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Am J Physiol Cell Physiol 290: C143-C151, 2006. First published August 24, 2005; doi:10.1152/ajpcell.00171.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages

Y. Osawa, H. T. Lee, C. A. Hirshman, D. Xu, and C. W. Emala

Department of Anesthesiology, College of Physicians and Surgeons, Columbia University, New York, New York

Submitted 11 April 2005 ; accepted in final form 19 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.7 cells) were treated with LPS in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, CaM, Gi proteins, and NF-{kappa}B translocation or transcription/translation. LPS effects on CaMKII phosphorylation and the expression of relevant adenylyl cyclase (AC) isoforms were measured. LPS caused a significant dose (5–10,000 ng/ml)- and time (1–8 h)-dependent increase in forskolin-stimulated AC activity that was abrogated by pretreatment with SN50 (an NF-{kappa}B inhibitor), actinomycin D, or cycloheximide, indicating that the effect is mediated via NF-{kappa}B-dependent transcription and new protein synthesis. Furthermore, LPS decreased the phosphorylation state of CaMKII, and pretreatment with a CaM antagonist attenuated the LPS-induced sensitization of AC. LPS, cAMP, or PKA activation each independently decreased macrophage proliferation. However, inhibition of NF-{kappa}B had no effect on LPS-induced decreased proliferation, indicating that LPS-induced decreased macrophage proliferation can proceed via PKA-independent signaling pathways. Taken together, these findings indicate that LPS induces sensitization of AC activity by augmenting the stimulatory effect of CaM and attenuating the inhibitory effect of CaMKII on isoforms of AC that are CaMK sensitive.

cell culture; prostaglandin; phosphodiesterase; nuclear factor-{kappa}B; calcium/calmodulin-dependent kinase; calmodulin

MACROPHAGES PLAY A CENTRAL ROLE in orchestrating the immune response to foreign pathogens via phagocytosis and the secretion of cytokines. LPS, a major constituent of the gram-negative bacterial cell wall, is responsible in part for initiating macrophage responses, including the secretion of cytokines. Conversely, agents that elevate cAMP are known to reverse LPS-induced cytokine release, but whether LPS modulates cAMP, which could feed back to modulate further cytokine release, is unknown.

There is growing recognition that immunosuppression during the later phases of sepsis contributes to adverse clinical outcomes (11). LPS reduces macrophage proliferation (5, 21), and LPS coincubated with IFN-{gamma} increases the rate of cell death in a macrophage-like cell line (RAW264.7) (14). Ayala et al. (2) showed increased peritoneal macrophage apoptosis in vivo in a cecal ligation and puncture sepsis model.

In addition to the secretion of cytokines, immune cell proliferation (or the lack thereof) is considered critical to a host’s successful response to sepsis. The elevation of intracellular cAMP by dibutyryl adenosine 3',5'-cyclic monophosphate or PGE2 has been shown to suppress proliferation of guinea pig peritoneal macrophages (15) and murine bone marrow-derived macrophages (28). Furthermore, Aronoff et al. (8) showed that cAMP inhibition of macrophage function did not activate PKA but instead activated a recently characterized signaling pathway involving the exchange protein directly activated by cAMP-1, Epac-1 (1).

cAMP is synthesized by a nine-member family of mammalian transmembrane adenylyl cyclase (AC) isoforms that are differentially regulated in part by Ca2+/CaM or by phosphorylation by diverse kinases, including CaMK, PKA, PKC, raf-1 kinase (9, 29, 30), and tyrosine kinases (25). One well-defined mechanism of increased activity of certain isoforms of AC is "supersensitization" (also termed cAMP overshoot, supersensitivity, superactivation, or heterologous sensitization), which is classically described after chronic activation of Gi protein-coupled receptors and is thought to be mediated by raf-1 kinase-mediated phosphorylation of AC (9, 29). However, amplified AC responses also have been described after chronic exposure to classic mediators of sepsis, including TNF-{alpha} (19, 22) and IL-1{beta} (3, 19). Because the LPS receptor (Toll-like receptor 4, TLR4) has been known to share a common signaling cascade with the IL-1 receptor, we hypothesized that exposure to LPS would sensitize AC activity in a murine macrophage cell line and that LPS-induced cAMP accumulation would induce inhibition of macrophage proliferation.

