Epac-1 in primary macrophages and a role for this protein in a key aspect of host defense, FcR-mediated phagocytosis. We further show that cAMP-dependent immunomodulatory effects on the AM result from the activation of distinct PKA- and/or Epac-1-mediated pathways. These studies are, to our knowledge, the first to identify specific roles for PKA vs Epac in the regulation of phagocyte function. Our findings have important implications for efforts to pharmacologically modulate inflammatory and innate immune processes. Future experiments are necessary to understand the mechanistic basis for the specificity of these distinct cAMP effectors in modulating various macrophage functions and to determine the involvement of Epac in the phagocytic and microbicidal actions of other leukocytes, such as neutrophils.
Acknowledgments
We thank Teresa Marshall for technical assistance, and Dr. Michael Coffey for human AM lysates.
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 National Institutes of Health Grants HL007749 and HL058897, the Parker B. Francis Foundation, Conselho Nacional de Pesquisa (Brazil), and Coordena??o de Aperfei?oamento de Pessoal de Nível Superior.
2 D.M.A. and C.C. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Marc Peters-Golden, 6301 Medical Science Research Building III, Box 0642, 1150 West Medical Center Drive, University of Michigan Health System, Ann Arbor, MI 48109-0642. E-mail address: petersm@umich.edu
4 Abbreviations used in this paper: AM, alveolar macrophage; ROI, reactive oxygen intermediate; PKA, protein kinase A; Epac, exchange protein directly activated by cAMP; LT, leukotriene; EIA, enzyme immunoassay; PKI14-22, myristoylated PKA inhibitory peptide 14-22; 6-Bnz-cAMP, N6-benzoyladenosine-3',5'-cAMP; 8-pCPT-2'-O-Me-cAMP, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP.
Received for publication September 28, 2004. Accepted for publication November 1, 2004.
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cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14–22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3',5'-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B4 and TNF-, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H2O2 production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.
Introduction
Since its discovery, cAMP remains the archetypal "second messenger" responsible for directing cellular responses to extracellular signals. In the alveolar macrophage (AM),4 cAMP has largely inhibitory effects on a variety of components of cell activation, including phagocytosis (1), reactive oxygen intermediate (ROI) generation (2), and the production of inflammatory mediators such as TNF- (3). Modulation of macrophage activation is important for immunoregulation, yet the downstream mechanisms involved in these cAMP effects are incompletely defined. Classically, cAMP signaling involves the immediate activation of protein kinase A (PKA), which phosphorylates myriad downstream targets, such as the CREB. However, PKA-independent actions of cAMP have been recognized in various experimental systems. Recently, novel targets for cAMP signaling have been described that affect changes in cell function independently of PKA. These include cyclic nucleotide gated channels involved in the transduction of olfactory and visual signals and the guanine exchange proteins directly activated by cAMP (Epac-1 and -2) (4, 5).
Epac expression has been described in diverse cell types. A functional role for Epac-1 in integrin-mediated adhesion has been shown in nonmyeloid cell lines (6, 7), whereas Epac-2 appears to be important for pancreatic cell insulin granule exocytosis (8). In leukocytes, Epac-1 mRNA transcripts were detected in circulating human B cells, but not in peripheral blood T cells, monocytes, or neutrophils (9). Apart from this, no information about Epac-1 expression in primary myeloid cells, or its role in such cells, is available. We sought to examine the roles of Epac-1 and PKA in macrophage function. We focused on AMs, because these cells serve important functions as the resident immune effector cell in the distal lung. We now describe for the first time the presence of Epac-1 in primary phagocytic cells, and characterize the respective roles of Epac-1 vs PKA in the inhibitory effects of cAMP on various aspects of cell activation.
Materials and Methods
Animals and reagents
Wistar rats (Charles River Laboratories; 125–150 g, female) were 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. Myristoylated PKA inhibitory peptide 14-22 (PKI14–22) and Escherichia coli (055:B5) LPS were from Sigma-Aldrich. PGE2 was from Cayman Chemical. Calcium ionophore A23187, forskolin, and the PKA inhibitors H-89 and KT-5720 were from Calbiochem. cAMP analogs were from Biolog LSI. Experimental compounds showed no adverse effects on cell viability as determined by a trypan blue exclusion assay (not shown).
