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Originally published online as doi:10.1189/jlb.0104005 on June 3, 2004

Published online before print June 3, 2004
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(Journal of Leukocyte Biology. 2004;76:641-647.)
© 2004 by Society for Leukocyte Biology

cAMP protects neutrophils against TNF-{alpha}-induced apoptosis by activation of cAMP-dependent protein kinase, independently of exchange protein directly activated by cAMP (Epac)

Camilla Krakstad, Anne Elisabeth Christensen and Stein Ove Døskeland1

Cell Biology Research Group, Department of Biomedicine, Section of Anatomy and Cell Biology, University of Bergen, Norway

1 Correspondence: University of Bergen, Department of Biomedicine, Section of Anatomy and Cell Biology, Jonas Lies vei 91, N-5009 Bergen, Norway. E-mail: stein.doskeland{at}iac.uib.no


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is unclear by which receptor cyclic adenosine monophosphate (cAMP) acts to promote neutrophil survival. We found that 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, a specific activator of the recently discovered cAMP receptor, cAMP-regulated guanosine 5'-triphosphate exchange protein directly activated by cAMP, failed to protect human neutrophils from cell death. In contrast, specific activators of cAMP-dependent protein kinase type I (cA-PKI) could protect against death receptor [tumor necrosis factor receptor 1 (TNFR-1), Fas]-mediated apoptosis as well as cycloheximide-accelerated "spontaneous" apoptosis. A novel "caged" cA-PK-activating analog, 8-bromo (8-Br)-acetoxymethyl-cAMP, was more than 20-fold more potent than 8-Br-cAMP to protect neutrophils challenged with TNF-{alpha} against apoptosis. This analog acted more rapidly than forskolin (which increases the endogenous cAMP production) and allowed us to demonstrate that cA-PK must be activated during the first 10 min after TNF-{alpha} challenge to protect against apoptosis. The protective effect was mediated solely through cA-PK activation, as it was abolished by the cA-PKI-directed inhibitor Rp-8-Br-cAMPS and the general cA-PK inhibitor H-89. Neutrophils not stimulated by cAMP-elevating agents showed increased apoptosis when exposed to the cA-PK inhibitors Rp-8-Br-cAMPS and H-89, suggesting that even moderate activation of cA-PK is sufficient to enhance neutrophil longevity and thereby contribute to neutrophil accumulation in chronic inflammation.

Key Words: apoptosis • inflammation • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are terminally differentiated polymorphonuclear leukocytes with a central role in the defense against bacterial and fungal infections (for recent reviews, see refs. [1 2 3 4 ]). About 1011 mature neutrophils are produced daily, implying that a similar number of neutrophils are eliminated per day [5 ]. The longevity of these cells in vivo is dependent on survival factors and death-inducing peptides. In vitro, and presumably in some in vivo situations, the rate of neutrophil apoptosis is accelerated by ligands that engage death receptors of the tumor necrosis factor receptor (TNFR)/nerve growth factor receptor superfamily, among which Fas [6 , 7 ], TNFR1, and TNFR2 [8 ] and the receptor for TNF-related apoptosis-inducing ligand [9 ] are expressed in neutrophils [2 ]. Circulating neutrophils have a short half-life (less then 20 h), which is extended at the inflamed site by prosurvival and proinflammatory cytokines [4 , 10 , 11 ]. One mechanism is through increased production of cyclic adenosine monophosphate (cAMP) through stimulation of adenylyl cyclase by, e.g., prostaglandins [6 , 12 13 14 ]. Other proposed mechanisms are through stimulation of the mitogen-activated protein kinase pathway or phosphoinositol-3-kinase/Akt pathway, both of which lead to activation of nuclear factor-{kappa}B, which is believed to act by enhancing the transcription of genes coding for survival proteins [15 16 17 ].

Enhanced apoptosis of granulocytes at inflamed sites promotes the resolution of chronic inflammation [1 , 18 ]. Elucidating the molecular mechanisms that regulate granulocyte apoptosis has therefore significant therapeutic implication. Particularly interesting drug targets are pathways such as the cAMP-signaling system, which serve to extend neutrophil life at the inflamed site. Knowledge of the mediator of cAMP action will not only help future studies aimed at pinpointing the details of the pathway downstream of the primary mediator but will also provide opportunity to target directly the cAMP mediator. Most effects of cAMP are mediated through activation of cAMP-dependent protein kinase (cA-PK) or the newly discovered cAMP-regulated guanosine 5'-triphosphate exchange protein directly activated by cAMP (Epac) family of exchange proteins [19 20 21 ]. The mediator of the antiapoptotic effect of cAMP is not known with certainty [6 , 22 ]; for a recent review, see ref. [3 ]. It has recently been proposed that cAMP protects against neutrophil apoptosis by a non-cA-PK mechanism [3 , 22 ].

