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The Journal of Immunology, 2005, 174: 8090-8096.
Copyright © 2005 by The American Association of Immunologists

Apoptotic Pathways Are Inhibited by Leptin Receptor Activation in Neutrophils1

Andreina Bruno, Sébastien Conus, Inès Schmid and Hans-Uwe Simon2

Department of Pharmacology, University of Bern, Bern, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leptin regulates food intake as well as metabolic, endocrine, and immune functions. It exerts proliferative and antiapoptotic activities in a variety of cell types, including T cells. Leptin also stimulates macrophages and neutrophils, and its production is increased during inflammation. In this study, we demonstrate that human neutrophils express leptin surface receptors under in vitro and in vivo conditions, and that leptin delays apoptosis of mature neutrophils in vitro. The antiapoptotic effects of leptin were concentration dependent and blocked by an anti-leptin receptor mAb. The efficacy of leptin to block neutrophil apoptosis was similar to G-CSF. Using pharmacological inhibitors, we obtained evidence that leptin initiates a signaling cascade involving PI3K- and MAPK-dependent pathways in neutrophils. Moreover, leptin delayed the cleavage of Bid and Bax, the mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase, as well as the activation of both caspase-8 and caspase-3 in these cells. Taken together, leptin is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutrophils are important effector cells of the immune system that represent the body’s first line of defense against invading microorganisms. Delayed neutrophil apoptosis has been associated with several infectious diseases (1) and often associated with overexpression of neutrophil survival factors such as G-CSF, GM-CSF (2), and macrophage migration inhibitory factor (3). These survival cytokines have been described to block the mitochondria-dependent death pathway in neutrophils (3, 4). Accordingly, the expression of members of the Bcl-2 family appears to be regulated by antiapoptotic cytokines in these cells (5).

Leptin is a pleiotropic cytokine involved in different biological systems. It shares structural similarities with some cytokines, including IL-6, IL-11, IL-12, IL-15, as well as with G-CSF, oncostatin M, ciliary neurotrophic factor, and leukemia-inhibitory factor (6). Leptin binds to short and long forms of leptin receptors, which are generated by differential splicing (7). The leptin receptors are expressed in multiple cells and tissues, including kidney, lung, adrenal gland, hemopoietic precursor cells, and bone marrow, as well as in neutrophils, monocytes, and T cells (8). The signal transduction pathways regulated by leptin are diverse and include those characteristic for both cytokine and growth factor receptor signaling (9).

Leptin plays a key role in the regulation of body weight, but also exerts other biological functions, which modulate hemopoiesis, angiogenesis, and immune responses (8). Interestingly, leptin production is increased in patients suffering from sepsis and other inflammatory diseases (10, 11). Moreover, leptin mediates both proliferative and antiapoptotic activities in a variety of cell types, including T cells (12) and monocytes (13). In this study, we demonstrate that leptin delays neutrophil apoptosis in vitro. Analyzing the intracellular events in these cells revealed that leptin activates both PI3K and MAPK signaling pathways, resulting in the inhibition of the mitochondrial death pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Recombinant human leptin, G-CSF, and goat polyclonal anti-human Bid Ab were purchased from R&D Systems (distributed by Bühlmann). The anti-human Fas (CH11) and anti-human caspase-8 mAbs were from Lab-Force and Cell Signaling Technology (Bioconcept), respectively. Anti-GAPDH mAb was from Chemicon International. Rabbit polyclonal anti-human Bax and anti-human caspase-3 Abs, as well as anti-cytochrome c and anti-human CD15 mAbs were from BD Biosciences. The anti-Cox1 mAb and polyclonal anti-second mitochondria-derived activator of caspase (anti-Smac)3 Ab were purchased from Molecular Probes (distributed by Invitrogen Life Technologies) and Stratagene Europe, respectively. The goat polyclonal anti-leptin receptor (sc-1834) was from Santa Cruz Biotechnology (Lab-Force). Mouse and rabbit HRP-conjugated secondary Abs were obtained from Amersham Biosciences. The pharmacological inhibitors SB 203580 (inhibitor of p38 kinase) and LY 294002 (inhibitor of PI3K) as well as polymyxin B sulfate were from Calbiochem. All other reagents were, unless stated otherwise, from Sigma-Aldrich.

Cells

Mature peripheral blood neutrophils were purified from healthy normal individuals by Ficoll-Hypaque centrifugation, as previously described (14, 15). The resulting cell populations contained <5% contaminating cells. Cell purity was assessed by staining with Diff-Quik (Medion) and light microscopy analysis. Written informed consent was obtained from all patients and control individuals, and the study was approved by the ethics committee of the Canton Bern.