Cell isolation and culture
NR8383 rat AMs and RAW 264.7 murine macrophages (American Type Culture Collection) were maintained in RPMI 1640 containing 10% FBS and 1% antibiotics (complete medium) before use. Human AMs were obtained from healthy individuals by bronchoalveolar lavage as reported previously (10). Resident AMs from rats were obtained via ex vivo lung lavage as described (11) and were cultured in complete medium overnight before use, whereas cell lines were used on the day of subculturing.
Immunoblot analysis
Western blots were performed as previously described (12). Protein samples (90 μg for Epac-1, 50 μg for Epac-2) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with commercially available rabbit polyclonal Epac-1 (1:500; Upstate) or Epac-2 (1:300; Santa Cruz Biotechnology) Abs, followed by HRP-conjugated anti-rabbit secondary Abs and ECL Plus chemiluminescence detection reagents (Amersham Biosciences). Positive control cerebellar tissue lysate for Epac-2 came from Santa Cruz Biotechnology.
Phagocytosis and bacterial killing assays
Phagocytosis of either IgG-opsonized, FITC-labeled E. coli (Molecular Probes) or IgG-opsonized SRBC by rat AMs was assessed as previously described (1). The ability of Klebsiella pneumoniae to survive intracellularly following phagocytosis was assessed using a tetrazolium dye (MTT) reduction assay as described elsewhere (13). Preliminary experiments established that this assay provided similar results as did conventional CFU-based killing assays (not shown).
Assessment of AM H2O2 production
A solution containing 50 μM Amplex Red reagent (Molecular Probes), 10 U/ml HRP, and 3% rat immune serum-opsonized K. pneumoniae was prepared in PBS. AMs (5 x 105 cells/well) were pretreated for 30 min with compounds of interest as indicated in the figures before the addition of 0.1 ml of the above solution (50 bacteria per AM). Plates were incubated (37°C for 1 h), and the H2O2 concentration was determined according to the manufacturer’s instructions.
Leukotriene (LT)B4 and TNF- measurements
Rat AMs or NR8383 cells were cultured in 96-well plates (1 x 105 cells/well) in RPMI 1640. For LTB4 experiments, cultures were incubated for 15 min in the presence or absence of compounds of interest and then exposed to calcium ionophore A23187 (10 μM) for an additional 30 min to stimulate LTB4 production. LTB4 levels were quantified in culture supernatants by enzyme immunoassay (EIA; Assay Designs). Separately, cells were treated with compounds of interest for 60 min before the addition of LPS (100 ng/ml) in RPMI 1640 containing 1% FBS for 16 h. TNF- levels were quantified in supernatants by EIA (Assay Designs).
PKA activation assay
Rat AMs or NR-8383 cells (3 x 106 per well) were treated with vehicle, N6-benzoyladenosine-3',5'-cAMP (6-Bnz-cAMP) (2 mM), or 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP (8-pCPT-2'-O-Me-cAMP) (2 mM) for 15 min, and PKA activity was determined in whole-cell lysates using the SignaTECT PKA assay kit (Promega).
Statistical analysis
Data are represented as mean ± SE. Comparisons were performed with ANOVA followed by the Bonferroni test. Differences were considered significant if p < 0.05. Experiments were performed on 3 separate occasions.
Results and Discussion
cAMP-mediated inhibition of FcR-mediated phagocytosis in AMs is PKA independent
We previously found that PGE2 inhibited FcR-mediated phagocytosis by AMs through a cAMP-dependent mechanism (1). We sought to determine whether pharmacological inhibition of PKA could prevent the inhibitory effects of PGE2. As expected (Fig. 1A), pretreatment with PGE2 inhibited the ingestion of IgG-opsonized E. coli by rat AMs. However, preincubation of AMs with the standard PKA inhibitors H-89 and KT-5720 failed to prevent this inhibition. Because both H-89 and KT-5720 may inhibit kinases other than PKA, we used the highly specific, myristoylated peptide PKA inhibitor PKI14–22. As illustrated, PKI14–22 also failed to prevent inhibition of phagocytosis by PGE2. We verified these results using IgG-opsonized SRBC as an alternative phagocytic target (Fig. 1B). In addition, PKA inhibition did not prohibit the ability of forskolin, a direct activator of adenylate cyclase, from inhibiting FcR-mediated phagocytosis by AMs (Fig. 1A). Each of the three PKA inhibitors by itself had a small suppressive effect on phagocytosis (10% inhibition; data not shown), suggesting a requirement for PKA in FcR-mediated phagocytosis, which has also been observed in human neutrophils (14). These data suggest that an alternative, PKA-independent signaling pathway must be responsible for inhibiting phagocytosis.
