Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages

Angie L. Hertz, Andrew T. Bender, Kimberly C. Smith, Mark Gilchrist, Paul S. Amieux, Alan Aderem, and Joseph A. Beavo
PNAS December 22, 2009 106 (51) 21978-21983; https://doi.org/10.1073/pnas.0911684106

  1. Contributed by Joseph A. Beavo, October 14, 2009

  2. ↵1A.L.H. and A.T.B. contributed equally to this work. (received for review April 14, 2009)

Abstract

Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE2). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.

Cyclic nucleotides are ubiquitous second messengers that can differentially regulate multiple cell processes via their various downstream effectors. cAMP can exert its effects through any of several distinct pathways, including cyclic nucleotide-gated ion channels, cAMP-activated protein kinases (PKA), or exchange proteins directly activated by cAMP (Epac) (1, 2). Phosphodiesterases (PDEs) control the amplitude and duration of the cAMP signal through hydrolysis to 5′-AMP, and so can regulate all of these cellular processes through control of the availability of this second messenger (3). Current studies in the field indicate that there are distinct cAMP-signaling microdomains within many cells that are controlled by specific PDEs, which can differentially regulate independent processes controlled by this common second messenger (4–7).

cAMP plays a key role in regulation of activated macrophage inflammatory responses. Increases of cAMP through either PDE4 inhibition, PDE4 gene knockout, or cAMP-agonist administration have been widely shown to dramatically decrease much of the inflammatory response of macrophages to activating agents such as lipopolysaccharide (LPS), including cytokine expression, adhesion, and FcγR-mediated phagocytosis (8, 9). This recognition has led to a great interest in PDE4 inhibitors as therapeutic agents (10). Previous work in this laboratory has shown that both cAMP and cGMP can arrest differentiation from a monocyte to dendritic cell (11). While some work has been carried out on the effects of cAMP and PDEs during monocytic differentiation to macrophages (12–14), the roles of these regulators in the long-term macrophage responses of the body remain insufficiently answered.

Monocytes provide a first line of defense against infection. As such, they constantly circulate in the blood and enter tissues, where they are exposed to a distinct milieu of cytokines and growth factors that affect their differentiation and activation states (15–17). For example, monocytes differentiated in the presence of macrophage-colony stimulating factor (CSF) carry a much different PDE profile than those differentiated in granulocyte macrophage-colony stimulating factor (GM-CSF) (18). In the lung, infiltrating monocytes are exposed to high levels of GM-CSF, causing them to differentiate into an alveolar-type macrophage, characterized by low levels of CD11b and high levels of IL-10 and nitric oxide (19, 20). In the inflamed lung, as seen with disorders such as asthma or chronic obstructive pulmonary disease (COPD), the levels of cAMP-increasing agonists like PGE2, histamine, and adenosine are high (21–23). Administration of a PDE4 inhibitor, such as those now in clinical trials for asthma and COPD treatment, can drive intracellular cAMP levels even higher, potentially altering both the activation and differentiation states of monocytes and macrophages. However, the efficacy of PDE4 inhibition in these trials has been limited by concerns about the doses that can be safely administered (24).

In the present study we determined the effects of cAMP on monocyte to macrophage differentiation, in the absence of macrophage-activating factors like LPS, and found that several proinflammatory chemokine mRNAs and protein levels were increased in a PDE4-dependent manner. Further analysis showed that this increase in chemokine expression depended on both Epac-mediated cAMP-signaling microdomains that respond to PDE4, as well as NF-κB and ATF3-dependent transcription factor mechanisms.

Results

DNA Microarray Analysis of Forskolin-Treated Monocyte/Macrophages.

We wished to evaluate the role of elevated cAMP in the GM-CSF-induced macrophage to determine its potential relevance to current PDE4-inhibitor treatments for inflammatory disorders. To look at changes in gene expression increased by cAMP, we differentiated monocytes for 6 days with GM-CSF (50 ng/ml) in the presence or absence of 50 μM forskolin and performed a DNA microarray analysis. The two conditions were compared using a large-oligo human gene array as described in Materials and Methods (see also SI Materials and Methods). Of the 8,530 genes that were reliably detected (seen in 8 of 10 samples), a total of 334 genes had expression increased greater than 2-fold, while 356 genes had expression decreased greater than 2-fold upon treatment with forskolin (see Table S1).