Cell cultures

Human mature neutrophils were cultured at 1 x 106 per ml in complete culture medium (RPMI 1640 containing 10% FCS) in the presence and absence of rG-CSF (25 ng/ml), anti-Fas mAb (1 µg/ml), and leptin (0.0001–5 µM, usually 0.5 µM) for the indicated times. In some experiments, we added 15 µg/ml polymyxin B. Leptin-blocking experiments were performed with anti-human leptin receptor mAb (80–320 µM). SB 203580 and LY 294002 (both at 25 and 50 µM) were added 30 min before adding leptin and left in the culture.

Determination of cell death and apoptosis

Neutrophil death was assessed by uptake of 1 µM ethidium bromide and flow cytometric analysis (FACSCalibur) (16, 17, 18). To determine whether cell death was apoptosis, DNA fragmentation and redistribution of phosphatidylserine (PS) were measured (16, 17, 18).

Enzymatic caspase-3 assay

Caspase-3-like activity was measured using a commercial kit (QuantiZyme caspase-3 cellular activity kit; BIOMOL), according to the manufacturer’s instructions and as previously described (17, 18).

Immunoblotting

Neutrophils (1 x 106/ml) were cultured for 6 and 9 h, respectively, washed with cold PBS supplemented with a protease inhibitor mixture (Sigma-Aldrich), and lysed by using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM EGTA, and 0.25% sodium deoxycholate supplemented with a protease inhibitor mixture) with frequent vortexing on ice for 15 min. Equal amounts of cell lysates were mixed with running buffer, boiled, and subjected to gel electrophoresis on 12% NuPage-Gels (NOVEX, distributed by Invitrogen Life Technologies). Separated proteins were electrotransferred onto polyvinylidene difluoride membranes (Immobilion-P; Millipore). The filters were incubated with blocking solution (TBS/0.1% Tween 20, 5% nonfat dry milk) at room temperature for 1 h. The primary Abs anti-Bid (1/1000), anti-Bax (1/1000), anti-caspase-3 (1/1000), and anti-caspase-8 (1/1000), respectively, were incubated in blocking solution overnight at 4°C. For loading controls, stripped filters were incubated with anti-GAPDH mAb (1/3000). Filters were washed in TBS/0.1% Tween 20 for 30 min and incubated with the appropriate HRP-conjugated secondary Ab (1/3000) at room temperature for 1 h. After another washing step, filters were developed by an ECL technique (ECL kit; Amersham), according to the manufacturer’s instructions.

RT-PCR

Total RNA was isolated from neutrophils using TRIzol solution (Invitrogen Life Technologies), according to the manufacturer’s instructions. First-strand synthesis was performed using total RNA, oligo(dT) 15 primer (Promega, distributed by Catalys), and Superscript reverse transcriptase (Invitrogen Life Technologies). Primers for the long isoform of human leptin receptor (5'-GAA GAT GTT CCG AAC CCC AAG AAT TG-3' and 5'-CTA GAG AAG CAC TTG GTG ACT GAA C-3') (19), the short isoform of human leptin receptor (5'-CCA TTG AGA AGT ACC AGT TCA GTC TTT ACC-3' and 5'-GGG AAG TTG GCA CAT TGG GTT CA-3') (19), and GAPDH (5'-CCC CTT CAT TGA CCT CAA CTA C-3' and 5'-GAG TCC TTC CAC GAT ACC AAA G-3') (20) amplifications were synthesized (MWG Biotec). The cycling parameters for leptin receptor cDNA amplification were as follows: 95°C for 5 min, 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s, followed by 7 min at 72°C. Leptin receptor short isoform (329 bp), leptin receptor long isoform (427 bp), and GAPDH (417 bp) PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining.

Confocal laser-scanning microscopy

Cytospins were prepared from freshly purified neutrophils and neutrophils, which were cultured in the presence or absence of leptin, G-CSF, and anti-Fas mAb for 13 h. Cells were fixed and permeabilized, as previously described (3, 17). Immunofluorescent stainings were performed at room temperature for 1 h using the following primary Abs diluted in PBS plus 3% BSA plus 2% normal goat serum: anti-cytochrome c mAb (1/100), polyclonal anti-leptin receptor Ab (1/100), anti-Cox1 mAb (1/500), and polyclonal anti-Smac Ab (1/1500). Incubation with appropriate tetramethylrhodamine isothiocyanate- and FITC-conjugated secondary Abs was performed in the dark at room temperature for 1 h. The antifading agent Slowfade (Molecular Probes) was added, and the cells were covered by coverslips.

Immunofluorescent stainings were also performed on 5-µm-thick paraformaldehyde-fixed paraffin-embedded tissue sections from appendicitis and ulcerative colitis patients, as previously described (21). Immunostainings were performed at 4°C overnight using goat polyclonal anti-leptin receptor (1/100) together with anti-CD15 mAb (1/20) in blocking buffer. Incubation with secondary Abs and mounting was performed, as described above. All slides were analyzed by confocal laser-scanning microscopy (LSM 510; Zeiss) equipped with Ar and HeNe lasers.