We have developed novel cAMP analogs that selectively activate Epac without activating cA-PK in vitro and in vivo [23 , 24 ] and have shown that six modified cAMP analogs preferentially activate cA-PK [19 , 23 ]. These analogs, together with Rp-8-bromo (8-Br)-cAMPS, which we previously showed to be a preferential inhibitor of cA-PK type I (cA-PKI) isozyme [25 ], provide powerful tools to study the possible involvement of cA-PK or Epac in neutrophil apoptosis.

In the present study, we show that cA-PK-specific analogs can substitute for the second messenger cAMP to inhibit TNF-{alpha}- and anti-Fas (a-Fas)-induced, as well as "spontaneous" neutrophil apoptosis. The Epac-specific activator failed to protect against apoptosis. Additionally, we show that cA-PK must be active during early stages of the apoptotic process to prevent the cell from dying. Thus, the death-inhibitory effect of cAMP is mediated via the cA-PK signaling pathway through cA-PK activation in the preapoptotic stage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and cell preparation
Antihuman Fas immunoglobulin M antibody (Clone CH-11) was from Upstate Biotechnology (Waltham, MA). Hoechst 33342, forskolin, 3-isobutyl-1-methylxanthine (IBMX), cycloheximide (CHX), H-89, and human TNF-{alpha} were from Sigma Chemical Co. (St. Louis, MO). Rp-8-Br-cAMPS, 8-(4-chlorophenylthio; 8-pCPT)-2'-O-methyl (Me)-cAMP, N6-monobutyryl-cAMP (N6-MB-cAMP), 2-Cl-8-methylamino-cAMP (2-Cl-8-MA-cAMP), 8-Br-cAMP-acetoxymethyl (8-Br-cAMP-AM), and 8-pCPT-cAMP were from Biolog Life Science Institute (Bremen, Germany).

Determination of the binding affinity for sites A and B and the equilibrium-binding inhibition constant of analog relative to cAMP (K'i) values for 2-Cl-8-MA-cAMP for cA-PK regulatory subunits I and II as well as Epac were performed as described [23 ].

Human neutrophil granulocytes were obtained from peripheral blood of normal, nonsmoking donors. EDTA blood was diluted 1:1 in phosphate-buffered saline (PBS) containing serum albumin (5 mg/ml) and glucose (1 mg/ml), and the neutrophils isolated by sedimentation through a lymphoprep/polymorphprep (Nycomed Pharma, Norway) gradient. Erythrocytes were removed by hypotonic lysis in ammonium chloride (8 mg/ml), and the remaining cells (95% neutrophils) were washed in PBS.

Induction and evaluation of neutrophil apoptosis
Neutrophils were diluted in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS) and were seeded in 96-well culture plates at a concentration of 40,000 cells/well. For apoptosis induction, cells were incubated with TNF-{alpha} (25 ng/ml) in combination with the translation inhibitor CHX (1 µg/ml) at 37°C for up to 3 h. Alternatively, cells were treated with a-Fas receptor-activating antibody (500 ng/ml) for 3 h. In some cases, the cells were treated with CHX alone or without any apoptogens and left for up to 24 h to study CHX-induced and spontaneous apoptosis.

To study the modulatory effect of cAMP analogs on apoptosis, the cells were pretreated routinely with analogs for 30 min before the addition of apoptosis inducer. In one series of experiments, the analogs were added at various time-points before and after apoptogen addition to determine the critical time-point of action. The incubations were terminated by fixing the cells in 2% glutaraldehyde containing 1 µg/ml Hoechst 33342 (for visualization of chromatin). Apoptosis was determined by Hofmann differential interference contrast and fluorescence microscopy (Nikon Diaphot 300). The percentage of apoptotic cells was determined by counting at least 300 cells/well. Photomicrographs were taken with a digital (Sensys) camera fitted with IPLab Spectrum 3.1a software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha}- and a-Fas-induced neutrophil apoptosis is counteracted by cyclic nucleotides selectively activating cA-PK but not by Epac-specific cAMP analogs
a-Fas and TNF-{alpha}/CHX induced neutrophil death rapidly (within 2 h) to at least 90% (Fig. 1 ). The commonly used cAMP analog 8-pCPT-cAMP delayed TNF-{alpha}/CHX-induced and a-Fas-induced apoptosis. It was more efficient against apoptosis induced by TNF-{alpha}/CHX than against a-Fas (Fig. 1) .



