Specific Role of 4B in Lipopolysaccharide-Induced Signaling in Mouse Macrophages

This information is current as S.-L. Catherine Jin, Linda Lan, Maria Zoudilova and Marco of September 24, 2021. Conti J Immunol 2005; 175:1523-1531; ; doi: 10.4049/jimmunol.175.3.1523 http://www.jimmunol.org/content/175/3/1523 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Specific Role of Phosphodiesterase 4B in Lipopolysaccharide-Induced Signaling in Mouse Macrophages1

S.-L. Catherine Jin, Linda Lan, Maria Zoudilova, and Marco Conti2

Cyclic nucleotide signaling functions as a negative modulator of inflammatory responses, and type 4 (PDE4) are important regulators of this pathway. In this study, we provide evidence that only one of the three PDE4 expressed in mouse peritoneal macrophages is involved in the control of TLR signaling. In these cells, LPS stimulation of TLR caused a major up-regulation of PDE4B but not the paralogs PDE4A or PDE4D. Only ablation of PDE4B impacted LPS signaling and TNF-␣ production. TNF-␣ mRNA and protein were decreased by >50% in PDE4B؊/؊, but not in PDE4A؊/؊ or PDE4D؊/؊ macrophages. The PDE4 selective inhibitors and roflumilast had no additional inhibitory effect in macrophages deficient in PDE4B, but suppressed the TNF-␣ response in the other PDE4 null cells. The inhibition of TNF-␣ production that follows either

genetic ablation or acute inhibition of PDE4B is cAMP-dependent and requires protein kinase A activity. However, no global Downloaded from changes in cAMP concentration were observed in the PDE4B؊/؊ macrophages. Moreover, ablation of PDE4B protected mice from LPS-induced shock, suggesting that altered TLR signaling is retained in vivo. These findings demonstrate the highly specialized function of PDE4B in macrophages and its critical role in LPS signaling. Moreover, they provide proof of concept that a PDE4 inhibitor with subtype selectivity retains useful pharmacological effects. The Journal of Immunology, 2005, 175: 1523–1531.

he second messenger cAMP plays a key role in the reg- these isoenzymes are redundant or each subtype has its own spe- http://www.jimmunol.org/ ulation of most cellular functions. In inflammatory cells, cialized function. The phenotypes of different PDE4 null mice sug- T activation of cAMP signaling has negative modulatory gested that each PDE4 has unique functions. PDE4D null effects on numerous steps required for immune and inflammatory mice display a broad spectrum of pleiotropic phenotypes (6), responses, including T cell activation and proliferation, cytokine whereas PDE4B null mice are healthy in general and display a release, and recruitment of leukocytes (1). Given these broad in- deficit in TNF-␣ production (7). PDE4A null mice have been re- hibitory effects, pharmacological manipulation of cAMP levels is cently generated, but phenotypes derived from the ablation of this viewed as a promising approach for the treatment of chronic in- gene are still under investigation. flammatory conditions such as asthma, chronic obstructive pulmo- Peripheral blood leukocytes derived from the PDE4B null mice nary disease, and inflammatory bowel disease. Together with ac- produce very little TNF-␣ in response to LPS, where the response by guest on September 24, 2021 tivation of G protein-coupled receptors with agonists, inhibition of of PDE4D null leukocytes is not affected (7). This observation 3 cyclic nucleotide phosphodiesterases (PDEs), the that constitutes an initial indication for a nonredundant function of degrade and inactivate cyclic nucleotides, is one strategy to in- PDE4s. However, a question that could not be addressed in that crease cAMP levels in these cells and to suppress inflammatory study is whether the differential effect of PDE4 ablation on LPS responses (1–3). signaling is due to differences in PDE4B and PDE4D expression in The phosphodiesterase 4 (PDE4) family consists of four paralog these cells or is the consequence of a specialized function of each genes (PDE4A–D), and each gene encodes multiple variants gen- PDE4 gene. Because monocytes, the major producer of TNF, can- erated from alternate splicing and different transcriptional promot- not be recovered in sufficient number to allow biochemical studies, ers (4, 5). Thus far, a total of at least 20 transcripts derived from the four genes have been identified, and the corresponding proteins in this study we have used peritoneal resident macrophages to can be usually distinguished by their unique N-terminal sequences. determine which PDE4 is expressed in this naive, nonelicited cell These variants, particularly those belonging to the PDE4A, population and whether ablation of different PDE4 subtypes ex- PDE4B, and PDE4D subtypes, are widely expressed and often pressed within the same cell indeed produces distinct effects. The coexist in different tissues and cells. It is largely unknown whether characterization of this naive macrophage model has also allowed us to define the mechanism by which different PDE4 ablation af- fects cyclic nucleotide signaling. Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stan- Given their pattern of expression and involvement in the control ford University School of Medicine, Stanford, CA 94305 of inflammatory cell responses, PDE4s have received considerable Received for publication April 8, 2005. Accepted for publication May 13, 2005. attention as targets for anti-inflammatory drugs. PDE4 inhibitors The costs of publication of this article were defrayed in part by the payment of page are being developed for chronic lung inflammatory diseases; some charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. compounds have showed considerable efficacy and are being tested in humans for asthma and chronic obstructive pulmonary disease 1 This work was supported by a National Institutes of Health-Specialized Center of Research grant (NIH-HL67674) and the Sandler Foundation for Asthma Research. (8, 9). However, the pharmacological potential of this class of 2 Address correspondence and reprint requests to Dr. Marco Conti, Division of Re- drugs is limited by their narrow therapeutic window (10). Dosing productive Biology, Department of Gynecology and Obstetrics, Stanford University of PDE4 inhibitors sufficient to produce anti-inflammatory effects School of Medicine, Stanford, CA 94305. E-mail address: [email protected] in humans is associated with side effects, particularly in the CNS. 3 Abbreviations used in this paper: PDE, cyclic nucleotide phosphodiesterase; PDE4, type 4 cAMP-specific PDE; TG, thioglycollate; AP, alkaline ; PKA, pro- Thus, new strategies are being actively sought to improve the ther- tein kinase A; GEF, guanine nucleotide exchange factor. apeutic index of these PDE4 inhibitors.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1524 PDE4B IN LIPOPOLYSACCHARIDE SIGNALING IN MACROPHAGES