One class of genes in particular stood out because of their very large and significant fold increases. This group, listed in Table 1, contained a number of chemokine gene products that primarily bind to either of two distinct chemokine receptors, CXCR2 or CCR2. These chemokine receptors largely regulate recruitment of neutrophils and monocytes, respectively, and their production would be expected to increase the number of these leukocytes trafficking to an area of inflammation. Additionally, several of these chemokines, including CCL18 and CCL13, also attract eosinophils and T cells. These large increases in chemokine production induced with forskolin treatment were particularly unexpected, because cAMP is widely known to have anti-inflammatory effects on cytokine and chemokine production by activated macrophages. As such, we decided to focus on the CXCR2 and CCR2 classes of chemokines for subsequent experiments.

View this table:
Table 1.

Genes with chemokine function showing 10-fold or greater change in mRNA expression

PDE4 Controls Surface Marker and Chemokine Expression.

Using isozyme-selective PDE activity analysis, we identified PDE3 and -4 as the major PDEs controlling cAMP degradation in these macrophages (see Fig. S1). Therefore, we treated differentiating monocytes with combinations of PDE3- or PDE4-specific inhibitors or the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and forskolin to determine which specific functional compartments of cAMP and their associated PDEs were important for controlling expression of these genes. The selective PDE inhibitors used were cilostamide (PDE3) and rolipram (PDE4) (3). In the presence of a low dose of forskolin, a high, but selective, dose of PDE inhibitor (a dose 10-times larger than the EC50) should have the effect of shifting the dose-response curve to the left. One expects that when a specific PDE is inhibited, there should be an increase in cAMP levels in the compartments to which it is localized, resulting in a larger change in gene expression to the same low dose of agonist.

We first looked at expression of a number of surface markers to determine the macrophage phenotype (Fig. S2). We found two surface markers that were up-regulated with forskolin treatment, CD14 and CD163. By carrying out dose-response curves on these cells, we found that low doses of forskolin were 5 to 10 μM (Fig. S3). Treatment with 5 μM forskolin showed a slight increase in expression of these surface markers after 6 days (Fig. 1A). When the monocytes were treated with forskolin plus PDE-specific inhibitors, we observed a marked potentiation of surface marker expression with the PDE4 inhibitor, rolipram, but minimally increased expression with PDE3 inhibitors. We also treated the cells with IBMX alone to inhibit multiple PDEs, and saw a modest increase in expression. However, when used with forskolin, IBMX causes massive cell death, implying some additional PDE may affect cell viability. From these data we can conclude that PDE4, but not PDE3, coordinates a signaling microdomain involved in regulation of CD14 and CD163 expression.

Fig. 1.
Fig. 1.

PDE control of gene expression. (A) Cells were treated for 6 days as indicated and analyzed by flow cytometry for surface marker expression. Cil, Cilostamide (1 μM); FSK, forskolin (5 μM); IBMX, isobutylmethylxanthine (100 μM); Rol, Rolipram (10 μM). n = 4–5. (B and D) Cells were treated for 6 days as indicated and the mRNA was analyzed by RT-PCR for changes in chemokine expression and expressed as fold-change induction over control cells. n = 4–12. (C and E) ELISA measurements of secreted protein expression in the supernatant of cells treated for 6 days as indicated. (n = 4–12). PGE2, prostaglandin E2 (10 nM); FSK (10 μM); *, P < 0.05; **, P < 0.01 vs. control cells; #, P < 0.05 vs. FSK (C) or vs. PGE2 (E).

We also evaluated the regulation by PDEs of the expression of the C-X-C and C-C classes of chemokines using specific PDE inhibitors. The PDE4 inhibitors were of particular interest not only because this family of PDEs appears to be important for both differentiation and function of the macrophages, but also because they are a class of drugs currently being evaluated clinically as treatment for COPD and several other inflammatory disorders. Because several of the chemokines up-regulated by forskolin are found in high levels in the lungs of patients with COPD (23, 25), we wanted to determine if a PDE4 could be regulating one or more signaling microdomains that control the expression of these chemokines. We saw an increase in both the mRNA (Fig. 1 B and D) and protein expression (Fig. 1 C and E) of several of these chemokines over basal upon treatment with either forskolin or the endogenous cAMP agonist PGE2 when coadministered with rolipram.

In contrast, the PDE3-selective inhibitor cilostamide showed little or no potentiation of expression for either chemokine when given in conjunction with a low dose of forskolin or PGE2, demonstrating a minimal involvement of PDE3 in controlling chemokine expression (Fig. S4). Therefore, it would appear that PDE4 is a major regulator of the cAMP-dependent pathways that can increase chemokine expression.