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  		Figure 1. Death receptor-mediated neutrophil apoptosis is inhibited by the nondiscriminating cAMP analog 8-pCPT-cAMP. Freshly isolated human neutrophils cultured in DMEM with 10% FCS were incubated with TNF-{alpha} (25 ng/ml)/CHX (1 µg/ml) or a-Fas (500 ng/ml) in the absence and presence of the nondiscriminative cAMP agonist 8-pCPT-cAMP (0.7 mM). Apoptosis was determined by fluorescence microscopy. Further details are given in Materials and Methods. The data are given as mean values ± SEM of at least three separate experiments.

 
The mediator(s) of the antiapoptotic effect of the second messenger cAMP has not been unequivocally identified [6 , 22 ]. The cAMP analog 8-pCPT-cAMP, used in the experiments shown in Figure 1 , is not able to discriminately activate cA-PK or Epac. This cAMP analog is commonly used as an activator of cA-PK but has a very high affinity for Epac (Table 1 ) and is a potent Epac activator [23 ].


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  		Table 1. Analog Affinity (Rel. to cAMP) for Epac-1 and cA-PKI

 
We have recently found that the cAMP analog N6-MB-cAMP is a preferential activator of cA-PK and that 8-pCPT-2'-O-Me-cAMP is a specific activator of Epac [23 , 24 ] (Table 1) . These analogs were therefore used to find whether cA-PK or Epac mediated the protection against apoptosis. It appeared that N6-MB-cAMP gave strong protection against TNF-{alpha}-induced apoptosis, whereas 8-pCPT-2'-O-Me-cAMP was inefficient (Fig. 2A and 2B ). This suggested strongly that cA-PK rather than Epac was the mediator of the cAMP effect. To further substantiate this conclusion, we tested whether Rp-8-Br-cAMPS and H-89 could counteract the effect of N6-MB-cAMP. Rp-8-Br-cAMPS is a preferential inhibitor of cA-PK isozyme I [25 ], which is the dominating isoenzyme in human neutrophils [6 ]. H-89 acts as a competitive inhibitor of adenosine 5'-triphosphate at the active site of the cA-PK [27 ]. H-89 (Fig. 2C) and Rp-8-Br-cAMPS (Fig. 2A and 2B) abolished the protective effect of N6-MB-cAMP, indicating that N6-MB-cAMP acted via cA-PK, most probably cA-PKI.



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  		Figure 2. The effect of cAMP on TNF-{alpha}-induced neutrophil apoptosis is mediated through cAMP-kinase rather than Epac. To identify the cAMP pathway responsible for inhibition of human neutrophil apoptosis, selected cAMP analogs were added 30 min prior to TNF-{alpha} (25 ng/ml)/CHX (1 µg/ml) or CHX alone (control). Cells were fixed and stained with fluorescent DNA-binding dye (Hoechst 33342) 2 h thereafter and scored for apoptosis. (A) The left panels show that the apoptotic morphology (note particularly the hypercondensed chromatin) induced by TNF-{alpha} was counteracted by the cAMP-kinase activator N6-MB-cAMP (1 mM) but not by the specific Epac activator 8-pCPT-2-O'-Me-cAMP (1 mM). The right panels show that the cAMP-kinase antagonist Rp-8-Br-cAMPS (0.7 mM) could abolish the protective effect of N6-MB-cAMP. The apoptotic score, based on microscopy of cells as shown in A, is given in B. The kinase inhibitor H-89 prevents TNF-{alpha}-induced neutrophil apoptosis in a dose-dependent manner (C). For full inhibition of TNF-{alpha}-induced apoptosis, a high and cA-PK-specific concentration (60 µM) is required. The data shown represent the mean of two to four separate experiments. SEM values are given when more than three experiments were conducted.

 
It is interesting that Rp-8-Br-cAMPS and H-89 enhanced the TNF-{alpha}-induced death (Fig. 2A 2B 2C ; data not shown), suggesting that even basal cA-PK activity could provide some protection against cell death.

We noted that the ability of H-89 to abolish the effect of N6-MB-cAMP decreased rapidly upon dilution, 60–80 µM being required for full antagonism (Fig. 2C ; data not shown).

Activation of the cA-PKI is responsible for the inhibitory effect of cAMP
To activate preferentially cA-PKI, we exploited the fact that the two cAMP-binding sites of cA-PK (A, B) act synergistically to activate the kinase [28 29 30 ]. Among available cAMP analogs, N6-MB-cAMP has the highest relative site AI specificity and 2-Cl-8-MA-cAMP, the highest site BI specificity. The combination of N6-MB-cAMP and 2-Cl-8-MA-cAMP therefore gives a strong synergy for cA-PKI activation. For comparison, the analog pair 8-pCPT-cAMP and 8-AHA-cAMP [6 ] has less predicted synergy (Table 2 ). Furthermore, N6-MB-cAMP is, unlike 8-pCPT-cAMP, a poor Epac activator (Table 1) . This decreases the possibility that N6-MB-cAMP enhances the action of 2-Cl-8-MA-cAMP through Epac activation. When 2-Cl-8-MA-cAMP was combined with a low concentration (0.05 or 0.10 mM) of N6-MB-cAMP, a marked synergism was noted for protection against TNF-{alpha} (Fig. 3 ). This lent further support to the contention that cA-PKI was responsible for the protective effect of cAMP against apoptosis.