The presence of four PDE4 genes led to the proposal that gen- (pH 7.4), 150 mM sodium chloride, 5% glycerol, 10 mM sodium fluoride, eration of novel PDE4 inhibitors with some selectivity may have 1 mM EDTA, 0.2 mM EGTA, 10 mM sodium pyrophosphate, 1 mM so- the distinct advantage of maintaining useful therapeutic effects dium orthovanadate, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Roche), 1 tablet/10 ml of protease inhibitor mixture (Roche), 1% Nonidet while decreasing the side effects (11). At present, this hypothesis P-40, and 5 mM 2-ME). After sonication with 20 bursts, the lysate was cannot be tested pharmacologically because with few exceptions centrifuged at 4°C for 20 min at 16,000 ϫ g. The supernatant was assayed all PDE4 inhibitors synthesized are nonselective, i.e., they inhibit for both total PDE activity and rolipram-insensitive PDE activity in the all PDE4 subtypes with comparable potencies. It is then difficult to presence of 10 ␮M rolipram. The PDE assay was performed according to the method of Thompson and Appleman (12) as detailed previously (13). dissect functions of individual PDE4 subtypes using a pharmaco- The rolipram-sensitive activity (i.e., PDE4 activity) was obtained by sub- logical approach. tracting the rolipram-insensitive activity from the total activity. Protein In this study, we have used a genetic approach to demonstrate concentration was determined according to the Bradford (14) method. that only one of the three PDE4s expressed in macrophages is involved in the control of LPS signaling. More importantly, we Immunoprecipitation and PDE assay provide evidence that PDE4 inhibitors block LPS-induced TNF-␣ Following incubation with LPS (100 ng/ml) for 4 h, the macrophages were production in macrophages only by inhibiting this PDE4 subtype. washed once with PBS and then scraped in the above-mentioned lysis The other PDE4s expressed in the same cells are not involved in buffer. After sonication with 20 bursts, the lysate was centrifuged at 4°C for 20 min at 16,000 ϫ g. Aliquots of the supernatant were incubated with regulation of this response. PDE4A-, PDE4B-, or PDE4D-specific Ab (AC55, K118, and M3S1, re- spectively) that was preincubated with protein A-Sepharose (Zymed Lab- Materials and Methods oratories) or protein G-Sepharose (Amersham Biosciences). On the basis of