Thus the effect of LPS on macrophage cAMP levels could modulate two critical events in the macrophages’ responses during sepsis: proliferation and cytokine release. Therefore, we addressed the following questions: 1) Does LPS sensitize AC activity in macrophages? 2) If so, does the mechanism involve a) prostaglandin release, b) phosphodiesterase inhibition, c) kinase-mediated phosphorylation of AC, d) altered expression of AC isoforms, e) CaM/CaMKII regulation of AC, f) NF-{kappa}B-mediated gene transcription, or f) new transcription/translation? Finally, we questioned whether the LPS-induced reduction in macrophage proliferation is related to LPS-induced sensitization of AC activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Cell culture reagents were obtained from GIBCO-BRL (Grand Island, NY). The bicinchoninic acid protein assay reagent was obtained from Pierce (Rockford, IL). [{alpha}-32P]ATP (800 Ci/mmol), [3H]cAMP (32 Ci/mmol), and [3H]thymidine (6.7 Ci/mmol) were obtained from MP Biochemicals (Irvine, CA). Sp-8-bromoadenosine 3',5'-cyclic monophosphate (Sp-8-BrcAMP; adenosine 3',5'-cyclic monophosphorothioate, 8-bromo, Sp-isomer, sodium salt), Rp-8-BrcAMP 3',5'-cyclic monophosphorothioate (adenosine 3',5'-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt), Raf-1 kinase inhibitor I, SN50, SN50M, geldanamycin, and W-7 were obtained from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma (St. Louis, MO).

Cell culture. The murine monocyte/macrophage RAW264.7 cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were grown in a humidified incubator at 37°C and 5% CO2.

Inhibitors of signaling intermediate strategies. Murine macrophages were grown to confluence in 24-well plates. After overnight serum starvation, LPS and indicated inhibitors were added at the indicated concentrations and times. After treatment, cells were washed twice in warm PBS and used directly for AC assays.

To investigate a potential role of prostaglandins in LPS-induced AC sensitization, we used an inhibitor of prostaglandin synthesis (10 µM indomethacin added 30 min pretreatment) or prostaglandin receptor antagonists [prostaglandin type E (EP)2 or EP4 antagonists AH6809 or AH23848, respectively, 10 µM, added 30 min pretreatment]. To evaluate the potential role of phosphodiesterase inhibition in the LPS-induced increase in intracellular cAMP, we treated some cells with 10 µM IBMX 30 min pretreatment. To evaluate the potential role of the heterotrimeric protein Gi in the LPS-induced sensitization of AC, some cells were pretreated with pertussis toxin (100 ng/ml; 4 h) before LPS exposure and AC measurements.

To investigate the potential role of specific kinases in the LPS-induced sensitization of AC, cells were pretreated with Rp-8-BrcAMP (PKA inhibitor, 100 µM, 1 h), GF-109203X (PKC inhibitor, 100 nM, 1 h), Raf-1 kinase inhibitor I (100 nM, 1 h), herbimycin A (tyrosine kinase and NF-{kappa}B inhibitor, 1 µM, 1 h), and genistein (tyrosine kinase inhibitor, 50 µM, 1 h). To investigate the potential role of CaM in the LPS-induced sensitization of AC, cells were pretreated with W-7 (CaM antagonist, 25 µM, 1 h).

To investigate the potential requirement for new gene transcription and translation in the LPS-induced sensitization of AC activity, some cells were pretreated with inhibitors of transcription or translation (actinomycin D, 5 µg/ml, 4 h, or cycloheximide, 0.5 µg/ml, 30 min, respectively). To examine specifically the transcriptional factor NF-{kappa}B in the LPS-induced sensitization of AC, some cells were pretreated with an inhibitor of NF-{kappa}B nuclear translocation (SN50, 20 µM, 1 h) or its inactive control peptide (SN50M, 20 µM, 1 h).

Heat shock protein 90 (HSP90) is a protein involved in the receptor complex responsible for LPS signaling. To determine whether LPS-induced sensitization was due to specific activation of the LPS-HSP90 receptor complex, some cells were pretreated with an inhibitor of HSP90 before LPS treatment (geldanamycin, 5 µM, 1 h).

AC assays. Basal, forskolin (10 µM)-, or PGE2 (10 µM)-stimulated AC activity was determined by measuring the conversion of [{alpha}-32P]ATP to [32P]cAMP according to the method of Salomon et al. (23). In brief, AC assays were performed for 15 min at 37°C in a total volume of 150 µl containing 50 mM HEPES, pH 8.0, 50 mM NaCl, 0.4 mM EGTA, 1 mM cAMP, 7 mM MgCl2, 0.1 mM ATP, 7 mM creatine phosphate, 50 U/ml creatine phosphokinase, 0.1 mg/ml BSA, and 10 µCi/ml [{alpha}-32P]ATP (12). Preliminary experiments confirmed the linearity of AC activity at the cell density and incubation times used. The reactions were terminated by addition of 100 µl of stop buffer (50 mM HEPES, pH 7.5, 2 mM ATP, 0.5 mM cAMP, 2% SDS, and 1 µCi/ml [3H]cAMP). The synthesis of [32P]cAMP was determined by performing sequential column chromatography over Dowex (Bio-Rad Laboratories, Hercules, CA) and alumina (23). Recovery of [3H]cAMP from each column was used to calculate column recovery rates, which ranged from 75% to 90%. Data are expressed as the percentage of cAMP accumulation in treated cells relative to that in control cells treated with vehicle.