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  		Table 2. Predicted cA-PKI Synergy between Pairs of cAMP Analogs

 


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  		Figure 3. The site-specific analogs N6-MB-cAMP and 2Cl-8-MA-cAMP act synergistically to activate type I cAMP-kinase. Low concentrations of N6-MB-cAMP strongly enhanced the ability of the complementary cA-PKI activatory analog [28 ] 2-Cl-8-MA-cAMP to protect against TNF-{alpha}/CHX-induced apoptosis. The data shown represent the mean of two to four separate experiments. SEM values are given when more than three experiments were conducted.

 
The cA-PK target responsible for inhibition of TNF-{alpha}/CHX-induced apoptosis is phosphorylated at an early, preapoptotic time-point
We wanted to know when, in relation to the addition of TNF-{alpha}, the cA-PK had to be activated. We decided first to find a highly potent cA-PK activator, which could be used subsequently to activate rapidly the neutrophil cA-PK. We found that 8-Br-cAMP-AM, which is a membrane-permeable precursor of 8-Br-cAMP [31 ], was 25-fold more potent than its parent compound 8-Br-cAMP and also more potent than 8-pCPT-cAMP, N6-MB-cAMP, and dibutyryladenosine (DB)-cAMP (Fig. 4 ).



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  		Figure 4. Comparison of cAMP analogs as antagonists of TNF-{alpha}-induced neutrophil apoptosis. Neutrophils, preincubated with various concentrations of cAMP analogs for 30 min, were treated (2 h) with TNF-{alpha}. The cells were fixed and scored for apoptosis as described in Materials and Methods. The broadly acting analogs 8-Br-cAMP [inhibitory concentration 50% (IC50)=0.84 mM)] and 8-CPT-cAMP (IC50=0.32 mM) were found to be less potent inhibitors than N6-MB-cAMP (IC50=0.146 mM) and DB-cAMP (IC50=0.096 mM), whereas the caged cAMP agonist 8-Br-cAMP-AM was the most potent inhibitor (IC50=0.034 mM). The data shown represent the mean of two to four separate experiments. SEM values are given when more than three experiments were conducted.

 
To determine at what time cAMP was required to prevent cell death, cAMP-elevating agents and/or cAMP analogs were added prior to, at the same time as, or after the addition of TNF-{alpha} (Fig. 5 ). To elevate the endogenous cAMP level, the neutrophils were incubated with the adenylate cyclase stimulator forskolin (50 µM) and the phosphodiesterase inhibitor IBMX (300 µM). Cells pretreated with forskolin and IBMX 15 min before the addition of TNF-{alpha}/CHX were still completely protected against death 2 h later. When forskolin/IBMX was added 10 min after TNF-{alpha}/CHX, only a marginal protection was achieved (Fig. 5A) . This shows that stimulants of endogenous cAMP accumulation must be present prior to or close to the point of death receptor activation to delay death. As the stimulation of cAMP accumulation inevitably takes time, we used the extremely cell-permeable AM-ester analog of 8-Br-cAMP to further pinpoint the time when cAMP was required to protect against death. Using this analog, the cells could be nearly completely protected even 3–6 min after the addition of death stimulator (Fig. 5B) . This suggests that cAMP does not have to be present before the death stimulus but rather that it acts at a step occurring a few minutes downstream of the initial death receptor stimulation. 8-Br-cAMP-AM appeared to give a maximal cAMP effect, as no further protection was observed when it was combined with forskolin, IBMX, and a cocktail of other cAMP analogs (N6-MB-cAMP, 8-pCPT-cAMP, 2-Cl-8-MA-cAMP, and 8-pCPT-2'-O-Me-cAMP), as shown in Figure 5C . We conclude that cAMP must be present at the latest a few minutes after death receptor stimulation to optimally delay apoptosis.