Statistical analysis

Statistical analysis was performed by using the Mann-Whitney U test. If no original data are provided, the figures show mean levels ± SD. A probability value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Leptin receptor expression by neutrophils

We measured the expression of leptin receptor at mRNA and protein levels in freshly isolated and G-CSF-stimulated (5-h stimulation) blood neutrophils from normal donors. In agreement with previously published work (22), we observed that freshly isolated neutrophils express mRNA for the short, but not the long form of leptin receptor (Fig. 1A). G-CSF stimulation of neutrophils appeared to increase the mRNA expression of the short form of the leptin receptor, but the long form was again not detectable. Freshly purified PBMC and PHA-activated PBMC (5-h stimulation) served as controls. Fresh PBMC expressed both short and long leptin receptors, but PHA activation appeared to decrease the levels of the long form (Fig. 1A). To determine whether the expression of mRNA correlates with protein expression, we performed immunofluorescence analysis on freshly purified blood neutrophils. Neutrophils demonstrated a ring-like staining pattern, suggesting leptin receptor surface expression (Fig. 1B). To demonstrate leptin receptor expression on neutrophils under in vivo conditions, we analyzed neutrophils in tissue sections of patients with acute appendicitis and ulcerative colitis by a double immunofluorescence technique. Infiltrating neutrophils were identified using an anti-CD15 mAb. Neutrophils expressed leptin receptors, demonstrated by a ring-like staining pattern and its colocalization with CD15, consistent with its expression on the cell surface (Fig. 1C).



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  		FIGURE 1. Neutrophils express leptin receptors. A, RT-PCR. Freshly purified neutrophils express detectable levels of the short form of leptin receptor (329 bp). Following short-term stimulation (5 h) with G-CSF, the levels of expression appeared to increase. In contrast, the long form (427 bp) was not detectable in these cells in the presence and absence of G-CSF. Positive control for both forms of the receptor was cDNA from PBMC. B, Immunofluorescence. Freshly purified neutrophils express leptin surface receptors (ring-like staining pattern). Control Abs were used as negative controls. The bars represent 10 µm. Lower left corner, Magnification of one cell. C, Immunofluorescence. Leptin surface receptors were readily detected on neutrophils (open arrows), which infiltrated in appendicitis and ulcerative colitis tissues. Neutrophils were detected using an anti-CD15 mAb. The bars represent 10 µm. Control Abs were used as negative controls and demonstrated no significant fluorescence signals (data not shown).

 
Leptin delays neutrophil apoptosis

Because leptin delivers antiapoptotic signals in T cells (12) and monocytes (13), we investigated whether leptin delays apoptosis of neutrophils that spontaneously occurs following culturing these cells (1). Leptin delayed spontaneous neutrophil death in a dose- and time-dependent manner (Fig. 2A). Maximal inhibition of neutrophil death was reached with 0.5 µM; higher concentrations had no further effect (Fig. 2B). The EC50 of leptin used for these experiments was ~0.1 µM. Optimal concentrations of leptin and G-CSF had similar antideath effects on neutrophils (Fig. 2C). Anti-Fas stimulation induced neutrophil death in this system, as expected (3, 16, 17). To demonstrate specificity of leptin actions, an anti-leptin receptor mAb was used. This mAb dose dependently inhibited the survival effect of leptin, but not of G-CSF (Fig. 2D). The anti-leptin receptor mAb had no effect on neutrophil viability when used in the absence of cytokine stimulation. Moreover, a control mAb had no effect and leptin effects were not blocked when optimal concentrations of polymyxin B were added, excluding any potential nonspecific effects via LPS (data not shown).



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  		FIGURE 2. Leptin delays neutrophil apoptosis in vitro. A, Dose-dependent inhibition of neutrophil death by leptin. Mean ± SD of six independent experiments are shown. B, Concentration-effect curve of leptin in 24-h cultures (n = 6). Maximal antideath effects were seen at 0.5 µM. Higher leptin concentrations did not further increase this effect. The EC50 for leptin was ~0.1 µM. C, Leptin (0.5 µM, n = 6) and G-CSF (25 ng/ml, n = 6) had very similar efficacy regarding maintaining neutrophil survival. Anti-Fas mAb treatment (1 µg/ml, n = 6) accelerated neutrophil death. D, Anti-leptin receptor Ab (but not control Ab; data not shown) dose dependently abolished the antideath effect of leptin (0.1 µM), but not of G-CSF (25 ng/ml). This panel shows data from a 24-h neutrophil culture and is representative of six independent experiments. *, p < 0.05; **, p < 0.01.