Epac Mediates Much of the cAMP-Stimulated Increase in Chemokine Expression.

It seemed likely that cAMP is acting through either the Epac or PKA signaling pathways to exert its effects on chemokine expression. To determine which isoform of Epac was present in these cells, we examined the microarray data and found that mRNA for Epac1, but not Epac2, was present in our differentiated macrophages. To address how chemokine expression is regulated, we treated monocytes for 6 days during differentiation with cAMP analogues that can specifically activate either PKA or Epac. Using the nonhydrolyzable Epac-selective activator, Sp-8-pCPT-2′-O-Me-cAMPS (Sp-8-pCPT), we observed a dose-dependent increase in CXCL7, CXCL5, and CCL2 expression at both the mRNA (Fig. 2A, C, and E) and protein levels (Fig. 2 B, D, and F) in monocyte-differentiated macrophages. The magnitude of the increase was usually as great as or greater than the maximal seen with either forskolin or PGE2. When we used the PKA-specific activator, N6-Benzoyladenosine-3′,5′-cyclic monophosphate (6-Bnz-cAMP), at a concentration of 1 μM, we also saw small increases in chemokine mRNA levels, but no significant increases in secreted protein levels. Unfortunately, higher levels of 6-Bnz-cAMP caused cell death when present during differentiation, and therefore could not be tested. This suggests that chemokine expression is largely controlled by Epac, with a possible smaller involvement of PKA. However, we have noted that the magnitude of induction of these chemokines in response to treatment with Sp-8-pCPT was highly variable among patients, perhaps suggesting an additional level of regulation or alteration of basal activation state in these cells.

Fig. 2.
Fig. 2.

Role of Epac and PKA in chemokine signaling. Chemokine levels in cells treated for 6 days with indicated agonist. Sp-8-pCPT = Sp-8-pCPT-2′-O-Me-cAMPS, 6Bz = 6-Bnz-cAMP (1 μM), FSK = forskolin (25 μM). mRNA levels analyzed by RT-PCR for CXCL7 (A), CXCL5 (C) and CCL2 (E) Secreted protein levels after 6 days measured from cell supernatant by ELISA for CXCL7 (B), CXCL5 (D) and CCL2 (F). *, P < 0.05; **, P < 0.01 vs. control cells. n = 4–12.

cAMP Controls Surface Marker Expression in an NF-κB-Dependent Manner.

To determine mechanistically how cAMP/Epac might be affecting the expression of these genes, the putative promoter regions of many of the genes identified by clustering analysis of the array data were searched for common transcription-factor binding sites. The sequence 2,000 bp upstream of the start codon for 64 of the immune relevant genes, including all of the chemokines, identified in the microarray analysis, was searched for transcription-factor binding sites using the program Clover (26). Through comparison to a background list of unchanged genes, six transcription-factor binding sites were found to be over-represented (Fig. 3A), with a P value indicating the statistical significance of over-representation and a score indicating the strength of the factor's presence in the whole sequence set. The NF-κB family represents the top three sites, suggesting that this transcriptional regulator might be common to many of the genes regulated by forskolin. We also graphed the motif against its corresponding score for each of three up-regulated groups: those increased more than 5-fold, those increased 3- to 5-fold, and those increased 2- to 3-fold (Fig. 3B). Again, we found that the most highly up-regulated genes contained NF-κB sites in their promoters.

Fig. 3.
Fig. 3.

NF-κB control of gene expression. (A) Promoter analysis of transcription factor binding sites for all genes up-regulated greater than 5-fold on the microarray. (B) Transcription factor binding-site prediction grouped by fold induction. (C) The forskolin-induced increase in CD163 and CD14 expression was dose-dependently inhibited by the NF-κB inhibitor, SN50, after 6 days of treatment. n = 5. (D) The increase in chemokine expression seen with 25 μM forskolin treatment was not inhibited by treatment with SN50 (50 μg/ml) after 6 days of treatment. n = 4. (E) Cells were differentiated for 6 days in the presence of GM-CSF, the media changed and either FSK (25 μM) or FSK + BMS-345541 (10 μM) was added for 6 h and the mRNA harvested. CXCL7 and CXCL5 mRNA levels were calculated relative to GAPDH. n = 4. *, P < 0.05; **, P < 0.01.

