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Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by cAMP-1 This information is current as of October 2, 2021. David M. Aronoff, Claudio Canetti, Carlos H. Serezani, Ming Luo and Marc Peters-Golden J Immunol 2005; 174:595-599; ; doi: 10.4049/jimmunol.174.2.595

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JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by cAMP-11 David M. Aronoff,2*† Claudio Canetti,2† Carlos H. Serezani,†‡ Ming Luo,† and Marc Peters-Golden3†

cAMP has largely inhibitory effects on components of mac- tion of protein kinase A (PKA), which phosphorylates myriad rophage activation, yet downstream mechanisms involved downstream targets, such as the CREB. However, PKA-indepen- in these effects remain incompletely defined. Elevation of dent actions of cAMP have been recognized in various experimen- cAMP in alveolar macrophages (AMs) suppresses Fc␥R- tal systems. Recently, novel targets for cAMP signaling have been mediated phagocytosis. We now report that protein kinase described that affect changes in cell function independently of Downloaded from A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA. These include cyclic nucleotide gated channels involved in PKA inhibitory peptide 14–22) failed to prevent this sup- the transduction of olfactory and visual signals and the guanine expression in rat AMs. We identified the expression of the change proteins directly activated by cAMP (Epac-1 and -2) (4, 5). alternative cAMP target, exchange protein directly acti- Epac expression has been described in diverse cell types. A functional role for Epac-1 in integrin-mediated adhesion has vated by cAMP-1 (Epac-1), in human and rat AMs. been shown in nonmyeloid cell lines (6, 7), whereas Epac-2 ap- http://www.jimmunol.org/ Using cAMP analogs that are highly specific for PKA (N6- pears to be important for pancreatic ␤ cell insulin granule exo- -benzoyladenosine-3؅,5؅-cAMP) or Epac-1 (8-(4-chloro -؅ ؅ ؅ cytosis (8). In leukocytes, Epac-1 mRNA transcripts were de phenylthio)-2 -O-methyladenosine-3 ,5 -cAMP), we found tected in circulating human B cells, but not in peripheral blood that activation of Epac-1, but not PKA, dose-dependently T cells, monocytes, or neutrophils (9). Apart from this, no in- suppressed phagocytosis. By contrast, activation of PKA, formation about Epac-1 expression in primary myeloid cells, or but not Epac-1, suppressed AM production of leukotriene its role in such cells, is available. We sought to examine the roles ␣ B4 and TNF- , whereas stimulation of either PKA or of Epac-1 and PKA in macrophage function. We focused on

Epac-1 inhibited AM bactericidal activity and H2O2 pro- AMs, because these cells serve important functions as the resi- by guest on October 2, 2021 duction. These experiments now identify Epac-1 in pri- dent immune effector cell in the distal lung. We now describe mary macrophages, and define differential roles of Epac-1 for the first time the presence of Epac-1 in primary phagocytic vs PKA in the inhibitory effects of cAMP. The Journal of cells, and characterize the respective roles of Epac-1 vs PKA Immunology, 2005, 174: 595–599. in the inhibitory effects of cAMP on various aspects of cell activation. ince its discovery, cAMP remains the archetypal “second messenger” responsible for directing cellular responses to 4 Materials and Methods S extracellular signals. In the alveolar macrophage (AM), Animals and reagents cAMP has largely inhibitory effects on a variety of components of Wistar rats (Charles River Laboratories; 125–150 g, female) were treated ac- cell activation, including phagocytosis (1), reactive oxygen inter- cording to National Institutes of Health guidelines for the use of experimental mediate (ROI) generation (2), and the production of inflamma- animals with the approval of the University of Michigan Committee for the Use tory mediators such as TNF-␣ (3). Modulation of macrophage ac- and Care of Animals. Myristoylated PKA inhibitory peptide 14-22 (PKI14–22) tivation is important for immunoregulation, yet the downstream and Escherichia coli (055:B5) LPS were from Sigma-Aldrich. PGE2 was from Cayman Chemical. Calcium ionophore A23187, forskolin, and the PKA in- mechanisms involved in these cAMP effects are incompletely de- hibitors H-89 and KT-5720 were from Calbiochem. cAMP analogs were from fined. Classically, cAMP signaling involves the immediate activa- Biolog LSI. Experimental compounds showed no adverse effects on cell viability