 
We next investigated whether the antideath effect mediated by leptin was due to inhibition of apoptosis. Leptin reduced redistribution of PS, a characteristic feature of apoptotic neutrophils (16, 17, 18), with the same efficacy as G-CSF did (Fig. 3A). In contrast, anti-Fas stimulation accelerated neutrophil apoptosis in this in vitro system. We also analyzed DNA fragmentation, another hallmark of apoptotic cells. Again, leptin and G-CSF significantly blocked apoptosis, whereas anti-Fas stimulation resulted in increased DNA fragmentation (Fig. 3B). In conclusion, leptin exerts antiapoptotic activities on neutrophils in vitro.



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  		FIGURE 3. Leptin delays neutrophil apoptosis in vitro. A, Leptin (0.5 µM) reduced PS redistribution in neutrophil membranes (8-h cultures, n = 3). Right, Representative examples of flow cytometric analysis. The numbers indicate the percentage of apoptotic and necrotic cells, respectively. B, Leptin reduced the formation of hypoploid DNA in neutrophils (20-h cultures, n = 3). Right, Representative examples of flow cytometric analysis. *, p < 0.05.

 
Leptin inhibits caspase activation as well as Bid and Bax cleavage

Caspase-3 is a critical effector caspase in neutrophil apoptosis (3, 17). Moreover, there is evidence that neutrophil apoptosis is associated with the activation of caspase-8 (3, 17), although the mechanism of activation of this initiator caspase in neutrophils is unclear. In agreement with these earlier findings, spontaneous apoptosis was associated with both caspase-3 and caspase-8 cleavage. In both cases, culturing of the cells revealed in the appearance of the apparent active enzymes (17-kDa fragment of caspase-3 and 18-kDa form of caspase-8) (Fig. 4). Caspase cleavage was accelerated in anti-Fas-stimulated neutrophils. In contrast, leptin and G-CSF prevented the occurrence of the active forms of both caspase-3 and caspase-8.



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  		FIGURE 4. Leptin delays cleavage of caspase-8, Bid, Bax, and caspase-3 in cultured neutrophils. Delay (or prevention) of cleavage was also seen in G-CSF-treated neutrophils. Accelerated cleavage was seen in anti-CD95 mAb-treated neutrophils. The filters were reprobed with an anti-GAPDH mAb to ensure equal loading of the gels. Results of 6- and 9-h cultures are shown. The immunoblots are representative of three independent experiments.

 
Leptin and G-CSF also suppressed caspase-3-like DEVDase activity in neutrophils (Fig. 5), supporting the assumption that caspase-3 activation is blocked in the presence of these cytokines. Moreover, Bid cleavage was prevented by leptin and G-CSF (Fig. 4), in agreement with the observation that active 18-kDa caspase-8 is not generated under these conditions. Bax, a target of calpain-1 in neutrophils (17), was also not cleaved in the presence of leptin or G-CSF (Fig. 4).



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  		FIGURE 5. Leptin blocks enzymatic caspase-3-like activity in cultured neutrophils. Leptin (0.5 µM) and G-CSF (25 ng/ml) had equal efficacy. In contrast, Fas receptor activation resulted in an increase of caspase-3-like activity compared with cells cultured in medium. Results of 25-h cultures (n = 3) are shown. *, p < 0.05.

 
Leptin inhibits the mitochondrial release of cytochrome c and Smac

Mitochondria have been implicated in the regulation of neutrophil apoptosis (3, 4, 5, 17). The observed cleavage of Bid and Bax in this study supports the concept that mitochondria are involved in this process. We therefore analyzed the mitochondrial release of two proapoptotic factors in cultured neutrophils in the presence and absence of leptin and G-CSF, respectively, by fluorescence immunostaining and microscopic analysis. Both cytochrome c and Smac were present in mitochondria of freshly purified neutrophils, as indicated by the colocalization with the mitochondrial marker protein Cox1 (23). In these cells, a punctate staining pattern was observed (Fig. 6A). Culturing of neutrophils for 13 h demonstrated evidence for cytochrome c and Smac release (= diffuse pattern) in a subgroup of cells. Both leptin and G-CSF preserved the punctate pattern in the majority of the cells, whereas anti-Fas treatment revealed a diffuse pattern of both cytochrome c and Smac in almost all cells. No difference was observed when cytochrome c and Smac stainings were compared. A statistical analysis of these experiments is given in Fig. 6B. Leptin and G-CSF had the same efficacy to block the transition into a diffuse cytosolic staining pattern associated with neutrophil apoptosis.