Immunoblot analysis. Whole cell lysates were electrophoresed (10% SDS-PAGE) and immunoblotted using antibodies directed against phospho-CaMKII Thr286 (1:1,000 dilution; Upstate Cell Signaling Solutions, Charlottesville, VA) and {beta}-actin (1:2,000 dilution; Sigma). Epitopes were visualized using horseradish peroxidase-conjugated secondary antibodies (1:1,000–1:5,000 dilution; Amersham Biosciences, Arlington Heights, IL) using ECL or ECL Plus (Amersham Biosciences) and developed on light film (BioMax; Kodak, Rochester, NY). Film was developed such that band intensities were within the linear range of film responses, and band intensities were quantified using Quantity One software (Bio-Rad Laboratories). Levels of phospho-CaMKII immunoreactivity were normalized to {beta}-actin to control for total protein levels. Data are presented as means ± SE.

RNA isolation and RT-PCR. Total RNA was isolated from confluent RAW macrophages in T75 flasks and from whole mouse brain (positive control for all 9 AC isoforms) using the RNAWIZ RNA isolation reagent (Ambion, Austin, TX) according to the manufacturer’s recommendations. Using the Advantage RT-PCR kit (BD Biosciences, Palo Alto, CA), we reverse transcribed 1 µg of total RNA at 42°C for 1 h in 20 µl of solution, including 200 U of Moloney murine leukemia virus reverse transcriptase, 20 U of RNase inhibitor, 20 pmol oligo(dT) primer, and 0.5 mM 2-deoxynucleotide 5'-triphosphate (dNTP) mix in reaction buffer (50 mM Tris·HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2).

We initially determined which isoforms of AC were expressed in RAW macrophages. PCR was performed by adding 5 µl of newly synthesized cDNA to a 45-µl reaction mixture, yielding final concentrations of 0.2 mM of each dNTP, 1x Advantage 2 polymerase mix, PCR buffer (BD Biosciences, Palo Alto, CA), and 0.4 µM of both sense and antisense primers (Sigma) for all nine AC isoforms (Table 1). PCR was performed using a PTC-200 Peltier thermal cycler (MJ Research, Waltham, MA) after an initial denaturation step at 94°C for 2 min, 35 cycles of PCR were performed, including denaturation at 94°C for 10 s, annealing at 64°C for 30 s, extension at 72°C for 1 min, and a final extension of 72°C for 5 min.


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  		Table 1. Adenylyl cyclase isoform-specific primer and GAPDH primer sequences

 
Functional assays suggested that LPS-induced sensitization of AC involved CaMK-dependent isoforms (classically known to be AC isoforms I, III, and VIII). Because AC isoform I was not expressed in RAW macrophages, we focused on potential LPS-induced changes in the mRNA expression of AC isoform III or VIII using semiquantitative RT-PCR. Preliminary experiments were performed to determine the optimal number of PCR cycles that yielded a linear increase in band intensity. The number of PCR cycles chosen for each primer set (Table 1) was such that band intensities had not reached a plateau by the end of the amplification protocol; that is, PCR products were in an exponential phase of amplification (for a representative example of AC isoform III, see Fig. 9A). Amplification was performed using a two-step PCR including denaturation at 94°C for 10 s and primer annealing and extension at 68°C for 1.5 min. GAPDH was used as an internal control gene to control for relative RNA input. We used cycle 32 for AC III, cycle 34 for AC VIII, and cycle 19 for GAPDH. PCR products were electrophoresed on 5% nondenaturing polyacrylamide gel in 1x Tris-acetate-EDTA buffer. The gel was stained with SYBR Green I reagent (Molecular Probes, Eugene, OR), visualized using UV illumination, and analyzed using Quantity One software (Bio-Rad Laboratories).



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  		Fig. 9. A: representative gel images for AC III and GAPDH used to optimize PCR cycle number for semiquantitative RT-PCR are shown. Cycles 32 and 19 were chosen for subsequent semiquantitative analysis of AC III and GAPDH, respectively. B: representative gel image of the effect of LPS on mRNA expression of CaM-sensitive AC isoforms (III and VIII). Confluent RAW264.7 cells were incubated with LPS (100 ng/ml; 4 h). AC isoform and GAPDH (as internal control) mRNA were measured by performing RT-PCR. The PCR cycle number for each mRNA is indicated in brackets.