In a separate set of experiments, we were able to show that the forskolin-induced increases in CD14 and CD163 were dependent on NF-κB by using the cell-permeable peptide inhibitor SN50 to block the nuclear translocation of p50 NF-κB. The inhibitory effect of SN50 was dose-dependent for increasing concentrations of SN50 on both CD14 and CD163 surface marker expression (Fig. 3C). Surprisingly, SN50 had little or no effect on forskolin-induced chemokine expression (see Fig. 3D). One possibility to explain this lack of effect is that SN50 does not block the alternative p52/RelB pathway, which could also be regulated by cAMP. To address this possibility, we used BMS-345541, a selective allosteric inhibitor of the IκB kinases, IKK1 and IKK2 (27). When we treated fully differentiated macrophages with forskolin and a moderate dose (10 μM) of BMS-345541 for 6 h, we saw a marked decrease in CXCL5 and CXCL7 expression with inhibitor treatment, demonstrating that the NF-κB pathway also can control forskolin-induced expression of these chemokine genes.

Role of ATF3 in Transcriptional Regulation of CXCL7 Expression.

A further inspection of the down-regulated genes in the microarray suggested a possible additional mechanism for regulation of chemokine genes by forskolin: a 7-fold decrease in mRNA levels for the CREB-family transcription factor, ATF3. ATF3 has recently been shown to be a negative regulator of NF-κB acting through an increase in histone deacetylase activity (28, 29). Therefore, decreases in ATF3 levels should have the effect of increasing transcription of NF-κB-regulated genes. We confirmed the forskolin-dependent decrease in ATF3 mRNA expression using RT-PCR (Fig. 4A). Additionally, we performed ChIP analysis using an antibody to ATF3 and gene primers specific for the predicted ATF3 binding region in the CXCL7 promoter to measure the DNA binding activity of ATF3 in these cells. After 6 days of forskolin treatment, we observed a marked decrease in the amount of ATF3 bound to the promoter of CXCL7 (Fig. 4B). Our results are consistent with a relief of inhibition by ATF3, but more work will need to be done to determine the exact mechanism by which cAMP and ATF3 may be contributing to the increase in chemokine levels.

Fig. 4.
Fig. 4.

ATF3 mRNA expression and DNA binding activity. (A) Real-time PCR analysis of ATF3 mRNA levels, relative to 18 s mRNA in macrophages treated for 6 days with FSK (25 μM). n = 6–8. *, P < 0.05. (B) ATF3 binding on the CXCL7 promoter in macrophages was measured by ChIP assay using an anti-ATF3 antibody. Data shown is representative of three independent experiments.

Discussion

GM-CSF is the major cytokine driving differentiation of monocytes into macrophages in the lung and several other tissues. GM-CSF promotes the conversion of monocytes into macrophages that can then be strongly activated to promote inflammatory responses when they come in contact with external stimuli, such as LPS or cigarette smoke. To date, most studies on the effects of cAMP on macrophage function have focused on its anti-inflammatory effects on fully activated macrophages. For example, in LPS-activated macrophages, agents that increase cAMP levels decrease inflammatory cytokine release, inhibit phagocytosis, and reduce reactive oxygen and bactericidal activity (8). It is important to note that cAMP is itself not the primary mediator of these processes but is instead a modifying stimulus. From the present results, it appears that cAMP can also act as a positive regulator of a number of immune function genes, including some normally considered to be proinflammatory. It has been shown in several systems that LPS can increase cAMP in macrophages (9, 30), and in this regard a subset of cAMP-induced gene transcription could be viewed as part of a normal inflammatory response. If so, it points once again to the compartmentalized mechanisms by which cAMP functions in many cells, given that the same signaling molecule can have opposing effects within the same cell.

From the results of this study, it is clear that PDE4 inhibition can modulate monocyte to macrophage differentiation by potentiating the effects of forskolin and PGE2. Although PDE3 and PDE4 constitute nearly equal amounts of the cAMP PDE activity in these macrophages, only the PDE4 selective inhibitors had an appreciable effect on the macrophage phenotype observed.

PDE4 inhibitors have a long history as potential clinical agents and are currently under investigation for treatment of depression, memory disorders, and several inflammatory conditions (3, 24). These drugs can often act as anti-inflammatory agents against several types of activated immune cells both in vitro and in vivo. However, as it is very difficult to obtain material to measure monocyte- and macrophage-specific activity in humans treated in vivo, ex vivo stimulation of TNF-α release by LPS has been the predominant surrogate measure of their function.