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  		Figure 5. TNF-{alpha}-induced neutrophil apoptosis is counteracted at an early, preapoptotic stage by the caged cAMP agonist 8-Br-cAMP-MA. cAMP-elevating agents or cAMP analogs were added at different time-points relative to the addition of TNF-{alpha} (25 ng/ml)/CHX (1 µg/ml), and apoptosis was determined 2 h thereafter. Solid bars represent apoptosis induced by TNF-{alpha} alone. (A) Forskolin (50 µM) and IBMX (300 µM) were added to increase the endogenous cAMP level in the neutrophils. (B) The caged and highly lipophilic AM-ester of 8-Br-cAMP (0.3 mM) was added. (C) To ensure maximal stimulation of potential cAMP effectors, the concentration of 8-Br-cAMP-AM was increased to 0.5 mM and added together with forskolin, IBMX, 8-pCPT-cAMP (0.7 mM), the cA-PKI activators N6-MB-cAMP (1 mM) and 2-Cl-8-MA-cAMP (0.7 mM), and the potent and specific Epac activator 8-pCPT-2'-O-Me-cAMP (1 mM). The data shown represent the mean of two to four separate experiments. SEM values are given when more than three experiments were conducted. Note that 8-Br-cAMP-AM (B) prevented apoptosis at a later time-point than the stimulators of endogenous cAMP accumulation (A) and that additional cAMP agonists failed to further enhance the action of 8-Br-cAMP-AM (C).

 
The ability of cA-PK activation to protect against a-Fas and protracted challenge with TNF-{alpha} and CHX
To know if cA-PK activation could also protect against protracted challenge with apoptogens, we incubated human neutrophils with various concentrations of TNF-{alpha} in the presence of the cA-PK activator N6-MB-cAMP (and CHX) for 2 and 6 h (Fig. 6 ).



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  		Figure 6. The cA-PKI-specific activator N6-MB-cAMP prevents neutrophil apoptosis induced by physiological concentrations of TNF-{alpha}. Human neutrophils were preincubated (0.5 h) with 0.7 mM N6-MB-cAMP before incubation with various concentrations of TNF-{alpha} for 2 (A) or 6 h (B). Experimental details are as explained in the legend to Figure 1 . The data shown represent the mean of two to four separate experiments. SEM values are given when more than three experiments were conducted.

 
At 2 h, N6-MB-cAMP inhibited fully even the highest concentration (25 ng/ml) of TNF-{alpha} tested (Fig. 6A) . After 6 h of incubation, N6-MB-cAMP was still able to partially protect against moderate concentrations of TNF-{alpha}, 50% apoptosis being observed with 0.8 ng/ml in the absence of N6-MB-cAMP and 2 ng/ml in its presence (Fig. 6B) .

To know whether cA-PK activation could also protect against neutrophil apoptosis induced by other agents than TNF-{alpha}, neutrophils were induced to undergo apoptosis by incubation with a-Fas (2 h) or CHX (20 h). Cells treated with a-Fas (Fig. 7 , left panels) or CHX (Fig. 7 , right panels) in the presence of the cA-PK activator N6-MB-cAMP had normal morphology. The cA-PKI inhibitor Rp-8-Br-cAMPS abolished the protection by N6-MB-cAMP, suggesting that cA-PKI was able to also protect against Fas-mediated and CHX-induced cell death.



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  		Figure 7. cA-PK is responsible for the protection by cAMP against apoptosis induced by a-Fas or CHX. Neutrophils were induced to undergo apoptosis by exposure to 500 ng/ml a-Fas for 2 h or 1 µg/ml CHX for 20 h. The cA-PK-activating analog N6-MB-cAMP (1 mM) counteracted the apoptogenic effect of a-Fas or CHX. The protective effect of N6-MB-cAMP could be abolished when coincubating with Rp-8-Br-cAMPS (0.7 mM). The Epac-specific analog 8-pCPT-2-O'-Me-cAMP (1 mM) failed to prevent apoptosis. Values given represent percentage of apoptotic cells. Data shown represent a typical experiment. Values obtained from two other experiments differ from the presented results by less than 5%. For further experimental details, see Materials and Methods and the legend to Figure 2 .

 
The Epac-activating analog 8-pCPT-2'-O-Me-cAMP failed to protect against a-Fas- or CHX-induced apoptosis (Fig. 7) , further supporting the above conclusion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The pivotal role of the cA-PK as mediator of all cAMP actions in mammalian cells has recently been challenged by the discovery of cyclic nucleotide-gated ion channels and more recently, of a novel Epac family of guanine nucleotide exchange factors regulated directly by cAMP (for a recent review, see ref. [19 ]). There are cases where cAMP effects cannot be ascribed to active cA-PK alone, such as the stimulation of DNA replication in the thyroid [32 ], stimulation of exocytosis [33 ], integrin-mediated cell adhesion [34 ], and neuritogenesis [23 ]. In the latter three cases, the effect of cAMP is partly or completely mediated via Epac, as shown by the use of cAMP analogs able to distinguish between cA-PK and Epac [19 , 23 , 24 ].