 
Cell proliferation assay. Cell proliferation was quantified by the incorporation of [3H]thymidine into DNA. Murine macrophages were plated at a density of 2 x 105 cells/ml in 24-well plates and incubated for 24 h in complete medium containing 10% FBS, followed by serum starvation for another 24 h. Cells were treated in triplicate with or without LPS (100 ng/ml), forskolin (50 µM; a stimulator of AC), IBMX (500 µM; a phosphodiesterase inhibitor), or Sp-8-BrcAMP (an activator of PKA) for 6 h at 37°C, and [3H]thymidine (0.1 µCi/ml) was added for the last 4 h. The medium was removed, and cells were washed twice with 500 µl of cold PBS. After being washed twice in 500 µl of ice-cold 10% TCA and rinsed with 500 µl of 70% ethanol, the cells were solubilized in 250 µl of 0.2 N NaOH at room temperature for 15 min. This method allows for the recovery of [3H]thymidine incorporated into the DNA as opposed to measuring the total cellular uptake of [3H]thymidine and is therefore a more sensitive method of cellular (i.e., DNA) proliferation. Incorporated [3H]thymidine was quantified using scintillation counting, and results are expressed as means ± SE.

Cell viability. Cell viability after LPS exposure (100 ng/ml) for 6 or 24 h was measured in triplicate using Trypan blue dye exclusion. After incubation for 5 min with 0.2% Trypan blue in PBS, 300 cells from each group were counted and the cells that had internalized the dye were considered nonviable.

Statistics. Statistical analysis was performed using repeated-measures ANOVA, followed by a Bonferroni posttest comparison using Prism 4.0 software (GraphPad, San Diego, CA). Data are presented as means ± SE, and P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
AC activity after LPS treatment of murine macrophages. LPS demonstrated both concentration- and time-dependent sensitization of forskolin-stimulated AC activity. Significant sensitization of forskolin-stimulated AC activity was achieved within 1 h of LPS treatment (100 ng/ml), with peak sensitization occurring at 4 h (Fig. 1A). Sensitization of forskolin-stimulated AC activity occurred with concentrations of LPS ranging from 5 to 10,000 ng/ml with a peak effect at 100 ng/ml (487 ± 85% of control) (Fig. 1B). Therefore, subsequent experiments were performed with 100 ng/ml LPS for 4 h. AC activity decreased to below control levels at 24 h after treatment, presumably because of the cytotoxic effects of prolonged exposure to this concentration of LPS (see Cell viability subheading below).



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  		Fig. 1. A: time course of 10 µM forskolin-stimulated adenylyl cyclase (AC) activity in cultured RAW264.7 cells. Cells were treated with LPS (100 ng/ml) for the indicated times. B: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells pretreated with LPS at the indicated concentrations for 4 h (n = 4 within each experiment; values determined in triplicate). Data represent means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

 
LPS has been shown to inhibit phosphodiesterase activity (16). Therefore, we questioned whether the increased accumulation of [32P]cAMP was due to LPS-induced inhibition of phosphodiesterase activity as opposed to sensitization of AC activity. Preincubation of murine macrophages with 10 µM IBMX did not affect LPS-induced AC sensitization (data not shown), suggesting that the effects on AC activity and cAMP accumulation that we observed were independent of phosphodiesterase effects.

LPS has been shown to induce the release of prostaglandins in macrophages (6, 20). Prostaglandins also are known to modulate the activity of AC through EP receptors coupled to G proteins in a variety of cell types (7, 33). Therefore, we questioned whether LPS-induced AC sensitization involved prostaglandins in murine macrophages. We used three strategies. 1) We determined whether LPS-induced AC sensitization could be enhanced by acute stimulation with PGE2 (implying functionally coupled EP receptors). 2) We pretreated cells with indomethacin, an inhibitor of prostaglandin synthesis, before exposure to LPS. 3) We pretreated cells with inhibitors of prostaglandin receptors before exposure to LPS, because RAW264.7 cells have been reported to express EP2 and EP4 receptors, both of which couple to the stimulation of AC. LPS enhanced basal, forskolin (10 µM, 15 min)-, or PGE2 (10 µM, 15 min)-stimulated AC activity (Fig. 2A). However, preincubation with an inhibitor of prostaglandin synthesis, indomethacin (10 µM for 30 min) did not affect the sensitization of forskolin-stimulated AC activity induced by LPS, and indomethacin alone did not decrease AC activity compared with control (Fig. 2B). Thirty-minute pretreatment with an EP2 antagonist, AH6809 (10 µM), or an EP4 receptor antagonist, AH23848 (10 µM), failed to inhibit the LPS-induced sensitization of AC activity (data not shown). Taken together, these results suggest that the sensitization of AC activity by LPS is not mediated by prostaglandins in these cells.



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  		Fig. 2. A: AC activity in cultured RAW264.7 cells stimulated with vehicle (basal), forskolin (FSK; 15 min, 10 µM), or PGE2 (15 min, 10 µM) after 4-h LPS treatment (100 ng/ml) (n = 6 within each experiment; values determined in triplicate). B: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of indomethacin (10 µM; 30 min before LPS) (n = 3 within each experiment; values determined in triplicate). Data represent means ± SE. **P < 0.01, ***P < 0.001 vs. control.