Mice tests done with PDE4 recombinant proteins, the amount of Ab used in each Downloaded from immunoprecipitation was sufficient to completely pull down the specific Generation of PDE4B- and PDE4D-deficient mice has been described pre- PDE4 subtype at the concentration present in the sample. Aliquots of the viously (6, 7). Generation of PDE4A-deficient mice following a similar supernatant were also incubated with normal rabbit serum or purified strategy will be reported elsewhere. The homozygous null mice and their mouse IgG1 (Zymed Laboratories) to determine their nonspecific precip- wild-type littermates used in this study were 3–6 mo of age and had a itation of PDE4. After 1–2 h of incubation at 4°C, the Sepharose-Ab-Ag mixed genetic background of C57BL/6 and 129/Ola. All experimental pro- complexes were pelleted, washed twice with PBS containing 0.05% BSA, cedures involving animals were approved by the Administrative Panel on and resuspended in 40 mM Tris-Cl (pH 8) solution containing 1 mg/ml Laboratory Animal Care at Stanford University (Stanford, CA). BSA. The complexes were then assayed for PDE activity as described http://www.jimmunol.org/ above. Isolation and purification of resident and thioglycollate (TG)- elicited peritoneal macrophages TNF-␣ and IL-6 ELISA Naive mice were sacrificed, and cells in the peritoneal cavity were isolated Levels of TNF-␣ and IL-6 in macrophage culture supernatants were mea- by washing the cavity with 6–7 ml of cold HBSS (Invitrogen Life Tech- sured with commercially available ELISA kits (BioSource International). nologies). The cells were pelleted, resuspended in PBS supplemented with The sensitivities of the assays were 19.5 and 7.8 pg/ml, respectively. 2% heat-inactivated FBS (Invitrogen Life Technologies), and then trans- ␮ ferred onto a 10-cm petri dish that was pretreated with 35 g of goat RT-PCR and Southern blot analysis anti-mouse IgG ϩ IgM (H ϩ L) (Jackson ImmunoResearch Laboratories) in 10 ml of PBS containing 0.3 mg of goat ␥-globulin. After incubation at For semiquantitative measurements of LPS-induced expression of TNF-␣, by guest on September 24, 2021 4°C for 1 h, B cells attached to the dish by binding to the coated Igs. The total RNA was extracted from wild-type and PDE4-deficient macrophages. nonadherent cells containing mainly macrophages were collected and cen- After treatment with RNase-free DNase I (0.1 U/␮g total RNA; Boeh- trifuged at 160 ϫ g for 10 min. The cell pellet was resuspended in RPMI ringer Mannheim), first-strand cDNA was synthesized from 1 ␮g of total 1640 (Invitrogen Life Technologies) supplemented with 10% FBS, 100 RNA in the presence of oligo(dT) primer according to the manufacturer’s U/ml penicillin, and 100 ␮g/ml streptomycin (complete medium). Macro- protocol (SuperScript First-Strand Synthesis System for RT-PCR; Invitro- phages were counted and plated in petri dishes or tissue culture plates at a gen Life Technologies). For PCR, 2 ␮l of aliquots of the resulting 21 ␮lof ϫ 5 density of 2–3 10 /ml. After incubation at 37°C in 5% CO2 for 2 h, the cDNA were amplified 22 cycles in a 50-␮l reaction volume containing 1ϫ medium was aspirated to remove nonadherent cells, and the adherent mac- PCR buffer, 0.2 mM dNTP mix, 1 ␮M each specific primer, and1Uof rophages were cultured in complete medium overnight. The medium was TaqDNA polymerase. Oligonucleotide primers were as follows: TNF-␣, changed next day, and the cells were cultured for an additional hour before 5Ј-GTGACAAGCCTGTAGCCCA-3Ј and 5Ј-AAAGTAGACCTGCCCG LPS stimulation or drug treatment. GAC-3Ј (from base 419 to 437 and base 846 to 828, respectively; GenBank To culture TG-elicited macrophages, mice were given injections i.p. accession no. X02611), yielding a 428-bp product; and GAPDH, 5Ј- with 1.5 ml of TG medium (Sigma-Aldrich). Four days after injection, the TGAAGGTCGGTGTGAACGGATTTGGC-3Ј and 5Ј-CATGTAGGC mice were sacrificed, and the cells in the peritoneal cavity were isolated as CATGAGGTCCACCAC-3Ј (purchased from BD Clontech), yielding a describe above. Cells were washed once with complete medium and then 983-bp product. cultured at a density of 1 ϫ 106/ml. Two hours later, plates were washed The PCR products were fractionated by agarose gel electrophoresis and with medium to remove nonadherent cells. The remaining cells with at then transferred to nylon membrane (ICN Pharmaceuticals). Blots were least 95% macrophages were cultured overnight in complete medium be- hybridized with [␥-32P]ATP-labeled oligonucleotide probes corresponding fore LPS stimulation. to nucleotide sequences nested between the specific PCR primers. The sequences of the oligonucleotide probes were as follows: TNF-␣,5Ј-CAT Flow cytometry ACCAGGGTTTGAGCT-3Ј (from base 736 to 719; GenBank accession Ј Ј To determine the purity of macrophages prepared as described above, the no. X02611); and GAPDH, 5 -CAGTGGCAAAGTGGAGATT-3 (from adherent cells were gently scraped off the plates in PBS. Aliquots of ϳ1 ϫ base 112 to 130; GenBank accession no. M32599). The blots were then ϫ 106 cells were washed once with FACS buffer (2% FBS and 0.01% sodium washed in 1.5 SSC/0.1% SDS at 48–50°C, followed by autoradiography. azide in PBS), blocked with a mouse Fc blocker (BD Pharmingen) for 15 min, and then incubated for 40 min on ice with PE anti-mouse F4/80 Ab Cyclic AMP response to LPS in peritoneal naive macrophages (Caltag Laboratories) plus FITC anti-mouse CD11b Ab (BD Pharmingen). The macrophages were incubated in the presence of LPS (100 ng/ml) for After washing with FACS buffer, the cells were analyzed on the BD Bio- different times. To terminate the incubation, the cells were washed once sciences FACSscan flow cytometer using CellQuest software (BD Bio- with ice-cold PBS, and 5% TCA was added. After extraction on ice for 30 sciences). At least 95% of the adherent cells were macrophages (data not min, the cells were scraped off the plates and then centrifuged at 6000 ϫ shown). g for 20 min. The supernatant was subsequently extracted five times with PDE assay water-saturated ether, freeze-dried by lyophilization, and reconstituted in PBS. The cAMP content was measured by RIA as described by Harper and Following incubation with LPS (100 ng/ml) for 4 h, the macrophages were Brooker (15). The TCA pellet was resuspended in 1 N NaOH solution, and gently scraped off the culture dish in PBS, centrifuged, washed once with the aliquots were assayed for protein concentration according to the Lowry PBS, and then incubated on ice for 10 min in a lysis buffer (50 mM Tris-Cl method. The Journal of Immunology 1525