Divisions of *Infectious Diseases and †Pulmonary and Critical Care Medicine, Depart- 2 D.M.A. and C.C. contributed equally to this work. ment of Internal Medicine, University of Michigan Health System, Ann Arbor, MI 48109; 3 Address correspondence and reprint requests to Dr. Marc Peters-Golden, 6301 Medical and ‡Department of Immunology, Instituto de Cieˆncias Biome´dicas-IV, University of Saõ Science Research Building III, Box 0642, 1150 West Medical Center Drive, University of Paulo, Saõ Paulo, Brazil Michigan Health System, Ann Arbor, MI 48109-0642. E-mail address: Received for publication September 28, 2004. Accepted for publication November [email protected] 1, 2004. 4 Abbreviations used in this paper: AM, alveolar macrophage; ROI, reactive oxygen inter- The costs of publication of this article were defrayed in part by the payment of page charges. mediate; PKA, protein kinase A; Epac, exchange protein directly activated by cAMP; LT, This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. leukotriene; EIA, enzyme immunoassay; PKI14-22, myristoylated PKA inhibitory peptide Section 1734 solely to indicate this fact. 14-22; 6-Bnz-cAMP, N6-benzoyladenosine-3Ј,5Ј-cAMP; 8-pCPT-2Ј-O-Me-cAMP, 8-(4-chloro-phenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cAMP. 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 Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 596 CUTTING EDGE: Epac-1 AND PKA INHIBIT AM FUNCTIONS 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 Cul- ture 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). Res- ident 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 ␮g for Epac-1, 50 ␮g 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 Biotechnol- FIGURE 1. cAMP-induced suppression of FcR-mediated phagocytosis by ogy) Abs, followed by HRP-conjugated anti-rabbit secondary Abs and ECL AMs is PKA independent. Rat AMs were pretreated with the PKA inhibitors Plus chemiluminescence detection reagents (Amersham Biosciences). Positive ␮ H-89, KT-5720, or PKI14–22 (10 M each), or vehicle followed by PGE2 (1 control cerebellar tissue lysate for Epac-2 came from Santa Cruz Biotechnology. ␮M), forskolin (100 ␮M), or vehicle for 5 min, and then challenged with IgG- Phagocytosis and bacterial killing assays opsonized E. coli (A) or IgG-opsonized SRBC (B). Phagocytosis is expressed as percent inhibition compared with the control, to which no compounds were Downloaded from Phagocytosis of either IgG-opsonized, FITC-labeled E. coli (Molecular Probes) added. Results represent the mean (ϮSE) for three independent experiments .p Ͻ 0.05 vs control ,ء .or IgG-opsonized SRBC by rat AMs was assessed as previously described (1). performed in quadruplicate 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). http://www.jimmunol.org/ Assessment of AM H2O2 production KT-5720 failed to prevent this inhibition. Because both H-89 and KT-5720 may inhibit kinases other than PKA, we used the A solution containing 50 ␮M Amplex Red reagent (Molecular Probes), 10 U/ml HRP, and 3% rat immune serum-opsonized K. pneumoniae was prepared highly specific, myristoylated peptide PKA inhibitor PKI14–22. ϫ 5 in PBS. AMs (5 10 cells/well) were pretreated for 30 min with compounds As illustrated, PKI14–22 also failed to prevent inhibition of of interest as indicated in the figures before the addition of 0.1 ml of the above ϳ phagocytosis by PGE2. We verified these results using IgG-op- solution ( 50 bacteria per AM). Plates were incubated (37°C for 1 h), and the sonized SRBC as an alternative phagocytic target (Fig. 1B). In H2O2 concentration was determined according to the manufacturer’s instruc- tions. addition, PKA inhibition did not prohibit the ability of forsko- ␣ lin, a direct activator of adenylate cyclase, from inhibiting FcR- Leukotriene (LT)B4 and TNF- measurements