 
Role of Gi{alpha} in LPS-induced AC sensitization. AC sensitization has classically been described after the chronic activation of Gi{alpha}-coupled receptors (31). To determine whether LPS-induced sensitization of AC involved Gi proteins, murine macrophages were pretreated with pertussis toxin, which inactivates Gi/o proteins by ADP ribosylation, but pertussis toxin pretreatment had no effect on the LPS-induced sensitization of AC in murine macrophages (Fig. 3).



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  		Fig. 3. Forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of pertussis toxin (PT; 100 ng/ml for 4 h before 4-h LPS treatment) (n = 4 within each experiment; values determined in triplicate). Data represent means ± SE. ***P < 0.001 vs. control.

 
Role of CaM and CaMKII in LPS-induced AC sensitization. Certain AC isoforms (I, III, and VIII) are known to be stimulated by Ca2+/CaM and inhibited by CaMK isoform CaMKIV (AC I) or CaMKII (AC III) (30). Pretreatment of cells with the CaM antagonist W-7 attenuated LPS-induced sensitization of AC activity (Fig. 4A). This finding suggests that the mechanism of LPS-induced AC sensitization involves CaM and implicates AC isoforms III and/or VIII in these cells.



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  		Fig. 4. A: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of W-7 (25 µM; 1 h before LPS) (n = 4 within each experiment; values determined in triplicate). Data represent means ± SE. ***P < 0.001 vs. control. ##P < 0.01 vs. LPS. B: Western blot analysis of phospho-CaMKII expression in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of Na3VO4 (300 µM; 15 min before LPS). Results representative of 3 experiments are shown. C: relative band intensities of phospho-CaMKII from 3 separate immunoblots. The effect of each treatment on phospho-CaMKII expression is expressed as the percentage of phospho-CaMKII levels in the positive control. Values are means ± SE. *P < 0.05.

 
Phosphorylation of Thr286 of CaMKII is indicative of kinase activation, which is known to inhibit the function of CaMK-sensitive isoforms of AC. LPS treatment dramatically decreased the phosphorylation state of CaMKII in the presence of 300 µM orthovanadate (Fig. 4, B and C). These findings are consistent with the removal of an inhibitory effect of CaMKII on susceptible isoforms of AC and may mechanistically account in part for LPS-induced AC sensitization.

Role of protein kinases on LPS-induced AC sensitization. In addition to CaMK regulation of AC isoforms, multiple other kinases are known to regulate certain AC isoforms, including PKA (26), PKC (32), raf-1 kinase (9), and tyrosine kinases (25). Therefore, we pretreated cells with inhibitors of several classes of protein kinases in an attempt to identify any additional mechanisms of LPS-induced sensitization of AC. Pretreatment with the PKA inhibitor Rp-8-BrcAMP, the PKC inhibitor GF-109203X, or a raf-1 kinase inhibitor did not affect LPS-induced AC sensitization (Fig. 5A). In contrast, pretreatment with the tyrosine kinase inhibitor herbimycin A, but not with the tyrosine kinase inhibitor genistein, significantly reversed the increase of AC activity by LPS (Fig. 5B).



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  		Fig. 5. A: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of protein kinase inhibitors. Rp-cAMP (Rp) (100 µM), GF-109203X (GF) (100 nM), or Raf-1 kinase inhibitor I (Raf) (100 nM) was added 1 h before 4-h LPS treatment. B: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of tyrosine kinase inhibitors. Herbimycin A (H; 1 µM) or genistein (G; 50 µM) was added 1 h before 4-h LPS treatment (n = 4 within each experiment; values determined in triplicate). Data represent means ± SE. **P < 0.01, ***P < 0.001 vs. control. ###P < 0.001 vs. LPS.

 
Role of NF-{kappa}B in LPS-induced sensitization of AC activity. In addition to its inhibition of tyrosine kinases, herbimycin A has also been reported to inhibit NF-{kappa}B activation and block NF-{kappa}B-driven gene expression in T lymphocytes (13). Because many of the effects of LPS are known to be mediated via NF-{kappa}B, we determined whether NF-{kappa}B-dependent gene transcription was involved in LPS-induced sensitization of AC. Pretreatment of cells with SN50, a cell-permeable peptide inhibitor of NF-{kappa}B, completely attenuated the LPS-induced sensitization of AC (Fig. 6A). The inactive control peptide SN50M had no effect on LPS activation of AC (data not shown). Furthermore, pretreatment of cells with either the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide also attenuated the LPS-induced increase in AC activity (Fig. 6, B and C).