Recently, cAMP was reported to protect against neutrophil apoptosis through an uncharacterized cA-PK-independent pathway [3 , 22 ]. To know if this pathway could involve Epac activation, we applied novel cAMP analogs, able to discriminate between cA-PK and Epac. To our surprise, we found that cAMP protected neutrophils against apoptosis through activation of cA-PK alone and not Epac or Epac-like receptors. The cA-PK-preferring activator N6-MB-cAMP [23 , 26 ] protected neutrophils equally well as increased endogenous cAMP, and the Epac-specific activator 8-pCPT-2'-Me-cAMP failed to affect apoptosis. Furthermore, the protection by cAMP or cAMP analogs could be counteracted by the preferential cA-PKI inhibitor Rp-8-Br-cAMPS, as well as by the active, site-directed cA-PK inhibitor H-89. The conclusion that cA-PK mediated the antiapoptotic effect of cAMP was further supported by the synergistic protection obtained by combined treatment with the cAMP analogs 2-Cl-8-MA-cAMP and N6-MB-cAMP, which bind preferentially to site B and site A, respectively, of cA-PKI. The exclusion of cA-PK as protector against neutrophil death by Martin et al. [22 ] was based on the inability of 0.1 mM Rp-8-Br-cAMPS or 10 µM H-89 to reverse the effect of cAMP on neutrophil apoptosis. In the original publication describing Rp-8-Br-cAMPS, we found that it was efficient in concentrations from 0.05 to 0.8 mM, depending on the cell type studied [25 ]. As the mother compound 8-Br-cAMP was unusually weak as a cA-PK agonist in the neutrophils (Fig. 4) , it is not surprising if high concentrations are also required of Rp-8-Br-cAMPS. We found that 0.7 mM Rp-8-Br-cAMPS completely reversed the effect of N6-MB-cAMP. The inhibitor H-89 was, in our hands, inefficient at 10 µM but quite efficient at 30 µM and highly efficient at 60 µM (Fig. 2C) , in line with the results reported in the original publication describing this compound [27 ].

Stimulators of endogenous cAMP or conventional cAMP analogs acted too slowly to pinpoint the critical time when cAMP no longer could protect the cells. The highly cell-permeable "caged" analog 8-Br-cAMP-AM protected most of the cells when added 6–10 min after TNF-{alpha}, i.e., ~50 min before the onset of cell death. TNF-{alpha} is known to induce an oxidative burst in neutrophils with a latency of ~15 min, which is prevented by cAMP-elevating agents given early relative to TNF-{alpha} [14 ]. Similarly, apoptosis induced by the reactive oxygen species (ROS) generator NaN3 is only inhibited by cA-PK activators [19 ], when they are given early (C. Krakstad and S. O. Døskeland, unpublished data). The time-frame is therefore similar for cAMP protection against apoptosis and against ROS formation. One possible cA-PK target is cytochrome c oxidase. When this enzyme is phosphorylated by cA-PK, it becomes more resistant to stress-induced uncoupling of mitochondrial respiration and the ensuing ROS formation [35 ]. Another plausible target is Bad [36 , 37 ]. When phosphorylated by cA-PK, the p-Bad will release antiapoptotic Bcl-2 family members, which may prevent generation of ROS [38 ].

The main importance of the present study is to identify the cA-PK as the only mediator of the action of cAMP to protect neutrophils against TNF-{alpha}- and a-Fas-induced as well as spontaneous apoptosis. Mechanistically, it means that emphasis must be on identifying substrates of cA-PK rather than downstream targets of other cAMP receptors, such as the Epac family members, ion-channels directly activated by cAMP, or yet other putative cAMP mediators [39 ]. From a therapeutic standpoint, it means that agents directed at counteracting the protection by cAMP in chronic inflammation can be directed at cA-PK. We noted that the cA-PKI-directed inhibitor Rp-8-Br-cAMP blocked the protective effect of elevated cAMP. We noted also that it enhanced slightly the death of isolated neutrophils. Inhibitors of the antiapoptotic pathways in granulocytes are drug candidates to combat chronic inflammation [2 , 40 ]. In inflammation, the neutrophil is exposed to several cAMP-elevating agents such as prostaglandin and will therefore have an elevated level of cAMP. Inhibition of cA-PK will therefore be expected to help resolve chronic inflammation.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr. Alfred Halstensen and Steinar Sørnes at Haukeland Hospital, Bergen, Norway, for assistance with the preparation of human neutrophils. The excellent technical assistance of Nina Lied Larsen and Erna Finsås is highly appreciated. This work was supported by The Novo Nordic Foundation, The Norwegian Research Council, and The Norwegian Cancer Society.