Results Of the three PDE4 present in mouse peritoneal resident macrophages, LPS induces only PDE4B Macrophages were isolated from the peritoneal cavity of PDE4A, -B, and -D null mice and from corresponding wild-type littermates. After removal of B cells by IgG/IgM selection and nonadherent cells by culture, highly purified populations of macrophages were obtained, as judged by flow cytometry monitoring of F4/80 and CD11b surface marker (data not shown). Comparable numbers of macrophages were retrieved from the four genotypes with no dif- ferences in morphological appearance by light microscopy. In some experiments, macrophages recruited by i.p. injection of TG medium were used as an additional model (TG-elicited macrophages). FIGURE 1. PDE activity in mouse peritoneal macrophages. Peritoneal The pattern of PDE expression in wild-type macrophages was resident and TG-elicited macrophages from wild-type mice were incubated initially determined by measuring total PDE activity as well as the in the absence or presence of 100 ng/ml LPS for 4 h. Total and rolipram- activity sensitive to the PDE4 inhibitor rolipram. At a concentra- insensitive (RI) PDE activities in the cell extracts were measured using 1 ␮ tion of 10 M, rolipram has minimal inhibitory effects on other Downloaded from ␮M cAMP as substrate in the absence or presence of 10 ␮M rolipram. The PDEs (16, 17) and can be used as a pharmacological tool to quan- rolipram-sensitive activity (PDE4) was obtained by subtracting the RI ac- tify PDE4 activity (17). This analysis showed that ϳ40% of the Ϯ ϭ tivity from the total activity. Data are the mean SEM (n 5–16 mice/ basal cAMP-degrading activity in wild-type cells is rolipram-sen- .(p Ͻ 0.001 (compared with unstimulated cells ,ءء ;p Ͻ 0.05 ,ء .(group sitive (PDE4 activity), and the remaining 60% is rolipram-insen- sitive (Fig. 1). The rolipram-insensitive PDE activity likely reflects the expression of predominantly PDE1 and PDE3 (16, 18). After Western blot analysis http://www.jimmunol.org/ LPS stimulation for 4 h, total PDE activity was increased 2.0- to Following a 20-min incubation with or without LPS (100 ng/ml), the mac- 2.5-fold. This increase was due to a 4.0- to 4.5-fold increase in rophages were gently scraped off the culture dish in PBS, washed once with PDE4 activity, whereas the rolipram-insensitive activity was un- PBS, and then incubated on ice for 10 min in the 1% Nonidet P-40-con- taining lysis buffer as described above. After sonication with 20 bursts, the affected (Fig. 1). TG-elicited macrophages showed similar overall lysate was centrifuged at 4°C for 20 min at 16,000 ϫ g. Aliquots of the levels of PDE activity, but PDE4 accounted for only 20% of the supernatant were subjected to electrophoresis on 15% SDS-PAGE, and total activity. As a consequence of this relatively low PDE4 ex- then blotted onto an Immobilon membrane. Western blot analysis was per- pression, LPS stimulation produced only ϳ20–30% overall in- formed using an antiphosphorylated CREB Ab (Cell Signaling Technol- ogy) at a dilution of 1/1500. Immunoreactive bands were detected by using crease in PDE activity (Fig. 1B). a peroxidase-conjugated donkey anti-rabbit Ab (Amersham Biosciences) at Although several studies have investigated the PDE expression by guest on September 24, 2021 a dilution of 1/5000 and the ECL detection system (Amersham Bio- profile and activity in macrophages under different conditions (16, sciences). To monitor the level of total CREB in the cell, the blot was 18–23), the actual expression pattern of PDE4 subtypes and their subsequently washed extensively and then incubated with an anti-CREB activity in response to LPS have not been explored. Thus, to de- Ab (Cell Signaling Technology) at a dilution of 1/1000. Immunoreactive bands were detected by using an (AP)-conjugated termine which PDE4 subtypes contribute to the LPS-dependent goat anti-rabbit Ab at a dilution of 1/3000 and an Immun-Star AP substrate induction of PDE4, the PDE activity in peritoneal macrophages (Bio-Rad). was further characterized by a combined immunological and ge- High-dose LPS-induced shock model netic approach. PDE4 subtype-specific Abs were used for immu- noprecipitation of cell extracts of wild-type and PDE4-deficient Wild-type and PDE4B null mice were given injections i.p. with 800 ␮g/25 macrophages under basal conditions or after LPS stimulation. The g body weight LPS (Escherichia coli serotype 055:B5; Sigma-Aldrich). Animals were monitored every 12 h for death throughout the 7-day test immunoprecipitates were then used for PDE activity measurement. period. Mortality/survival was analyzed using the log-rank test (also known The results of these experiments are summarized in Fig. 2. Abs as the Kaplan-Meier test) and Wilcoxon test. specific to PDE4A (AC55), PDE4B (K118), and PDE4D (M3S1)

FIGURE 2. Effects of LPS on the activity of different PDE4s expressed in mouse peritoneal resident macrophages. Peritoneal macrophages from PDE4A-, PDE4B-, and PDE4D-deficient mice and their wild-type littermates were incubated in the absence or presence of 100 ng/ml LPS for 4 h. The cell lysate (pooled from five animals per group) was immunoprecipitated with Abs specific to PDE4A (AC55), PDE4B (K118), or PDE4D (M3S1), and PDE activity recovered in the immunoprecipitation pellet was measured using 1 ␮M cAMP as substrate. Arrows illustrate that the three Abs did not immunoprecipitate PDE4 activity in the corresponding PDE4-deficient macrophages, demonstrating the specificity of the Abs to each PDE4 subtype. Data shown are representative of two separate experiments. 1526 PDE4B IN LIPOPOLYSACCHARIDE SIGNALING IN MACROPHAGES immunoprecipitated significant PDE activity when compared with preimmune IgG. Conversely, they did not immunoprecipitate ap- preciable activity in the corresponding PDE4 null macrophages. Thus, under basal condition, activities of all three PDE4s are present in the extracts of wild-type macrophages (pmol/min/107 cells: PDE4A, 2.62 Ϯ 0.53; PDE4B, 5.38 Ϯ 0.28; PDE4D, 2.85 Ϯ 0.38; mean Ϯ SEM of three groups of five mice each). These measurements indicate that the ratio of the PDE4A:PDE4B: PDE4D activity in peritoneal resident macrophages is ϳ1:2:1. Af- ter LPS stimulation, PDE4B activity in the wild-type cells was increased ϳ5-fold, whereas PDE4A or PDE4D activities were marginally increased or not significantly affected (pmol/min/107 cells: PDE4A, 4.12 Ϯ 0.23; PDE4B, 26.45 Ϯ 1.45; PDE4D, 3.08 Ϯ 0.33; mean Ϯ SEM of three groups of five mice each; approximate ratio 1:7:1). Statistical analyses indicated that the in- crease in PDE4A activity after LPS had p values between 0.05 and 0.1. In agreement with the immunological data, the LPS-induced increase in PDE4 activity was absent only in the PDE4B null mac- rophages (Fig. 2). These data demonstrate that three PDE4 genes Downloaded from are expressed in peritoneal macrophages but that PDE4B is the major PDE4 subtype induced by LPS activation of these cells. Moreover, inactivation of one PDE4 gene did not produce a com- pensatory increase in expression of any other PDE4 genes, sug- gesting a lack of overlapping functions among the paralog genes.