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  		FIGURE 6. Leptin prevents the mitochondrial release of cytochrome c and Smac. Freshly isolated neutrophils and neutrophils from 13-h cultures were investigated by confocal microscopy. A, Cytochrome c and Smac colocalized with each other and with Cox1, demonstrating that they are mitochondrial proteins in freshly isolated neutrophils. Leptin preserved the punctate pattern in cultured neutrophils, whereas we observed diffuse staining in the absence of cytokine support or after anti-Fas mAb treatment. No detectable staining was observed using control Abs (data not shown). Results are representative of three independent experiments. B, Quantitative and statistical analysis of the experiments shown in A. *, p < 0.05.

 
The antiapoptotic effect of leptin is mediated via PI3K and MAPK pathways

To test whether activation of PI3K and/or MAPK pathways is involved in leptin-mediated antiapoptosis, neutrophils were preincubated with different concentrations of defined kinase inhibitors. Both SB 203580, a selective inhibitor of p38 MAPK, and LY 294002, an inhibitor of PI3K, accelerated spontaneous neutrophil death. Moreover, the antideath effect of leptin appeared to be partially blocked by each inhibitor, but leptin was still able to significantly increase neutrophil viability under these conditions. However, when SB 203580 and LY 294002 were used concurrently, leptin-induced survival was completely abrogated (Fig. 7). In these experiments, we also used PD 98059, an inhibitor of p42/44 MAPKs, that had no effect in this system. These data suggest that PI3K and p38 MAPK pathways are involved in transducing leptin-mediated antiapoptotic signals into neutrophils.



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  		FIGURE 7. Pharmacological inhibition of p38 MAPK and PI3K blocks antideath effects of leptin in cultured neutrophils. Neutrophils were cultured in the presence ({blacksquare}) and absence ({square}) of leptin (0.5 µM) for 24 h (n = 3). The indicated inhibitors were used at 25 µM and preincubated for 30 min before leptin stimulation. Both inhibitors accelerated spontaneous neutrophil death, but did only partially prevent the leptin-mediated antideath effect. Only combined treatment with SB 203580 and LY 294002 abolished the effect of leptin in this system. Same results were observed when the inhibitors were used at 50 µM and if neutrophils were cultured for 48 h (data not shown). *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Leptin has been implicated in the regulation of the immune system. Earlier studies suggested that leptin stimulates Th1 immunity (24). However, leptin also stimulates cells of the innate immune system such as neutrophils, which have previously been shown to express leptin receptors (22). However, whether these receptors are functional is a matter of debate. For instance, the effect of leptin on CD11b expression of neutrophils appeared to be indirect via monocyte-derived TNF-{alpha} (22). In contrast, it was reported that reactive oxygen production is the consequence of direct stimulation of neutrophils by leptin (25) and that leptin could induce the phagocytic activity in leptin-deficient purified mouse neutrophils (26). In this study, we investigated apoptotic pathways in neutrophils after stimulation with leptin. The following new findings are reported: 1) Leptin receptors are expressed on neutrophils under in vivo conditions. 2) Leptin activates directly neutrophils and delays spontaneous apoptosis of these cells. 3) Leptin appears to exert its antiapoptotic function by inhibiting proapoptotic events proximal to mitochondria. 4) The antiapoptotic function of leptin is mediated via PI3K and p38 MAPK signaling pathways.

Our study adds additional information on the potential role of leptin in regulating immune responses, which are often associated with elevated levels of leptin. Besides sepsis (10, 11), leptin has also been implicated in the pathogenesis of autoimmune diseases. For instance, in a mouse model of multiple sclerosis, it was found that leptin levels were particularly high before and at the start of clinical disease (27). In contrast, leptin-deficient mice did not develop this disease (28), and starvation, which reduced leptin levels, inhibited the progression of clinical symptoms (27). Neutrophils play an important pathogenic role in both infectious and autoimmune diseases. Therefore, the question as to whether leptin activates neutrophils, and how the functional response(s) of such an interaction might be, seems to be of great interest. Because leptin delayed spontaneous neutrophil apoptosis, it is possible that leptin contributes to neutrophil accumulation at inflammatory sites (2), and, perhaps, it may then also stimulate the release of proinflammatory mediators from these cells (1).

We confirm in this study previously published work, demonstrating that neutrophils express the short form of leptin receptors (22). Both forms have identical extracellular and transmembrane domains, but differ in their intracellular domains (7). Although initial studies suggested that the short form is unable to deliver signals into the cells (7), it is now clear that this is not the case. Despite lacking the STAT3 docking site, the short leptin receptor is still able to bind and activate Jak2, which subsequently activates the MAPK pathway (29). The data reported in this manuscript suggest that besides the MAPK pathway, PI3K is also activated in leptin-stimulated neutrophils. Both MAPK (30, 31) and PI3K (32, 33) activation have previously been shown to mediate neutrophil antiapoptosis.