FIGURE 1. cAMP-induced suppression of FcR-mediated phagocytosis by AMs is PKA independent. Rat AMs were pretreated with the PKA inhibitors H-89, KT-5720, or PKI14–22 (10 μM each), or vehicle followed by PGE2 (1 μM), forskolin (100 μM), or vehicle for 5 min, and then challenged with IgG-opsonized E. coli (A) or IgG-opsonized SRBC (B). Phagocytosis is expressed as percent inhibition compared with the control, to which no compounds were added. Results represent the mean (±SE) for three independent experiments performed in quadruplicate. *, p < 0.05 vs control.
Epac expression in macrophages
Because nothing is known about the presence or role(s) of Epac-1 or -2 in macrophages of any type, we assessed the presence of these isoforms in human and rat AMs. As demonstrated (Fig. 2A), only Epac-1 was expressed in these cells and its presence was also documented in the NR8383 rat AM cell line and RAW 264.7 macrophages. Epac-2 expression was documented in cerebellum, as expected (5), and to a minimal extent in RAW 264.7 cells, but not in AMs.
FIGURE 2. Expression of Epac proteins and their role in FcR-mediated phagocytosis. A, The presence of Epac-1 and -2 were determined by Western blotting using cell lysates from human and rat AMs, NR8383 cells, RAW 264.7 macrophages, and rat cerebellum. Representative results are shown. Bands are 115 kDa for Epac-1 and -2 proteins. B and C, Inhibition of FcR-mediated phagocytosis by cAMP analogs. B, Rat AMs were pretreated for 30 min with the dual PKA/Epac agonist (S)-p-8-(4-chloro-phenylthio)adenosine-3',5'-cAMP, the PKA specific agonist 6-Bnz-cAMP, or the Epac-1 specific agonist 8-pCPT-2'-O-Me-cAMP (2 mM each) or vehicle control, and then challenged with IgG-opsonized E. coli or SRBC. C, Rat AMs were pretreated for 30 min with 8-pCPT-2'-O-Me-cAMP or vehicle control, and then challenged with IgG-opsonized E. coli or SRBC. Phagocytosis is expressed as percent inhibition compared with the control, to which no compounds were added. Results show means ± SE for a representative experiment of three independent experiments performed in quadruplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control.
Selective Epac-1 activation inhibits FcR-mediated phagocytosis
Recently, phosphodiesterase-resistant cAMP analogs that are highly selective in their activation of either PKA or Epac have been developed (15). We used the best characterized of these compounds, the PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP (15). The specificity of these agents was confirmed by assessing the degree of PKA activation in both AMs and NR8383 cells. In these experiments, 8-pCPT-2'-O-Me-cAMP (2 mM) did not significantly alter PKA activity, whereas 6-Bnz-cAMP (2 mM) enhanced PKA activity in both AMs (2.67 ± 0.04-fold; p < 0.01; n = 3) and NR8383 cells (2.05 ± 0.04-fold; p < 0.01; n = 1) compared with untreated cells (data not shown). Such specificity of 8-pCPT-2'-O-Me-cAMP, even at millimolar concentrations, has been reported previously (16).
We investigated the effects of these cAMP analogs as well as the nonspecific (PKA and Epac) activator (S)-p-8-(4-chloro-phenylthio)adenosine-3',5'-cAMP on FcR-mediated phagocytosis. As shown (Fig. 2B), phagocytosis of IgG-opsonized targets was inhibited by the nonspecific PKA/Epac activator, whereas the PKA-specific activator 6-Bnz-cAMP failed to inhibit phagocytosis at concentrations as high as 2 mM. Mostnotably, the Epac activator suppressed phagocytosis to the same degree as the nonspecific cAMP analog, and this effect was dose dependent (Fig. 2C). These compounds clearly exerted distinctly different effects on phagocytosis at the indicated concentrations. The observed maximal degree of inhibition by PGE2 or 8-pCPT-2'-O-Me-cAMP differed between the two models of FcR-mediated phagocytosis used. We previously observed this difference for PGE2 (1), which likely reflects differences between the phagocytic targets used, different target-to-AM ratios, the source/type of opsonin used, and/or differences in assay sensitivity.