Despite their anti-inflammatory effects against monocytes and macrophages in vitro and in mouse models, to date the clinical trials for PDE4 inhibitors in the treatment of COPD and asthma have met with mixed success. One possible reason is that PDE4-inhibitor treatment in humans is dose-limited by side effects, such as emesis (24). However, a few earlier in vivo inflammation studies have suggested that under some conditions treatment with PDE4 inhibitors can actually promote macrophage accumulation, which could be counterproductive for resolving inflammation. For example, roflumilast treatment slightly increased the number of macrophages in lung bronchoalveolar lavage fluid using a mouse model of acute cigarette smoke exposure, although neutrophil numbers were decreased (31). Another study reported that roflumilast and piclamilast reduced KC (mouse CXCL1) and TNF-α release and cell infiltration into lungs of mice challenged with LPS. However, in the absence of LPS challenge, the inhibitors actually had the opposite effect by inducing release of KC and greatly increasing neutrophil infiltration into lung tissue (32). These investigators postulated that chemokine release was likely from endothelial cells, but the data shown here suggest that macrophages may also play an equally important role in PDE4-mediated chemokine release.

From the results presented, it seems quite possible that increased chemokine production by macrophages in the presence of a cAMP agonist and a PDE4 inhibitor may exacerbate the underlying chronic inflammation in lung diseases, such as asthma or COPD (33, 34). Given the large induction of chemokines generated by cAMP, it seems likely that many neutrophils, eosinophils, T cells, and monocytes, responding to CXCR2 and CCR2 receptors, would be recruited to sites of lung inflammation and contribute to underlying chronic inflammation through immune cell recruitment. These chemokines can also directly modulate immune cell functions, further contributing to the pathogenesis of these diseases. It will be of great interest to determine if combinations of other PDE inhibitors, perhaps working on PDEs in different functional compartments, can be identified that do not promote recruitment of immune cells but, rather, only target the anti-inflammatory functions of the activated macrophages (35). Finally, these results also suggest the possibility that PDE4 inhibitors may be more efficacious if used in conjunction with a glucocorticoid, one of the newly developed chemokine receptor antagonists (25, 36, 37) or, if they can be developed, Epac antagonists. One would expect that not only would the efficacy of each drug be increased by coadministration, but also that the safety margins and side-effect profiles of these PDE4 inhibitors may be improved by coadministration of such agents.

Mechanistically, these proinflammatory cAMP effects may be mediated in part through differential regulation of NF-κB activity. That increased cAMP can inhibit induction of many genes triggered by an inflammatory stimulus, such as LPS, is well established. We have shown here that several of the genes induced by forskolin have an NF-κB binding motif in their promoter regions and that NF-κB pathway inhibitors block chemokine and surface marker expression. Given the disparate data with BMS 345541 and SN50, the NF-κB pathway regulating the chemokine induction is likely to be the alternative, p100/p52 and RelB-mediated, pathway (38, 39).

Supporting this hypothesis is the fact that the transcription factor ATF3, which is down-regulated by forskolin treatment, has recently been reported as a negative regulator of NF-κB-dependent transcription (28). ATF3 has also been implicated in control of chemokine expression in a mouse asthma model (29). In these studies, Gilchrist et al (29) showed a marked increase in many of the same chemokines we see up-regulated with cAMP: CXCL1, -2, and -5, and CCL2 in the ATF3-null mice compared to wild-type controls.

Finally, it seems that the Epac signaling pathway plays a prominent role in cAMP regulation of chemokine expression in the GM-CSF macrophage differentiation model. In previous studies, it has been shown that many of cAMP's anti-inflammatory effects are mediated through the PKA pathway, although a role for Epac has been established in modulating microbicidal activity and FcγR-mediated phagocytosis (8, 29). Specific isoforms of PDE4, working through PKA, have been implicated in regulation of both cytokine and adhesion molecule expression using PDE4A, PDE4B, and PDE4D knockout mice (9, 40). Additionally, using a monocytic differentiation protocol similar to that used in this article, Bryn et al showed that TNF-α, IL-12, MIP1-β, integrin, and surface marker expression were controlled by PKA and not Epac (41). In these studies, Epac expression was increased upon differentiation; however, there were no major anti-inflammatory changes attributed to Epac alone. These data fit nicely with the present observations that activation of Epac appears to have proinflammatory effects, not anti-inflammatory effects, on monocyte/macrophages. It has also been reported that Epac can shuttle in and out of the nucleus, which may be relevant for how the changes in chemokine genes are regulated by Epac (42). Taken together, these data suggest an additional role for Epac in the inflammatory process and a possible additional site of therapeutic intervention, if Epac antagonists can be developed.