Received January 6, 2004; revised April 6, 2004; accepted May 4, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ward, I., Dransfield, I., Chilvers, E. R., Haslett, I., Rossi, A. G. (1999) Pharmacological manipulation of granulocyte apoptosis: potential therapeutic targets Trends Pharmacol. Sci. 20,503-509[CrossRef][Medline]
  2. Simon, H. U. (2003) Neutrophil apoptosis pathways and their modifications in inflammation Immunol. Rev. 193,101-110[CrossRef][Medline]
  3. Heasman, S. J., Giles, K. M., Ward, C., Rossi, A. G., Haslett, C., Dransfield, I. (2003) Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of inflammation J. Endocrinol. 178,29-36[Abstract]
  4. Akgul, C., Moulding, D. A., Edwards, S. W. (2001) Molecular control of neutrophil apoptosis FEBS Lett. 487,318-322[CrossRef][Medline]
  5. Walker, R. I., Willemze, R. (1980) Neutrophil kinetics and the regulation of granulopoiesis Rev. Infect. Dis. 2,282-292[Medline]
  6. Parvathenani, L. K., Buescher, E. S., Chacon-Cruz, E., Beebe, S. J. (1998) Type I cAMP-dependent protein kinase delays apoptosis in human neutrophils at a site upstream of caspase-3 J. Biol. Chem. 273,6736-6743[Abstract/Free Full Text]
  7. Kasahara, Y., Iwai, K., Yachie, A., Ohta, K., Konno, A., Seki, H., Miyawaki, T., Taniguchi, N. (1997) Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils Blood 89,1748-1753[Abstract/Free Full Text]
  8. Brockhaus, M., Schoenfeld, H. J., Schlaeger, E. J., Hunziker, W., Lesslauer, W., Loetscher, H. (1990) Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies Proc. Natl. Acad. Sci. USA 87,3127-3131[Abstract/Free Full Text]
  9. Renshaw, S. A., Parmar, J. S., Singleton, V., Rowe, S. J., Dockrell, D. H., Dower, S. K., Bingle, C. D., Chilvers, E. R., Whyte, M. K. (2003) Acceleration of human neutrophil apoptosis by TRAIL J. Immunol. 170,1027-1033[Abstract/Free Full Text]
  10. Colotta, F., Re, F., Polentarutti, N., Sozzani, S., Mantovani, A. (1992) Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products Blood 80,2012-2020[Abstract/Free Full Text]
  11. Lee, A., Whyte, M. K., Haslett, C. (1993) Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators J. Leukoc. Biol. 54,283-288[Abstract]
  12. Rossi, A. G., Cousin, J. M., Dransfield, I., Lawson, M. F., Chilvers, E. R., Haslett, C. (1995) Agents that elevate cAMP inhibit human neutrophil apoptosis Biochem. Biophys. Res. Commun. 217,892-899[CrossRef][Medline]
  13. Niwa, M., Hara, A., Kanamori, Y., Matsuno, H., Kozawa, O., Yoshimi, N., Mori, H., Uematsu, T. (1999) Inhibition of tumor necrosis factor-{alpha} induced neutrophil apoptosis by cyclic AMP: involvement of caspase cascade Eur. J. Pharmacol. 371,59-67[CrossRef][Medline]
  14. Ottonello, L., Morone, M. P., Dapino, P., Dallegri, F. (1995) Tumour necrosis factor {alpha}-induced oxidative burst in neutrophils adherent to fibronectin: effects of cyclic AMP-elevating agents Br. J. Haematol. 91,566-570[Medline]
  15. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., Greenberg, M. E. (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis Science 270,1326-1331[Abstract/Free Full Text]
  16. Gardner, A. M., Johnson, G. L. (1996) Fibroblast growth factor-2 suppression of tumor necrosis factor {alpha}-mediated apoptosis requires Ras and the activation of mitogen-activated protein kinase J. Biol. Chem. 271,14560-14566[Abstract/Free Full Text]
  17. Yang, K. Y., Arcaroli, J., Kupfner, J., Pitts, T. M., Park, J. S., Strasshiem, D., Perng, R. P., Abraham, E. (2003) Involvement of phosphatidylinositol 3-kinase {gamma} in neutrophil apoptosis Cell. Signal. 15,225-233[CrossRef][Medline]
  18. Savill, J., Fadok, V. (2000) Corpse clearance defines the meaning of cell death Nature 407,784-788[CrossRef][Medline]
  19. Kopperud, R., Krakstad, C., Selheim, F., Doskeland, S. O. (2003) cAMP effector mechanisms. Novel twists for an ‘old’ signaling system FEBS Lett. 546,121-126[CrossRef][Medline]
  20. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., Bos, J. L. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP Nature 396,474-477[CrossRef][Medline]
  21. Francis, S. H., Corbin, J. D. (1999) Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action Crit. Rev. Clin. Lab. Sci. 36,275-328[CrossRef][Medline]