The mechanism by which Epac-1 activation inhibits phagocytosis remains unclear. Epac-1 activates the small GTPases Rap1 and Rap2 (4), and Rap1 was found to associate with late endocytic/phagocytic compartments of J774.A1 macrophage-like cells (17). In addition, Rap1 was shown to play a positive role in complement-mediated phagocytosis by the same cell line, through the functional activation of the macrophage integrin, M2 (18). However, Rap1 overexpression or inhibition in the J774.A1 cells did not affect FcR-mediated phagocytosis (18). Furthermore, although we have observed that PGE2, forskolin, and 8-pCPT-2'-O-Me-cAMP can induce Rap1 activation in primary rat AMs, we also found that Rap1 was activated by selective stimulation of PKA in these cells (data not shown). Thus, it seems unlikely that Rap-1 activation alone provides the basis for the distinct effects of Epac-1 on FcR phagocytosis in the AM. Whether Rap-independent pathways are involved in our model (such as the activation of the stress-activated c-Jun protein kinase cascade (19)) remains to be explored.
PKA activation suppresses inflammatory mediator synthesis by AMs
PGE2 and other cAMP-elevating compounds can alter the production of cytokines, chemokines, and lipid mediators by inflammatory cells (20, 21). As illustrated (Fig. 3A), PGE2 suppressed ionophore-stimulated LTB4 production by primary rat AMs, and this was mimicked by the PKA activator, whereas the Epac agonist had no effect. A similar profile was observed for LPS-stimulated TNF- production in both NR8383 cells and rat AMs (Fig. 3, B and C). These data implicate PKA, but not Epac-1, in the regulation of inflammatory mediator synthesis by AMs and further highlight the specificity of these cAMP analogs. Although previous studies have defined a role for PKA in mediating the suppressive effects of cAMP on LTB4 and TNF- production (20, 22), the present studies are, to our knowledge, the first to examine, and exclude, the potential involvement of Epac-1 in this setting.
FIGURE 3. Regulation of AM production of LTB4 and TNF- by PKA. A, Rat AMs were pretreated for 15 min with PGE2 (1 μM), forskolin (100 μM), 8-pCPT-2'-O-Me-cAMP (2 mM), or 6-Bnz-cAMP (2 mM) followed by ionophore A23187 (10 μM) for 30 min. LTB4 levels were quantified by EIA and expressed as a percentage of the control to which no compounds were added. Shown are the mean (±SE) for three experiments performed in quintuplicate. B and C, NR8383 cells (B) or rat AMs (C) were cultured as described and then pretreated for 15 min with PGE2 (1 μM), 8-pCPT-2'-O-Me-cAMP, or 6-Bnz-cAMP at the concentrations noted. Cells were then incubated for 16 h at 37°C, and TNF- levels were quantified in the culture supernatants by EIA. Shown are the mean (±SE) for a representative of three independent experiments. *, p < 0.05; **, p < 0.01 vs control.