Compensation with rolipram-insensitive PDEs was not observed http://www.jimmunol.org/ either (Fig. 1). Similar results were obtained with TG-elicited mac- rophages, albeit with two major differences. PDE4A, PDE4B, and PDE4D were expressed at comparable levels under basal condi- tions, and the PDE4B stimulation was reduced (pmol/min/mg pro- tein: PDE4A, 2.89 Ϯ 0.09; PDE4B, 5.94 Ϯ 0.46; PDE4D, 2.46 Ϯ 0.29; mean Ϯ SEM of two groups of three mice each; ratio 1:2:1). It also should be pointed out that although the wild-type littermates for each genotype were analyzed separately, their properties and FIGURE 3. LPS-induced TNF-␣ and IL-6 production in peritoneal res- PDE4 expression were not significantly different in any of the ex- ident macrophages. Peritoneal macrophages from PDE4AϪ/Ϫ, PDE4BϪ/Ϫ, by guest on September 24, 2021 periments performed, confirming the robustness of the and/or PDE4DϪ/Ϫ mice and their wild-type littermates were incubated with measurements. 100 ng/ml LPS for the indicated times (A) or with increasing concentra- ␣ These analyses define a cellular model where three PDE4s are tions of LPS for6h(B and C). Accumulation of TNF- (A and B) and IL-6 Ϯ expressed at the same time, and cells derived from the three null (C) in the culture medium was determined by ELISA. Data are the mean p Ͻ 0.001 (compared with ,ءء ;p Ͻ 0.01 ,ء .(SEM (n ϭ 4–7 mice/genotype mice allow the investigation of the effect of inactivation of indi- the wild-type cells). vidual subtype on distal responses. LPS stimulation of TNF-␣ production is affected only in PDE4B-deficient macrophages ways show different sensitivity to cAMP inhibition, PDE4B spe- To assess the impact of different PDE4s on LPS activation of cy- cifically affecting only the TNF-␣ production but not IL-6. The tokine responses, the three PDE4 null macrophages and their cor- fact that distinct components are used in the two pathways is sug- responding wild-type macrophages were incubated with 100 ng/ml gested by analysis of the Rel knockout mice (24, 25). LPS for different times, and the levels of TNF-␣ released in the The decreased accumulation of protein in the medium was as- medium were measured by ELISA. As shown in Fig. 3A, LPS sociated with a decrease in TNF-␣ mRNA steady state in PDE4B induced TNF-␣ production in all wild-type macrophages, and the null macrophages after LPS stimulation (Fig. 4), suggesting that induction reached a maximum in 8 h and remained constant for up TNF-␣ transcription or mRNA stability is distal to PDE4B. to 24 h. PDE4A- and PDE4D-deficient macrophages responded to When using TG-elicited macrophages, only PDE4B ablation LPS in a manner indistinguishable from their wild-type counter- produced a significant decrease in TNF-␣ accumulation (data not parts. Conversely, LPS-induced TNF-␣ production was signifi- shown). In the three independent experiments performed with Ͼ10 cantly decreased in PDE4B null cells ( p Ͻ 0.01). With Ͼ30 mice mice, PDE4B null macrophages produced 52.5 Ϯ 7.8% TNF-␣ tested independently, TNF-␣ response in PDE4BϪ/Ϫ macrophages compared with wild-type cells. was decreased to 43.5 Ϯ 3.5% of control. This blunted TNF-␣ accumulation was not due to decreased sensitivity to LPS because Nonselective PDE4 inhibitors exert their pharmacological effects ␣ higher concentrations did not restore the full response in the on TNF- production by blocking only PDE4B activity PDE4B null cells (Fig. 3B). In addition, the decrease in TNF-␣ Numerous reports have shown that PDE4 inhibitors are effective in production is not caused by a generalized loss of viability or com- blocking TNF-␣ production in inflammatory cells (see review Ref. promised function of macrophages defective in PDE4B because 1), underscoring the anti-inflammatory potentials of this class of IL-6 accumulation was stimulated in PDE4B null cells to the same compounds (9, 26, 27). However, with few exceptions, all PDE4 extent as in wild-type cells (Fig. 3C). This latter observation sug- inhibitors thus far developed are nonselective, in that they block gests that distinct branches of LPS-activated TLR signaling path- the activity of every PDE4 with minimal differences in EC50 (9, The Journal of Immunology 1527

FIGURE 4. LPS regulation of TNF-␣ mRNA expression in peritoneal macrophages. Peritoneal macrophages from PDE4BϪ/Ϫ (A) and Ϫ/Ϫ