In summary, we suggest that the well-established effectiveness of PDE4 inhibitors to decrease acute inflammatory responses in activated macrophages may be limited by an opposing effect on chemokine induction. Addition of a chemokine receptor antagonist, glucocorticoid, or Epac inhibitor to the PDE4 inhibitor treatment may not only make the PDE4 inhibitor more efficacious, but also have the added benefit of reducing concerns about inflammatory side effects, such as the mesenteric vasculitis sometimes seen with chronic PDE4-inhibitor treatment.

Materials and Methods

Monocyte Isolation and Culture.

Monocytes were purified by positive selection with magnetic CD14 microbeads (Miltenyi Biotec) from buffy coats obtained from human donors with consent by the Portland Oregon Red Cross or the Stanford Blood Center, as previously described (13). Monocyte differentiation was carried out at a concentration of 5 × 105 cell/ml with 50 ng/ml recombinant human GM-CSF (leukine, Berlex) for 6 days; 10% volume of additional media was added on Day 3 if necessary. Reagents were added as indicated on Day 0 with GM-CSF, and allowed to incubate for 6 days without supplementation or added at Day 6 and incubated for 6 h.

FACS Staining and Analysis.

Fluorescently labeled antibodies were from either Beckman Coulter (mannose receptor, CD64, CD80, CD86,) or BD Biosciences (CD14, CD71, CD163). Cells were prepared for FACS and analyzed as previously described using a Becton Dickinson FACScanto flow cytometer and FACSdiva software (43).

RT-PCR Analysis.

Total RNA was isolated using QIAshredder columns and the RNeasy kit from Qiagen. Total RNA was transcribed to cDNA using the SuperScript III RT kit from Invitrogen according to the manufacturer's protocol. Real-time analysis of PCR was performed using SYBR Green (CXCL7, CCL2, CXCL5) from Applied Biosystems, or Taqman technology (ATF3), on a Stratagene MX3000P and analyzed using MxPro software. Data are expressed as fold-change using the delta-delta Ct method, or as an amount relative to either GAPDH or 18s mRNA.

Cytokine ELISA Measurements.

Duoset ELISA kits for CCL2, CXCL7, and CXCL5 (R&D Systems catalog #DY279, #DY393, and DY254, respectively) were used to assess secreted protein levels in cell-culture supernatants according to the protocol recommended by the manufacturer.

Chromatin Immunoprecipitation.

Monocytes were incubated for 6 days in the presence of GM-CSF +/− 25 μM forskolin. Cells were isolated as previously described, and the DNA was extracted and subjected to PCR analysis (28).

Reagents.

Cyclic nucleotide analogs were obtained from Biolog, Inc. and used at concentrations that promoted their relative selectivity for the cAMP effector molecules (44). Forskolin was obtained from Fisher Bioreagents (catalog # bp2520). SN50 was obtained from BioMol (catalog # P-600). BMS-345541 was obtained from Sigma (catalog #B-9935).

Statistical Analysis.

Data were analyzed using the two-tailed Student's t-test. P < 0.05 was considered significant.

Acknowledgments

We thank Biolog, Inc., for generously providing the cyclic nucleotide analogs. This study was supported in part by National Institutes of Health Trainee under Pharmaceutical Sciences Training Grant GM007750 (to A.L.H.), National Institutes of Health Trainee under University of Washington Pathology of Cardiovascular Disease Training Grant HL0732 (to A.T.B.), and National Institutes of Health Grants DK21723 and GM083296 (to J.A.B.), AI025032 (to A.A.), and HL092547 (to M.G.).

Footnotes

  • 2To whom correspondence should be addressed. E-mail: beavo{at}u.washington.edu

  • Author contributions: A.L.H., A.T.B., M.G., P.S.A., A.A., and J.A.B. designed research; A.L.H., A.T.B., K.C.S., and M.G. performed research; A.A. contributed new reagents/analytic tools; A.L.H., K.C.S., M.G., P.S.A., and J.A.B. analyzed data; and A.L.H. and J.A.B. wrote the paper.

  • The authors declare no conflict of interest.

  • Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE18654).

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0911684106/DCSupplemental.

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Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages
Angie L. Hertz, Andrew T. Bender, Kimberly C. Smith, Mark Gilchrist, Paul S. Amieux, Alan Aderem, Joseph A. Beavo
Proceedings of the National Academy of Sciences Dec 2009, 106 (51) 21978-21983; DOI: 10.1073/pnas.0911684106
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