Epac-1 and PKA are both involved in the suppression of AM microbicidal activity
Elevations in cAMP suppressed the microbicidal activity of THP-1 monocytes infected with Brucella in a PKA-dependent manner (23). However, the influence of Epac-1 activation on bacterial killing was not assessed and remains unknown. Furthermore, the ability of cAMP to regulate the bactericidal activity of AMs has not been determined. Thus, we assessed the effect of cAMP elevation and selective PKA or Epac-1 activation on the ability of rat AMs to kill immune serum-opsonized K. pneumoniae, which are ingested largely via FcR-mediated pathways (24). We previously found that cAMP elevation impaired the phagocytosis of opsonized K. pneumoniae (1). We now demonstrate that PGE2 also inhibited the ability of AMs to kill successfully ingested bacteria compared with untreated cells (Fig. 4A). Interestingly, both 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP inhibited AM microbicidal activity to the same degree as PGE2. The specific mechanisms whereby cAMP negatively regulates bacterial killing are unclear. The production of ROIs (such as H2O2) by NADPH oxidase represents a key bactericidal mechanism of the macrophage, and AM killing of K. pneumoniae depends on NADPH oxidase activity (our unpublished data). In addition, Dent et al. (2) showed that cAMP inhibited zymosan-stimulated H2O2 production by human AMs. As shown in Fig. 4B, infection with immune serum-opsonized K. pneumoniae provoked a 15-fold increase in AM H2O2 production. PGE2, 8-pCPT-2'-O-Me-cAMP, and 6-Bnz-cAMP each suppressed AM H2O2 production 30–40% compared with untreated, infected cells. Although effects on AM bactericidal activity and H2O2 production were parallel, it is uncertain whether cAMP-induced reduction in ROI production alone accounts for the impairment of bacterial killing. Nonetheless, these results implicate both Epac-1 and PKA activation in the suppression of AM microbicidal function and ROI generation.
FIGURE 4. Activation of PKA or Epac-1 suppresses AM microbicidal activity and H2O2 generation. A, Following infection and phagocytosis of immune serum-opsonized K. pneumoniae (multiplicity of infection, 50:1) for 30 min (37°C), rat AMs were either treated with PGE2 (100 nM), 8-pCPT-2'-O-Me-cAMP (2 mM), 6-Bnz-cAMP (2 mM), or vehicle for 90 min at 37°C (to allow bacterial killing), or were placed at 4°C (phagocytosis control) (13 ). The survival of ingested bacteria is expressed relative to the 4°C control (dashed line). As indicated, untreated cells killed 30% of phagocytosed bacteria. Data are the mean (±SE) of a representative of three experiments performed in quadruplicate. *, p < 0.05 vs control. B, AMs were pretreated with PGE2 (100 nM), 8-pCPT-2'-O-Me-cAMP (2 mM), 6-Bnz-cAMP (2 mM), or vehicle for 30 min followed by infection with opsonized K. pneumoniae. H2O2 production was assessed in culture supernatants. *, p < 0.05 vs uninfected cells; #, p < 0.05 vs infected but untreated AMs.
Conclusion
We demonstrate for the first time the presence of Epac-1 in primary macrophages and a role for this protein in a key aspect of host defense, FcR-mediated phagocytosis. We further show that cAMP-dependent immunomodulatory effects on the AM result from the activation of distinct PKA- and/or Epac-1-mediated pathways. These studies are, to our knowledge, the first to identify specific roles for PKA vs Epac in the regulation of phagocyte function. Our findings have important implications for efforts to pharmacologically modulate inflammatory and innate immune processes. Future experiments are necessary to understand the mechanistic basis for the specificity of these distinct cAMP effectors in modulating various macrophage functions and to determine the involvement of Epac in the phagocytic and microbicidal actions of other leukocytes, such as neutrophils.
Acknowledgments
We thank Teresa Marshall for technical assistance, and Dr. Michael Coffey for human AM lysates.
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 National Institutes of Health Grants HL007749 and HL058897, the Parker B. Francis Foundation, Conselho Nacional de Pesquisa (Brazil), and Coordena??o de Aperfei?oamento de Pessoal de Nível Superior.
2 D.M.A. and C.C. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Marc Peters-Golden, 6301 Medical Science Research Building III, Box 0642, 1150 West Medical Center Drive, University of Michigan Health System, Ann Arbor, MI 48109-0642. E-mail address: petersm@umich.edu
4 Abbreviations used in this paper: AM, alveolar macrophage; ROI, reactive oxygen intermediate; PKA, protein kinase A; Epac, exchange protein directly activated by cAMP; LT, leukotriene; EIA, enzyme immunoassay; PKI14-22, myristoylated PKA inhibitory peptide 14-22; 6-Bnz-cAMP, N6-benzoyladenosine-3',5'-cAMP; 8-pCPT-2'-O-Me-cAMP, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP.
Received for publication September 28, 2004. Accepted for publication November 1, 2004.
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