The leptin-initiated signaling cascades somehow block spontaneous neutrophil apoptosis before mitochondria release their proapoptotic factors. This is indicated by the observation that leptin delays cleavage of caspase-8, Bid, and Bax. Cleaved Bid (34, 35) and Bax (36) have previously been shown to be highly efficient in proapoptotic mitochondrial triggering. The delay of Bid and Bax cleavage by leptin was associated with inhibition of the release of cytochrome c into the cytosol. Cytochrome c forms a complex with Apaf-1 and caspase-9 that, in the presence of dATP, leads to the activation of caspase-3 (37). Smac is another proapoptotic factor released from mitochondria that inactivates members of the inhibitors of apoptosis protein family (38, 39). Leptin and G-CSF inhibited the release of both cytochrome c and Smac, explaining why these cytokines also blocked caspase-3 activation and subsequently apoptosis in neutrophils.

The efficacy of leptin to inhibit neutrophil apoptosis was similar to G-CSF. However, the potency appeared to be ~100-fold decreased (5 ng/ml G-CSF is usually sufficient to obtain optimal antiapoptotic effects in neutrophils). The low sensitivity of neutrophils toward leptin might be due to the fact that they express only the short form of the leptin receptor, which has been described as being less efficient in transducing leptin signals into cells compared with the long form (29). The question remains as to whether the high concentrations of leptin that are required for the antiapoptotic action on neutrophils in vitro occur under in vivo conditions. Leptin serum levels up to 400 ng/ml were reported in children with chronic renal failure (40). In addition, in obese subjects treated with leptin, serum levels of >700 ng/ml were measured (41). Even higher leptin serum levels might have been reached (based on our own calculations >0.1 µM) when mice were treated with 2 µg/g body weight leptin to improve wound healing (42). These levels are in the range in which significant antiapoptotic effects on neutrophils are observed under in vitro conditions. Moreover, it should be noted that recombinant leptin, which has been used in this study, is known to have a lower potency than native leptin, perhaps due to differences in glycosylation (43). In addition, it is possible that higher leptin concentrations are present in inflamed tissues that contain leptin-producing adipocytes. Therefore, leptin may indeed represent a neutrophil survival factor at sites of inflammation, but probably not in the circulation.

Besides neutrophils, leptin has been shown to exhibit antiapoptotic activity in T cells (12), monocytes (13), and neuroblastoma cells (44). In contrast, leptin has been reported to induce apoptosis in human bone marrow stromal cells (45). The reason for these contrasting responses in different cell types is unclear, but differences in the expression patterns of leptin receptors and associated signaling molecules may play a role. Despite these uncertainties, it seems that leptin, like other hormones, regulates apoptosis, and that cells of the immune system are targets of leptin. Further work is required to understand how the control of apoptosis by leptin influences innate and/or adaptive immunity.


   Acknowledgments
 
We thank E. Kozlowski (Department of Pharmacology, University of Bern) for excellent technical assistance, as well as Drs. D. Simon (Department of Dermatology, University of Bern) and M. Neef (Department of Clinical Pharmacology, University of Bern) for providing blood samples.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by grants from the Swiss National Science Foundation (31-58916.99 and 31-107526.04) and OPO-Foundation (Zurich). Back

2 Address correspondence and reprint requests to Dr. Hans-Uwe Simon, Department of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. E-mail address: hus{at}pki.unibe.ch Back