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  		Fig. 6. A: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of an NF-{kappa}B inhibitor (20 µM SN50 added 1 h before 4-h LPS treatment). B: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 1-h LPS treatment (100 ng/ml) in the presence or absence of actinomycin D (ActD; 5 µg/ml added 4 h before 1-h LPS treatment). C: forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 1-h LPS treatment (100 ng/ml) in the presence or absence of cycloheximide (CHX; 0.5 µg/ml was added 30 min before 1-h LPS treatment) (n = 3 within each experiment; values determined in triplicate). Data represent means ± SE. **P < 0.01, ***P < 0.001 vs. control. #P < 0.05, ##P < 0.01 vs. LPS.

 
Role of HSP90 in LPS-induced AC sensitization. Recently, HSP90 has been reported to be a member of a receptor complex associated with TLR4 and plays an important role in the specific activity of LPS’s binding to the TLR4 receptor in macrophages (27). Therefore, we used an inhibitor of the TLR4-HSP90 receptor complex to determine whether LPS-induced AC sensitization is mediated by specific binding of LPS to the TLR4-HSP90 receptor complex. Pretreatment with geldanamycin, a specific inhibitor of HSP90, significantly attenuated the LPS-induced sensitization of AC activity (Fig. 7).



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  		Fig. 7. Forskolin (10 µM)-stimulated AC activity in cultured RAW264.7 cells after 4-h LPS treatment (100 ng/ml) in the presence or absence of geldanamycin (geld; 5 µM, 1 h before LPS treatment) (n = 4 within each experiment; values determined in triplicate). Data represent means ± SE. **P < 0.01 vs. control. #P < 0.05 vs. LPS.

 
Detection of nine AC isoforms using type-specific primers. RT-PCR using total RNA from whole mouse brain yielded products of expected sizes for each of the nine known mammalian transmembrane AC (positive controls) (Fig. 8). RT-PCR using total RNA from RAW264.7 cells demonstrated products of expected sizes for AC III–AC IX but not for AC I or AC II (Fig. 8).



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  		Fig. 8. Representative RT-PCR products using isoform-specific primers of AC I–AC IX in total RNA from RAW264.7 cells (RAW) and whole mouse brain. Total RNA (1 µg) was used in each RT-PCR. Neither mRNA encoding AC I nor mRNA encoding AC II was detected in RAW264.7 cells, despite the fact that they were detected in whole mouse brain. mRNA encoding AC III–AC IX was detected in total RNA extracted from both RAW264.7 cells and whole brain.

 
Effect of LPS on CaM-sensitive AC isoform mRNA. To determine whether LPS was upregulating mRNA encoding specific AC isoforms, which in turn accounted for enhanced total AC activity, we used RT-PCR to quantitate mRNA. Because inhibition of CaM attenuated LPS-induced sensitization of total AC activity, we focused on AC isoforms known to be regulated by CaM (30) and shown in the present study to be expressed in RAW macrophages (i.e., AC isoforms III and VIII). We first established RT-PCR conditions that allowed for the relative quantitative assessment of mRNA for AC III and VIII. Figure 9A shows a representative gel image used to evaluate relative band intensities for AC III and GAPDH. Band intensities did not plateau before cycle numbers 32 and 19, respectively, which were the cycle numbers chosen for subsequent semiquantitative RT-PCR experiments. Four-hour treatment with LPS (100 ng/ml) did not upregulate mRNA expression of either AC III or AC VIII (Fig. 9B).

Proliferation effects of LPS, forskolin, IBMX, or activator of PKA. LPS treatment (100 ng/ml, 6 h) decreased [3H]thymidine incorporation in murine macrophages (66.0 ± 3.2% of control, n = 4; P < 0.001). In addition, increases in intracellular cAMP with forskolin or IBMX also decreased [3H]thymidine incorporation in these cells (62.3% ± 2.7 or 44.5% ± 0.5 of control, respectively, n = 4 each; P < 0.001) (Fig. 10A). Furthermore, direct activation of PKA with Sp-8-BrcAMP decreased proliferation of murine macrophages (46.8 ± 2.8% of control, n = 4; P < 0.001) (Fig. 10A). Combined application of forskolin and LPS additively decreased proliferation compared with the effect of a single agent (Fig. 10A). Thus LPS or activation of the AC-cAMP-PKA cascade at several levels was capable of inhibiting macrophage proliferation. We next questioned whether LPS-induced reduction in proliferation was mediated through a similar pathway mediating increased AC activity.