PDE4D (B) mice and their wild-type littermates were incubated in the Downloaded from absence or presence of 100 ng/ml LPS for 3 h. Total RNA was extracted from the cells (pooled from 7 to 11 animals/group) followed by RT-PCR using TNF-␣-specific primers. The amplified products were analyzed by Southern blot using an oligonucleotide probe with sequence nested be- tween the specific PCR primers. Amplification of a GAPDH fragment was included to monitor the amount of RNA in each sample. C, Densitometric scanning of Southern blots was conducted, and the ratios of TNF-␣ and http://www.jimmunol.org/ GAPDH density were compared between the wild-type and PDE4BϪ/Ϫ macrophages. Data are the mean Ϯ SEM of three independent experiments. ␣ -Significantly different from the wild-type cells (p Ͻ 0.05). FIGURE 5. Effect of PDE4 inhibitors on LPS-induced TNF- produc ,ء tion in PDE4-deficient peritoneal macrophages. A, Peritoneal macrophages from PDE4AϪ/Ϫ, PDE4BϪ/Ϫ, and PDE4DϪ/Ϫ mice and their wild-type littermates were incubated with 10 ␮M rolipram or vehicle (DMSO) for 30 26, 27). The cellular model characterized above allowed us to de- min before LPS (100 ng/ml) stimulation for 8 h. TNF-␣ accumulation in termine whether inhibition of a single PDE4 is sufficient to pro- the medium was determined by ELISA. No significant difference was ob- duce useful pharmacological effects. Macrophages of different ge- served between the control and rolipram-treated cells in PDE4BϪ/Ϫ mice. netic backgrounds were incubated with 10 ␮M rolipram, and the B, Wild-type and PDE4B-deficient macrophages were treated with increas- by guest on September 24, 2021 TNF-␣ response was evaluated. As shown in Fig. 5A, rolipram ing concentrations of rolipram or roflumilast for 30 min before LPS (100 inhibited LPS-stimulated TNF-␣ production in wild-type macro- ng/ml) stimulation. After 5 h, TNF-␣ accumulation in the medium was phages as well as PDE4A and PDE4D null cells but had no sig- measured. Data are the mean Ϯ SEM (n ϭ 4–7 mice/group). nificant effects in the PDE4BϪ/Ϫ cells. Similar results were ob- tained when rolipram or roflumilast, a PDE4 inhibitor in clinical trial (8, 27), was used at different concentrations (Fig. 5B). Col- lectively, these results demonstrate that the effects of PDE4B ab- cAMP could be detected with the ablation of either PDE4B or lation and rolipram inhibition on LPS-stimulated TNF-␣ produc- PDE4D. tion are not additive. In addition, they indirectly demonstrate that In light of the above findings, we evaluated the possibility that acute rolipram inhibition of PDE4A and PDE4D present in the PDE4B ablation may impact the TNF-␣ response by affecting PDE4BϪ/Ϫ macrophages has no efficacy in blocking TNF-␣ steps in the LPS cascade independent of cAMP accumulation. The response. involvement of cAMP signaling in the PDE4B phenotype was fur- ther evaluated in these macrophages. The adenylyl cyclase activa- Cyclic AMP signaling mediates the effects of PDE4B ablation tor forskolin and the protein kinase A (PKA) inhibitors H89 or A possible explanation of the above findings is that ablation of Rp-cAMPs were used, and the TNF-␣ production was compared in Ϫ Ϫ PDE4B, but not PDE4A or PDE4D, produces major changes in wild-type and PDE4B / macrophages. Preincubation of wild- cAMP accumulation in macrophages, hence the selective effect on type macrophages with 1 ␮M forskolin for 20 min followed by 5 TNF-␣ accumulation. To test this possibility, cAMP was measured h LPS stimulation led to a 64% decrease in TNF-␣ accumulation, Ϫ Ϫ in macrophages from wild-type and PDE4B or PDE4D null mice reaching levels similar to those observed in the PDE4B / cells during the course of LPS activation. LPS stimulation of macro- incubated with LPS alone (Fig. 7). Higher concentrations of for- phages is associated with a small but reproducible increase in skolin produced minor further inhibition of TNF-␣ accumulation cAMP accumulation in all genotypes tested. Cyclic nucleotide lev- in both the wild-type and PDE4B null cells. Rolipram had minimal els reached a maximum in 15–30 min and then returned to baseline additive effects to forskolin in wild-type macrophages and no ef- in ϳ3 h (Fig. 6A). However, ablation of either PDE4B or PDE4D fect in PDE4B null cells. In a slightly different experimental con- did not have detectable effects on cAMP accumulation (Fig. 6A). dition, forskolin added at the same time as LPS produced inhibi- Statistical analyses of the data showed no differences in the three tion of TNF-␣ production identical with that caused by PDE4B curves, even though a trend to an increase in cAMP at3hwas ablation with no further effect at higher doses (data not shown). sometimes observed in the PDE4B null cells. In addition, the level Cyclic AMP acts via activation of the effectors PKA, cAMP- of phosphorylation of CREB was comparable in wild-type and guanine nucleotide exchange factor (GEF), or cyclic nucleotide PDE4B null macrophages (Fig. 6B). Thus, no major changes in gated channels. However, cAMP-GEF does not appear to have a 1528 PDE4B IN LIPOPOLYSACCHARIDE SIGNALING IN MACROPHAGES