mediated phagocytosis by AMs (Fig. 1A). Each of the three by guest on October 2, 2021 Rat AMs or NR8383 cells were cultured in 96-well plates (1 ϫ 105 cells/well) in PKA inhibitors by itself had a small suppressive effect on phago- RPMI 1640. For LTB4 experiments, cultures were incubated for 15 min in the cytosis (ϳ10% inhibition; data not shown), suggesting a re- presence or absence of compounds of interest and then exposed to calcium ␮ quirement for PKA in FcR-mediated phagocytosis, which has ionophore A23187 (10 M) for an additional 30 min to stimulate LTB4 production. LTB4 levels were quantified in culture supernatants by enzyme immu- also been observed in human neutrophils (14). These data sug- noassay (EIA; Assay Designs). Separately, cells were treated with compounds of gest that an alternative, PKA-independent signaling pathway 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 must be responsible for inhibiting phagocytosis. (Assay Designs). Epac expression in macrophages PKA activation assay Because nothing is known about the presence or role(s) of ϫ 6 Rat AMs or NR-8383 cells (3 10 per well) were treated with vehicle, Epac-1 or -2 in macrophages of any type, we assessed the pres- N6-benzoyladenosine-3Ј,5Ј-cAMP (6-Bnz-cAMP) (2 mM), or 8-(4-chloro- phenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cAMP (8-pCPT-2Ј-O-Me- ence of these isoforms in human and rat AMs. As demonstrated cAMP) (2 mM) for 15 min, and PKA activity was determined in whole-cell (Fig. 2A), only Epac-1 was expressed in these cells and its pres- lysates using the SignaTECT PKA assay kit (Promega). ence was also documented in the NR8383 rat AM cell line and Statistical analysis RAW 264.7 macrophages. Epac-2 expression was documented in cerebellum, as expected (5), and to a minimal extent in RAW Data are represented as mean Ϯ SE. Comparisons were performed with ANOVA followed by the Bonferroni test. Differences were considered signifi- 264.7 cells, but not in AMs. cant if p Ͻ 0.05. Experiments were performed on Ն3 separate occasions. Selective Epac-1 activation inhibits FcR-mediated phagocytosis Results and Discussion Recently, phosphodiesterase-resistant cAMP analogs that are cAMP-mediated inhibition of FcR-mediated phagocytosis in AMs is highly selective in their activation of either PKA or Epac have PKA independent been developed (15). We used the best characterized of these We previously found that PGE2 inhibited FcR-mediated compounds, the PKA activator 6-Bnz-cAMP and the Epac ac- phagocytosis by AMs through a cAMP-dependent mechanism tivator 8-pCPT-2Ј-O-Me-cAMP (15). The specificity of these (1). We sought to determine whether pharmacological inhibi- agents was confirmed by assessing the degree of PKA activation tion of PKA could prevent the inhibitory effects of PGE2.As in both AMs and NR8383 cells. In these experiments, 8-pCPT- Ј expected (Fig. 1A), pretreatment with PGE2 inhibited the in- 2 -O-Me-cAMP (2 mM) did not significantly alter PKA activ- gestion of IgG-opsonized E. coli by rat AMs. However, prein- ity, whereas 6-Bnz-cAMP (2 mM) enhanced PKA activity in cubation of AMs with the standard PKA inhibitors H-89 and both AMs (2.67 Ϯ 0.04-fold; p Ͻ 0.01; n ϭ 3) and NR8383 The Journal of Immunology 597

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 inte- ␣ ␤ grin, M 2 (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-2Ј-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 Downloaded from 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 LTB production by primary rat FIGURE 2. Expression of Epac proteins and their role in FcR-mediated 4 phagocytosis. A, The presence of Epac-1 and -2 were determined by Western AMs, and this was mimicked by the PKA activator, whereas the

blotting using cell lysates from human and rat AMs, NR8383 cells, RAW 264.7 http://www.jimmunol.org/ macrophages, and rat cerebellum. Representative results are shown. Bands are ϳ115 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-3Ј,5Ј-cAMP, the PKA specific agonist 6-Bnz-cAMP, or the Epac-1 specific agonist 8-pCPT- 2Ј-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-2Ј-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 by guest on October 2, 2021 means Ϯ SE for a representative experiment of three independent experiments p Ͻ 0.001 vs ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .performed in quadruplicate control. cells (2.05 Ϯ 0.04-fold; p Ͻ 0.01; n ϭ 1) compared with untreated cells (data not shown). Such specificity of 8-pCPT-2Ј- 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-3Ј,5Ј-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. Most notably, 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 dis- ␣ FIGURE 3. Regulation of AM production of LTB4 and TNF- by PKA. A, tinctly different effects on phagocytosis at the indicated concen- ␮ ␮ Rat AMs were pretreated for 15 min with PGE2 (1 M), forskolin (100 M), trations. The observed maximal degree of inhibition by PGE2 8-pCPT-2Ј-O-Me-cAMP (2 mM), or 6-Bnz-cAMP (2 mM) followed by iono- Ј ␮ or 8-pCPT-2 -O-Me-cAMP differed between the two models phore A23187 (10 M) for 30 min. LTB4 levels were quantified by EIA and of FcR-mediated phagocytosis used. We previously observed expressed as a percentage of the control to which no compounds were added. Shown are the mean (ϮSE) for three experiments performed in quintuplicate. this difference for PGE2 (1), which likely reflects differences be- B and C, NR8383 cells (B) or rat AMs (C) were cultured as described and then tween the phagocytic targets used, different target-to-AM ra- ␮ Ј pretreated for 15 min with PGE2 (1 M), 8-pCPT-2 -O-Me-cAMP, or 6-Bnz- tios, the source/type of opsonin used, and/or differences in assay cAMP at the concentrations noted. Cells were then incubated for 16 h at 37°C, sensitivity. and TNF-␣ levels were quantified in the culture supernatants by EIA. Shown ,ء .The mechanism by which Epac-1 activation inhibits phago- are the mean (ϮSE) for a representative of three independent experiments .p Ͻ 0.01 vs control ,ءء ;cytosis remains unclear. Epac-1 activates the small GTPases p Ͻ 0.05 598 CUTTING EDGE: Epac-1 AND PKA INHIBIT AM FUNCTIONS