  22. Martin, M. C., Dransfield, I., Haslett, C., Rossi, A. G. (2001) Cyclic AMP regulation of neutrophil apoptosis occurs via a novel protein kinase A-independent signaling pathway J. Biol. Chem. 276,45041-45050[Abstract/Free Full Text]
  23. Christensen, A. E., Selheim, F., De Rooij, J., Dremier, S., Schwede, F., Dao, K. K., Martinez, A., Maenhaut, C., Bos, J. L., Genieser, H. G., Doskeland, S. O. (2003) cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension J. Biol. Chem. 278,35394-35402[Abstract/Free Full Text]
  24. Enserink, J. M., Christensen, A. E., De Rooij, J., Van Triest, M., Schwede, F., Genieser, H. G., Doskeland, S. O., Blank, J. L., Bos, J. L. (2002) A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK Nat. Cell Biol. 4,901-906[CrossRef][Medline]
  25. Gjertsen, B. T., Mellgren, G., Otten, A., Maronde, E., Genieser, H. G., Jastorff, B., Vintermyr, O. K., McKnight, G. S., Doskeland, S. O. (1995) Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 ß action J. Biol. Chem. 270,20599-20607[Abstract/Free Full Text]
  26. Rehmann, H., Schwede, F., Doskeland, S. O., Wittinghofer, A., Bos, J. L. (2003) Ligand-mediated activation of the cAMP-responsive guanine nucleotide exchange factor Epac J. Biol. Chem. 278,38548-38556[Abstract/Free Full Text]
  27. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., Hidaka, H. (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells J. Biol. Chem. 265,5267-5272[Abstract/Free Full Text]
  28. Christensen, A. E., Doskeland, S. O. (2003) Cyclic nucleotide analogs Handbook of Cell Signaling ,549-554 Academic/Elsevier Science San Diego, CA. Chapter 212
  29. Doskeland, S. O., Maronde, E., Gjertsen, B. T. (1993) The genetic subtypes of cAMP-dependent protein kinase–functionally different or redundant? Biochim. Biophys. Acta 1178,249-258[Medline]
  30. Francis, S. H., Corbin, J. D. (1994) Structure and function of cyclic nucleotide-dependent protein kinases Annu. Rev. Physiol. 56,237-272[CrossRef][Medline]
  31. Kruppa, J., Keely, S., Schwede, F., Schultz, C., Barrett, K., Jastorff, B. (1997) Bioactivatable derivatives of 8-substituted cAMP-analogues Bioorg. Med. Chem. Lett. 7,945-948
  32. Dremier, S., Pohl, V., Poteet-Smith, C., Roger, P. P., Corbin, J., Doskeland, S. O., Dumont, J. E., Maenhaut, C. (1997) Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells Mol. Cell. Biol. 17,6717-6726[Abstract]
  33. Kang, G., Joseph, J. W., Chepurny, O. G., Monaco, M., Wheeler, M. B., Bos, J. L., Schwede, F., Genieser, H. G., Holz, G. G. (2003) Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic ß-cells J. Biol. Chem. 278,8279-8285[Abstract/Free Full Text]
  34. Rangarajan, S., Enserink, J. M., Kuiperij, H. B., de Rooij, J., Price, L. S., Schwede, F., Bos, J. L. (2003) Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the ß 2-adrenergic receptor J. Cell Biol. 160,487-493[Abstract/Free Full Text]
  35. Lee, I., Bender, E., Kadenbach, B. (2002) Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase Mol. Cell. Biochem. 234-235,63-70[CrossRef][Medline]
  36. Yusta, B., Estall, J., Drucker, D. J. (2002) Glucagon-like peptide-2 receptor activation engages bad and glycogen synthase kinase-3 in a protein kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase J. Biol. Chem. 277,24896-24906[Abstract/Free Full Text]
  37. Harada, H., Becknell, B., Wilm, M., Mann, M., Huang, L. J., Taylor, S. S., Scott, J. D., Korsmeyer, S. J. (1999) Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A Mol. Cell 3,413-422[CrossRef][Medline]
  38. Gottlieb, E., Vander Heiden, M. G., Thompson, C. B. (2000) Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor {alpha}-induced apoptosis Mol. Cell. Biol. 20,5680-5689[Abstract/Free Full Text]
  39. Dremier, S., Kopperud, R., Doskeland, S. O., Dumont, J. E., Maenhaut, C. (2003) Search for new cyclic AMP-binding proteins FEBS Lett. 546,103-107[CrossRef][Medline]
  40. Pongracz, J., Webb, P., Wang, K., Deacon, E., Lunn, O. J., Lord, J. M. (1999) Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-{delta} J. Biol. Chem. 274,37329-37334[Abstract/Free Full Text]



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