FIGURE 7. Forskolin inhibits LPS-induced TNF-␣ production in peri- toneal macrophages. Macrophages from wild-type and PDE4BϪ/Ϫ mice Downloaded from were pretreated for 20 min with forskolin (FSK; 1 or 50 ␮M), a combi- nation of 50 ␮M FSK and 10 ␮M rolipram (Rol), or vehicle (DMSO) followed by LPS stimulation for 5 h. TNF-␣ accumulation in the medium -Sig ,ء .(was measured. Data are the mean Ϯ SEM (n ϭ 3–5 mice/group nificantly different from the wild-type control group (p Ͻ 0.01). http://www.jimmunol.org/ gene targeting are mediated by activation of cAMP signaling and FIGURE 6. LPS stimulation of cAMP accumulation and CREB phos- require PKA activity. phorylation in wild-type and PDE4-deficient macrophages. A, Peritoneal macrophages from PDE4BϪ/Ϫ and PDE4DϪ/Ϫ mice and their wild-type PDE4B-deficient mice are resistant to LPS-induced shock littermates were incubated with 100 ng/ml LPS for the indicated times. LPS Because TNF-␣ is a key mediator of endotoxin-induced toxicity in stimulation was terminated by adding 5% TCA immediately after cell wash vivo (29), the observation that macrophages deficient in PDE4B with ice-cold PBS. Concentration of cAMP was measured by RIA. B, ␣ Wild-type and PDE4B-deficient macrophages were incubated in the ab- exhibit a blunted TNF- response to LPS prompted a question of sence or presence of 100 ng/ml LPS for 20 min. Sixty micrograms of the whether PDE4B null mice were resistant to LPS-induced shock. by guest on September 24, 2021 cell extract were subjected to 15% SDS-PAGE and then electroblotted. The For this purpose, wild-type and PDE4B null mice were challenged phosphorylated CREB was detected by incubation of the membrane with a with a high dose of LPS (800 ␮g/25 g body weight), and survival phospho-CREB Ab and then a peroxidase-conjugated secondary Ab. Im- was monitored for 7 days. As shown in Fig. 9, this treatment munoreactive signals were developed using an ECL detection system. Sub- caused a significantly higher mortality in wild-type mice (52%) sequently, the membrane was incubated with an anti-CREB Ab, followed than in PDE4B null mice (27%). In addition, wild-type mice suc- by an AP-conjugated secondary Ab, and developed with an Immun-Star cumbed to shock mostly on day 1.5 after LPS injection, whereas AP detection system.

functional role in inflammatory cells (28). Indeed, the cAMP-GEF- specific activator 8-(4-chlorophenylthio)-2Ј-O-methyladenosine- 3Ј,5Ј-cyclic monophosphate had no significant effect on LPS-in- duced TNF-␣ accumulation in the peritoneal macrophages (data not shown). PKA inhibitors were then used to determine whether the effect of PDE4B ablation or rolipram inhibition requires PKA activity (Fig. 8). Consistent with the data described above, TNF-␣ accumulation was decreased after PDE4B ablation, and rolipram inhibited the TNF-␣ response in wild-type macrophages to the same degree as PDE4B inactivation but had no further effect in the PDE4B-deficient cells (Fig. 8). Preincubation of wild-type cells with 20 ␮M H89 for 20 min caused a significant increase in the TNF-␣ production. More importantly, H89 restored the TNF-␣ response in PDE4B null cells to levels not significantly different from that in wild-type cells treated with LPS alone. When the cells were incubated with a combination of H89 and rolipram, there was FIGURE 8. Effect of H89 on LPS-induced TNF-␣ production in wild- little or no rolipram inhibition of TNF-␣ production observed, in- type and PDE4B-deficient macrophages. Macrophages from wild-type and dicating that PKA is distal to PDE4B in the cAMP signaling cas- PDE4BϪ/Ϫ mice were pretreated for 20 min with 10 ␮M rolipram, 20 ␮M cade and that rolipram effects appeared to require an active PKA. H89, a combination of 10 ␮M rolipram and 20 ␮M H89, or vehicle Similar results were obtained with the cAMP analog Rp-cAMPS (DMSO) before LPS stimulation for 5 h. TNF-␣ accumulation in the me- used at 1 mM (data not shown). Thus, these experiments demon- dium was measured. Data are the mean Ϯ SEM (n ϭ 3–5 mice/group). A strate that effects of PDE4B inactivation by either rolipram or by representative experiment of the two performed is reported. The Journal of Immunology 1529

pituitary adenylate cyclase-activating peptide or vasoactive intes- tinal peptide decreases TNF-␣ production by blocking I␬B degra- dation and p65/Rel translocation, and/or rearrangement of the CREB/Jun/CBP complexes in the TNF-␣ promoter (34). In mac- rophages deficient in PDE4B, LPS-induced phosphorylation of CREB is not significantly increased, suggesting that change in complexes involving CREB in the TNF-␣ promoter is not the cause of decreased TNF-␣ accumulation. An additional interaction between NF-␬B and PKA has been described. The catalytic sub- unit of PKA is in a complex with NF-␬B/I␬B (35). Degradation of I␬B causes activation of the associated PKA catalytic subunit, phosphorylation of p65 at Ser 276, and increased transcriptional activity. This mechanism is likely not to be distal to PDE4B in- activation, in view of the finding that the above regulation is thought to be cAMP-independent (35). FIGURE 9. Survival after high-dose LPS treatment. Wild-type and We should emphasize that our experimental model is different PDE4B-deficient mice were injected i.p. with 800 ␮g/25 g body weight from that commonly used to explore the effect of cAMP on TLR p Ͻ 0.05 (com- signaling. The widely used forskolin and cAMP analogues produce ,ء .LPS. Mortality was monitored every 12 h for 7 days pared with the wild-type mice). generalized and massive increases in intracellular cAMP, whereas Downloaded from PDE4B ablation produces minor, if any, global cAMP changes. When strategies affecting overall cAMP are used, it is most likely PDE4B null mice died later, mostly on day 3.5. These results dem- that several redundant pathways are activated, rendering it difficult onstrated that ablation of PDE4B partially protects mice from to evaluate the hierarchies and intersections among the different LPS-induced shock. regulatory loops. In our model, instead, we believe that compart-