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  		Fig. 10. A: effect of LPS, cAMP-elevating agents, or PKA agonist on proliferation of RAW264.7 cells. RAW264.7 cells were treated with LPS (100 ng/ml), forskolin (50 µM), LPS with forskolin, IBMX (500 µM), or Sp-8-BrcAMP (Sp-cAMP; 100 µM) for 6 h. Cell proliferation was measured on the basis of the incorporation of [3H]thymidine during the last 4 h of treatment (n = 6 within each experiment; values determined in triplicate). Data represent means ± SE. B: effect of NF-{kappa}B inhibitor on the LPS-induced decreased RAW264.7 cell proliferation. RAW264.7 cells were treated with LPS (100 ng/ml) for 6 h in the presence or absence of NF-{kappa}B inhibitor (20 µM SN50 added 1 h before 6-h LPS treatment). Cell proliferation was measured by the incorporation of [3H]thymidine during the last 4 h of treatment (n = 6 within each experiment; values determined in triplicate). Data represent means ± SE. ***P < 0.001 vs. control. ##P < 0.01 vs. LPS with forskolin.

 
We evaluated the effect of the NF-{kappa}B inhibitor SN50 on LPS-induced decreases on [3H]thymidine incorporation. Although SN50 completely blocked the LPS-induced increase in AC activity (see above), it had no effect on LPS-induced decreases of [3H]thymidine incorporation (Fig. 10B), suggesting that LPS-induced reduction in proliferation can occur independently of the effects on the cAMP cascade.

Cell viability. Six-hour pretreatment with LPS (100 ng/ml), forskolin (50 µM), IBMX (500 µM), or Sp-8-BrcAMP (100 µM) did not affect cell viabilities of murine macrophages detected using Trypan blue exclusion. Viability was 96.4 ± 0.5%, 96.7 ± 0.4%, 96.1 ± 0.3%, or 96.4 ± 0.3% compared with control, respectively (n = 3; P > 0.05). In contrast, longer treatment (24 h) with 100 ng/ml LPS reduced the viability of murine macrophages to 33.6 ± 2.7% of control (n = 4; P < 0.001).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary findings of the present study are that LPS induced sensitization of CaM-regulated AC isoforms and decreased proliferation in a murine macrophage cell line, RAW264.7. Sensitization of AC activity, but not decreased proliferation by LPS, was NF-{kappa}B dependent, despite the finding that elevation of cAMP itself decreased proliferation. These findings suggest that both cAMP-dependent (activated by forskolin, IBMX, or Sp-8-BrcAMP) and cAMP-independent pathways (activated by LPS) are capable of attenuating proliferation of macrophages.

We demonstrated both time- and concentration-dependent effects of LPS on subsequently measured forskolin-stimulated AC activity. This finding is consistent with the observation that intracellular cAMP levels were increased by LPS treatment in RAW264.7 cells (6). LPS previously was shown to stimulate prostaglandin release (20) and to inhibit phosphodiesterase activity (16), two mechanisms that could indirectly account for elevated intracellular cAMP levels after LPS treatment. These mechanisms were excluded in the present study by the demonstration that 1) pretreatment with indomethacin to block prostaglandin synthesis had no effect on LPS-induced sensitization of AC activity, 2) prostaglandin receptor antagonists selective for the EP2 and EP4 receptors (AH6809 and AH23848, respectively) failed to affect the LPS-induced sensitization of AC, and 3) pretreatment with the phosphodiesterase inhibitor IBMX did not change the magnitude of subsequently measured LPS-induced sensitization of AC activity.

Okonogi et al. (17) reported that LPS inhibited PGE2- or forskolin-stimulated cAMP accumulation by increasing phosphodiesterase activity but that membranes prepared from LPS-treated peritoneal macrophages exhibited similar PGE2-stimulated AC activity. However, they used thioglycolate broth-elicited peritoneal macrophages, and they investigated brief periods of PGE2- or forskolin-stimulated cAMP accumulation after 1 h of LPS treatment. Thus their study differed from the present study with regard to 1) the origin of the macrophages studied, 2) pretreatment with LPS for 1 vs. 4 h, and 3) the duration of effector stimulation (PGE2 or forskolin for 2 or 5 min, respectively, compared with 15-min stimulation). However, in our study, inhibition of phosphodiesterases did not alter the LPS-induced sensitization of AC. Thus the peritoneal macrophages studied by Okonogi et al. were phenotypically different from the murine macrophages used in the present study with regard to prostaglandin and phosphodiesterase signaling, which likely accounts for the differences found in AC sensitization.

Isoforms of AC are known to be differentially stimulated or inhibited by a diverse array of intracellular signals, including G protein {alpha}- or {beta}{gamma}-subunits, Ca2+ concentration, CaM, and multiple isoforms of protein kinases, including PKA, PKC, raf-1 kinase (9, 29), and tyrosine kinases (25). Therefore, we attempted to block the LPS-induced sensitization of AC by pretreating cells with selective inhibitors, including W-7 (CaM inhibitor), Rp-8-BrcAMP (PKA inhibitor), GF-109203X (PKC inhibitor), raf-1 kinase I inhibitor, genistein (tyrosine kinase inhibitor), and herbimycin A (tyrosine kinase and NF-{kappa}B inhibitor).