3 Abbreviations used in this paper: Smac, second mitochondria-derived activator of caspase; PS, phosphatidylserine. Back

Received for publication January 26, 2005. Accepted for publication March 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Simon, H.-U.. 2003. Neutrophil apoptosis pathways and their modifications in inflammation. Immunol. Rev. 193: 101-110.[Medline]
  2. Dibbert, B., M. Weber, W. H. Nikolaizik, P. Vogt, M. H. Schöni, K. Blaser, H.-U. Simon. 1999. Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation. Proc. Natl. Acad. Sci. USA 96: 13330-13335.[Abstract/Free Full Text]
  3. Baumann, R., C. Casaulta, D. Simon, S. Conus, S. Yousefi, H.-U. Simon. 2003. Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway. FASEB J. 17: 2221-2230.[Abstract/Free Full Text]
  4. Maianski, N. A., F. P. J. Mul, J. D. van Buul, D. Roos, T. W. Kuijpers. 2002. Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils. Blood 99: 672-679.[Abstract/Free Full Text]
  5. Moulding, D. A., C. Akgul, M. Derouet, M. R. H. White, S. W. Edwards. 2001. BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis. J. Leukocyte Biol. 70: 783-792.[Abstract/Free Full Text]
  6. Faggioni, R., K. R. Feingold, C. Grunfeld. 2001. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J. 15: 2565-2571.[Abstract/Free Full Text]
  7. Lee, G. H., R. Proenca, J. M. Montez, K. M. Carroll, J. G. Darvishzadeh, J. I. Lee, J. M. Friedman. 1996. Abnormal splicing of the leptin receptor in diabetic mice. Nature 379: 632-635.[Medline]
  8. Fantuzzi, G., R. Faggioni. 2000. Leptin in the regulation of immunity, inflammation, and hematopoiesis. J. Leukocyte Biol. 68: 437-446.[Abstract/Free Full Text]
  9. Sweeney, G.. 2002. Leptin signalling. Cell. Signal. 14: 655-663.[Medline]
  10. Bornstein, S. R., J. Licinio, R. Tauchnitz, L. Engelmann, A. B. Negrao, P. Gold, G. P. Chrousos. 1998. Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm in cortisol and leptin secretion. J. Clin. Endocrinol. Metab. 83: 280-283.[Abstract/Free Full Text]
  11. Loffreda, S., S. Q. Yang, H. Z. Lin, C. L. Karp, M. L. Brengman, D. J. Wang, A. S. Klein. 1998. Leptin regulates proinflammatory immune responses. FASEB J. 12: 57-65.[Abstract/Free Full Text]
  12. Fujita, Y., M. Murakami, Y. Ogawa, H. Masuzaki, M. Tanaka, S. Ozaki, K. Nakao, T. Mimori. 2002. Leptin inhibits stress-induced apoptosis of T lymphocytes. Clin. Exp. Immunol. 128: 21-26.[Medline]
  13. Najib, S., V. Sanchez-Margalet. 2002. Human leptin promotes survival of human circulating blood monocytes prone to apoptosis by activation of p42/44 MAPK pathway. Cell. Immunol. 220: 143-149.[Medline]
  14. Yousefi, S., D. R. Green, K. Blaser, H.-U. Simon. 1994. Protein tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils. Proc. Natl. Acad. Sci. USA 91: 10868-10872.[Abstract/Free Full Text]
  15. Daigle, I., S. Yousefi, M. Colonna, D. R. Green, H.-U. Simon. 2002. Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils. Nat. Med. 8: 61-67.[Medline]
  16. Altznauer, F., S. von Gunten, P. Späth, H.-U. Simon. 2003. Concurrent presence of agonistic and antagonistic anti-CD95 autoantibodies in intravenous Ig preparations. J. Allergy Clin. Immunol. 112: 1185-1190.[Medline]
  17. Altznauer, F., S. Conus, A. Cavalli, G. Folkers, H.-U. Simon. 2004. Calpain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in neutrophil apoptosis. J. Biol. Chem. 279: 5947-5957.[Abstract/Free Full Text]
  18. Baumann, R., S. Yousefi, D. Simon, S. Russmann, C. Mueller, H.-U. Simon. 2004. Functional expression of CD134 by neutrophils. Eur. J. Immunol. 34: 2268-2275.[Medline]
  19. Bennett, B. D., G. P. Solar, J. Q. Yuan, J. Mathias, G. R. Thomas, W. Matthews. 1996. A role for leptin and its cognate receptor in hematopoiesis. Curr. Biol. 6: 1170-1180.[Medline]
  20. Heinisch, I. V. W. M., C. Bizer, W. Volgger, H.-U. Simon. 2001. Functional CD137 receptors are expressed by eosinophils from patients with IgE-mediated allergic responses but not by eosinophils from patients with non-IgE-mediated eosinophilic responses. J. Allergy Clin. Immunol. 108: 21-28.[Medline]
  21. Altznauer, F., S. Martinelli, S. Yousefi, C. Thürig, I. Schmid, E. M. Conway, M. H. Schöni, P. Vogt, C. Mueller, M. F. Fey, et al 2004. Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils. J. Exp. Med. 199: 1343-1354.[Abstract/Free Full Text]
  22. Zarkesh-Esfahani, H., A. G. Pockley, Z. Wu, P. G. Hellewell, A. P. Weetman, R. J. M. Ross. 2004. Leptin indirectly activates human neutrophils via induction of TNF-{alpha}. J. Immunol. 172: 1809-1814.[Abstract/Free Full Text]
  23. Perez-Martinez, X., S. A. Broadley, T. D. Fox. 2003. Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J. 22: 5951-5961.[Medline]