ments of signaling are disrupted only in a subtle way. PDE4A, http://www.jimmunol.org/ Discussion PDE4B, and PDE4D are expressed in resident and in TG-elicited Using a combination of genetic and pharmacological approaches, peritoneal macrophages, yet only ablation of PDE4B impacts we provide evidence that the function of PDE4s in macrophages is TNF-␣ production. The relative proportion of PDE4B vs all other highly specialized. Of the three PDE4 subtypes expressed in these PDEs expressed is not a predictor of the impact on TNF-␣ stim- cells, PDE4B is induced by activation of the TLR signaling and is ulation because inhibition of TNF-␣ is still present in TG-elicited the predominant PDE isoenzyme involved in the control of TNF-␣ macrophages where PDE4B relative expression is reduced to production. More importantly, we demonstrate that the pharmaco- ϳ20% of total PDE compared with naive macrophages (ϳ60%). logical effects of PDE4 inhibitors on macrophage TNF-␣ produc- Together with the absence of major global cAMP changes in cells tion are exerted exclusively through inhibition of PDE4B. More- deficient in PDE4B, this finding strongly suggests that a minor by guest on September 24, 2021 over, the altered TLR signaling is reflected by in vivo effects pool of cAMP regulated by PDE4B is involved in the control of because PDE4B null mice are protected in LPS-induced shock. TLR signaling. The rolipram inhibition of TNF-␣ production and These findings have considerable physiological and pharmacolog- the lack of additivity of the rolipram effects with PDE4B ablation ical implications, indicating a highly specialized, nonredundant further indicate that loss of PDE4B activity is responsible for the PDE function in a cell and providing the experimental basis for phenotype. Finally, the pharmacological data with forskolin and novel therapeutic strategies that use subtype-selective PDE4 PKA inhibitors indicate that both the acute and genetic PDE4B inhibitors. inactivation impact TNF-␣ accumulation via changes in cAMP That the effects of the genetic PDE4 inactivation are subtle and and PKA activity. Taken together, these findings define the fol- not due to generalized impairment of macrophage functions is doc- lowing regulatory loop of TLR signaling. Inactivation of PDE4B umented by at least two lines of evidence. First, IL-6 production is affects a minor pool of cAMP and a subpopulation of PKA, which unaffected in the PDE4B- and PDE4A-deficient macrophages, in turn interferes with steps downstream of TLR activation. Either demonstrating that the ability to respond to LPS stimulation re- physical sequestration of cAMP or the presence of macromolecu- mains intact in these cells. Secondly, that the effects of PDE4B lar complexes involving PDE4B2 is probably at the basis of this ablation are reversible is documented by the finding that TNF-␣ specific effect of PDE4B. It is possible that PDE4B2 and PKA are accumulation can be fully restored by inhibition of PKA. There- in a complex coordinated by an A-kinase anchor protein, as it has fore, the long-term removal of PDE4B has no generalized, dele- been shown for PDE4D (36, 37). If present, this complex must terious effects on the macrophage ability to generate TNF-␣. Fi- interact preferentially with components of the TLR signaling. A nally, the observation that acute PDE inhibition with rolipram corollary of this hypothesis is that PDE4D and PDE4A expressed closely reproduces the effects of PDE4B ablation argues that the in these cells are engaged in macromolecular complexes or are impaired TNF-␣ response is a well-defined phenotype linked to the localized in compartments distinct from those involving PDE4B2 loss of PDE4 activity. and somehow removed from TLR signaling. In support of this Many reports have addressed the effect of cAMP on TNF-␣ hypothesis, it has been reported that PDE4B2 is compartmental- accumulation, and a plethora of possible interaction points be- ized in T cells and that the subcellular localization differentially tween the two pathways have been identified (30–33). Neverthe- impacts T cell responses (38). less, the exact sites where cAMP signaling interferes with TNF-␣ Given the facts that activation of the TLR pathways induces the are still debated. In some cases, a decrease in expression and activity of PDE4B and ablation of this gene pro- signaling through the MEK kinase pathway has been observed duces a blunted TNF-␣ response in monocytes/macrophages, we following an increase in cAMP (33). In other cases, the transacti- have proposed that PDE4B induction is a positive feedback mech- vating properties of p65 have been indicated as the target of PKA anism necessary to overcome the negative modulation of cAMP activation (32). Activation of cAMP signaling in macrophages by and to finely control TNF-␣ production in time and in response to 1530 PDE4B IN LIPOPOLYSACCHARIDE SIGNALING IN MACROPHAGES different extracellular environments (7). Consistent with the pres- Acknowledgments ence of a tonic cAMP inhibition in these macrophages, we show in We are indebted to Drs. Dale Umetsu and Stephen Galli for critical reading this study that inhibition of PKA in wild-type cells causes a sig- of the manuscript and Dr. Susumu Nakae for the FACS analysis. nificant increase in TNF-␣ accumulation. This positive feedback may be operating to oppose the inhibitory effects of prostaglan- Disclosures dins, vasoactive intestinal peptide/pituitary adenylate cyclase- Dr. M. Conti is a consultant for Pfizer Inc. and has received a research grant activating peptide, or other deactivating cytokines and chemokines from Altana Pharma. released at the site of inflammation. In view of the differential expression of PDE4 during monocyte-macrophage differentiation References and marked differences in sensitivity to PDE4 inhibitors, this feed- 1. Torphy, T. J. 1998. Phosphodiesterase isozymes: molecular targets for novel back probably loses its function after macrophages are terminally antiasthma agents. Am. J. Respir. Crit. 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