Me-cAMP inhibited AM microbicidal activity to the same degree as PGE2. The specific mechanisms whereby cAMP nega- tively 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 unpub- lished 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 pro- Ј duction. PGE2, 8-pCPT-2 -O-Me-cAMP, and 6-Bnz-cAMP ϳ 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 ac- counts 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. Downloaded from Conclusion FIGURE 4. Activation of PKA or Epac-1 suppresses AM microbicidal ac- We demonstrate for the first time the presence of Epac-1 in pri- tivity and H2O2 generation. A, Following infection and phagocytosis of im- mary macrophages and a role for this protein in a key aspect of mune serum-opsonized K. pneumoniae (multiplicity of infection, 50:1) for 30 host defense, FcR-mediated phagocytosis. We further show min (37°C), rat AMs were either treated with PGE (100 nM), 8-pCPT-2Ј-O- 2 that cAMP-dependent immunomodulatory effects on the AM

Me-cAMP (2 mM), 6-Bnz-cAMP (2 mM), or vehicle for 90 min at 37°C (to http://www.jimmunol.org/ allow bacterial killing), or were placed at 4°C (phagocytosis control) (13). The result from the activation of distinct PKA- and/or Epac-1-me- survival of ingested bacteria is expressed relative to the 4°C control (dashed diated pathways. These studies are, to our knowledge, the first line). As indicated, untreated cells killed ϳ30% of phagocytosed bacteria. Data to identify specific roles for PKA vs Epac in the regulation of are the mean (ϮSE) of a representative of three experiments performed in qua- phagocyte function. Our findings have important implications Ͻ ء druplicate. , p 0.05 vs control. B, AMs were pretreated with PGE2 (100 for efforts to pharmacologically modulate inflammatory and in- nM), 8-pCPT-2Ј-O-Me-cAMP (2 mM), 6-Bnz-cAMP (2 mM), or vehicle for nate immune processes. Future experiments are necessary to 30 min followed by infection with opsonized K. pneumoniae.H2O2 production -p Ͻ 0.05 vs uninfected cells; #, p Ͻ 0.05 understand the mechanistic basis for the specificity of these dis ,ء .was assessed in culture supernatants vs infected but untreated AMs. tinct cAMP effectors in modulating various macrophage functions and to determine the involvement of Epac in the phago- by guest on October 2, 2021 cytic and microbicidal actions of other leukocytes, such as Epac agonist had no effect. A similar profile was observed for neutrophils. LPS-stimulated TNF-␣ production in both NR8383 cells and rat AMs (Fig. 3, B and C). These data implicate PKA, but not Acknowledgments Epac-1, in the regulation of inflammatory mediator synthesis We thank Teresa Marshall for technical assistance, and Dr. Michael Coffey for by AMs and further highlight the specificity of these cAMP an- human AM lysates. alogs. Although previous studies have defined a role for PKA in References mediating the suppressive effects of cAMP on LTB4 and ␣ 1. Aronoff, D. M., C. Canetti, and M. Peters-Golden. 2004. Prostaglandin E2 inhibits TNF- production (20, 22), the present studies are, to our alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated in- knowledge, the first to examine, and exclude, the potential increase in intracellular cyclic AMP. J. Immunol. 173:559. 2. Dent, G., M. A. Giembycz, K. F. Rabe, B. Wolf, P. J. Barnes, and H. Magnussen. volvement of Epac-1 in this setting. 1994. Theophylline suppresses human alveolar macrophage respiratory burst through phosphodiesterase inhibition. Am. J. Respir. Cell Mol. Biol. 10:565. Epac-1 and PKA are both involved in the suppression of AM 3. Rowe, J., J. J. Finlay-Jones, T. E. Nicholas, J. Bowden, S. Morton, and P. H. Hart. microbicidal activity 1997. Inability of histamine to regulate TNF-␣ production by human alveolar macrophages. Am. J. 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Among circulating hematopoietic cells, B-CLL uniquely expresses functional EPAC1, kill successfully ingested bacteria compared with untreated cells but EPAC1-mediated Rap1 activation does not account for PDE4 inhibitor-induced (Fig. 4A). Interestingly, both 6-Bnz-cAMP and 8-pCPT-2Ј-O- apoptosis. Blood 103:2661. The Journal of Immunology 599

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