W-7 attenuated LPS-induced AC sensitization, which suggested that CaM-sensitive AC isoforms likely play an important role in sensitization induced by LPS. Three isoforms have been reported to be CaM sensitive, namely, AC I, AC III, and AC VIII (30). Among these isoforms, we found evidence for mRNA expression of AC III and AC VIII in RAW264.7 cells. In addition, there is evidence that CaM can indirectly inhibit AC III via activation and phosphorylation of CaMKII, which phosphorylates AC III, thereby inhibiting its activity (30). In the present study, LPS decreased the phosphorylation of CaMKII, the active form of this enzyme. Thus LPS reduction in CaMKII activity could remove a negative regulatory effect on a susceptible AC isoform, resulting in increased AC activity.

Herbimycin A, but not genistein, attenuated the LPS-induced sensitization of AC activity. To distinguish herbimycin A’s blockade of tyrosine kinases from its known inhibitory effects on NF-{kappa}B, separate experiments were performed with a specific inhibitor of NF-{kappa}B nuclear translocation, SN50. SN50, but not its inactive peptide control, was effective in attenuating LPS-induced AC activation. These data suggest that a transcriptional event mediated by NF-{kappa}B is required. This finding is not surprising, because a myriad of LPS-induced effects have been attributed to NF-{kappa}B-mediated events (18). To further confirm the requirement for new transcription and translation, we performed studies in which murine macrophages were pretreated with actinomycin D and cycloheximide, and both pretreatments blocked LPS-induced sensitization of AC.

Sensitization of AC has been described most widely after chronic stimulation of a Gi protein-coupled receptor. Classically, after chronic exposure to an agonist that acutely inhibits AC activity, subsequent stimulation of AC activity results in overshoot, or supersensitization, of AC activity. This Gi protein-required sensitization of AC is pertussis toxin sensitive. Therefore, in the present study, we questioned whether Gi proteins could be intermediates in the LPS-induced sensitization of AC, but pretreatment with pertussis toxin failed to reverse LPS’s effect. Therefore, the cellular mechanisms of AC sensitization by chronic Gi activation vs. LPS exposure are different. Furthermore, AC activity has been enhanced by prior exposure to TNF-{alpha} (19, 22) or to IL-1{beta} (3, 19). Similar to our presently reported findings in murine macrophages, IL-1{beta} induced sensitization of forskolin-stimulated cAMP accumulation, which was blocked by cycloheximide (3).

The requirement for new protein synthesis in the LPS-induced sensitization of AC led us to question whether LPS may directly increase AC expression. To determine whether the expression of AC was effected by LPS pretreatment, we quantified mRNA for AC isoforms III and VIII. These specific isoforms were chosen for evaluation because our functional data indicated a role for CaMK-regulated AC (30), of which AC isoforms III and VIII were found to be expressed using RT-PCR. We found no evidence for an increase in mRNA encoding AC isoforms III or VIII after LPS treatment, indicating that a change in AC expression is unlikely to account for the increased activity measured.

There is evidence that HSP90 plays an important role in LPS recognition. Byrd et al. (4) reported that HSP90 bound to LPS and mediated the activation of macrophages by LPS. Moreover, a signaling complex of receptors comprising HSP70, HSP90, chemokine receptor 4, and growth differentiation factor 5 has been reported to form after LPS stimulation and is thought to associate with TLR4 to mediate LPS signaling (27). In the present study, geldanamycin, a specific inhibitor of the HSP90 family, attenuated LPS-induced sensitization of AC. This result suggests that LPS’s effect on AC was mediated via specific activation of the LPS receptor complex.

Interestingly, even though blockade of NF-{kappa}B completely abrogated LPS-induced AC sensitization, it had no effect on LPS-induced decreased proliferation, despite the fact that direct stimulation of AC with forskolin, elevation of cAMP with IBMX, or stimulation of PKA with Sp-8-BrcAMP was each capable of inhibiting proliferation. Furthermore, combined application of LPS and forskolin caused the additive effect of inhibition of proliferation. Taken together, these results suggest that LPS’s effects on decreased proliferation can proceed through a cAMP-PKA-independent pathway in these murine macrophages.

Cyclic AMP has inhibitory effects on the macrophage immune response, including the production of inflammatory mediators such as TNF-{alpha} and interleukins (1, 10, 24). Thus modulation of cAMP levels appears to have an important role in the regulation of innate immune responses. Sensitization of CaM/CaMKII-regulated isoforms of AC could function in an intracellular negative feedback loop to attenuate the LPS-induced release of cytokines or phagocyte function, which in turn could limit the macrophage’s successful response to sepsis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by an intramural grant from the Department of Anesthesiology, Columbia University, New York, NY.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons, Columbia Univ., 630 W. 168th St., P&S Box 46, New York, NY 10032 (e-mail: cwe5{at}columbia.edu)

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


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