  24. Lord, G. M., G. Matarese, J. K. Howard, R. J. Baker, S. R. Bloom, R. I. Lechler. 1998. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394: 897-901.[Medline]
  25. Caldefie-Chezet, F., A. Poulin, A. Tridon, B. Sion, M. P. Vasson. 2001. Leptin: a potential regulator of polymorphonuclear neutrophil bactericidal action?. J. Leukocyte Biol. 69: 414-418.[Abstract/Free Full Text]
  26. Moore, S. I., G. B. Huffnagle, G.-H. Chen, E. S. White, P. Mancuso. 2003. Leptin modulates neutrophil phagocytosis of Klebsiella pneumoniae. Infect. Immun. 71: 4182-4185.[Abstract/Free Full Text]
  27. Sanna, V., A. Di Giacomo, A. La Cava, R. I. Lechler, S. Fontana, S. Zappacosta, G. Matarese. 2003. Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses. J. Clin. Invest. 111: 241-250.[Medline]
  28. Matarese, G., A. Di Giacomo, V. Sanna, G. M. Lord, J. K. Howard, A. Di Tuoro, S. R. Bloom, R. I. Lechler, S. Zappacosta, S. Fontana. 2001. Requirement for leptin in the induction and progression of autoimmune encephalomyelitis. J. Immunol. 166: 5909-5916.[Abstract/Free Full Text]
  29. Bjorbaek, C., S. Uotani, B. da Silva, J. S. Flier. 1997. Divergent signaling capacities of the long and short isoforms of the leptin receptor. J. Biol. Chem. 272: 32686-32695.[Abstract/Free Full Text]
  30. Frasch, S. C., J. A. Nick, V. A. Fadok, D. L. Bratton, G. S. Worthen, P. M. Henson. 1998. p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J. Biol. Chem. 273: 8389-8397.[Abstract/Free Full Text]
  31. Aoshiba, K., S. Yasui, M. Hayashi, J. Tamaoki, A. Nagai. 1999. Role of p38-mitogen-activated protein kinase in spontaneous apoptosis of human neutrophils. J. Immunol. 162: 1692-1700.[Abstract/Free Full Text]
  32. Klein, J. B., M. J. Rane, J. A. Scherzer, P. Y. Coxon, R. Kettritz, J. M. Mathiesen, A. Buridi, K. R. McLeish. 2000. Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. J. Immunol. 164: 4286-4291.[Abstract/Free Full Text]
  33. Cowburn, A. S., K. A. Cadwallader, B. J. Reed, N. Farahi, E. R. Chilvers. 2002. Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. Blood 100: 2607-2616.[Abstract/Free Full Text]
  34. Li, H., H. Zhu, C. Xu, J. Yuan. 1998. Cleavage of Bid by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491-501.[Medline]
  35. Luo, X., I. Budihardjo, H. Zou, C. Slaughter, X. Wang. 1998. Bid, a Bcl-2 interacting protein, mediates cytochrome c release in response to activation of cell surface receptors. Cell 94: 481-490.[Medline]
  36. Gao, G., Q. P. Dou. 2000. N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome c release and apoptotic cell death. J. Cell. Biochem. 80: 53-72.[Medline]
  37. Li, P., D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase 9 complex initiates an apoptotic protease cascade. Cell 91: 479-489.[Medline]
  38. Du, C., M. Fang, Y. Li, X. Wang. 2000. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP-inhibition. Cell 102: 33-42.[Medline]
  39. Verhagen, A. M., P. G. Ekert, M. Pakusch, J. Silke, L. M. Connolly, G. E. Reid, R. L. Moritz, R. J. Simpson, D. L. Vaux. 2000. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102: 43-53.[Medline]
  40. Daschner, M., B. Tonshoff, W. F. Blum, P. Englaro, A. M. Wingen, F. Schaefer, E. Wuhl, W. Rascher, O. Mehls. 1998. Inappropriate elevation of serum leptin levels in children with chronic renal failure: European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. J. Am. Soc. Nephrol. 9: 1074-1079.[Abstract]
  41. Fujioka, K., J. Patane, J. Lubina, D. Lau. 1999. CSF leptin levels after exogenous administration of recombinant methionyl human leptin. J. Am. Med. Assoc. 282: 1517-1518.[Free Full Text]
  42. Goren, I., H. Kämpfer, M. Podda, J. Pfeilschifter, S. Frank. 2003. Leptin and wound inflammation in diabetic ob/ob mice: differential regulation of neutrophil and macrophage influx and a potential role for the scab as a sink for inflammatory cells and mediators. Diabetes 52: 2821-2832.[Abstract/Free Full Text]
  43. Cohen, S. L., J. L. Halaas, J. M. Friedman, B. T. Chait, L. Brennett, D. Chang, R. Hecht, F. Collins. 1996. Human leptin characterization. Nature 382: 589.[Medline]
  44. Russo, V. C., S. Metaxas, K. Kobayashi, M. Harris, G. A. Werther. 2004. Antiapoptotic effects of leptin in human neuroblastoma cells. Endocrinology 145: 4103-4112.[Abstract/Free Full Text]
  45. Kim, G. S., J. S. Hong, S. W. Kim, J.-M. Koh, C. S. An, J.-Y. Choi, S.-L. Cheng. 2003. Leptin induces apoptosis via ERK/cPLA2/cytochrome c pathway in human bone marrow stromal cells. J. Biol. Chem. 278: 21920-21929.[Abstract/Free Full Text]



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