Apoptotic Pathways Are Inhibited by
                                     Leptin Receptor Activation in
The Journal of Immunology

Apoptotic Pathways Are Inhibited by Leptin Receptor
Activation in Neutrophils1

Andreina Bru...
The Journal of Immunology                                                                                                 ...
8092                                                                                     LEPTIN IS A NEUTROPHIL SURVIVAL F...
The Journal of Immunology                                                                                                 ...
8094                                                                                        LEPTIN IS A NEUTROPHIL SURVIVA...
The Journal of Immunology                                                                                                 ...
8096                                                                                                    LEPTIN IS A NEUTRO...
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Neutrophils Leptin Receptor Activation in Apoptotic Pathways ...

  1. 1. Apoptotic Pathways Are Inhibited by Leptin Receptor Activation in Neutrophils This information is current as of October 19, 2010 Andreina Bruno, Sébastien Conus, Inès Schmid and Hans-Uwe Simon J. Immunol. 2005;174;8090-8096 http://www.jimmunol.org/cgi/content/full/174/12/8090 Downloaded from www.jimmunol.org on October 19, 2010 References This article cites 45 articles, 25 of which can be accessed free at: http://www.jimmunol.org/cgi/content/full/174/12/8090#BIBL 12 online articles that cite this article can be accessed at: http://www.jimmunol.org/cgi/content/full/174/12/8090#otherarti cles Subscriptions Information about subscribing to The Journal of Immunology is online at http://www.jimmunol.org/subscriptions/ Permissions Submit copyright permission requests at http://www.aai.org/ji/copyright.html Email Alerts Receive free email alerts when new articles cite this article. Sign up at http://www.jimmunol.org/subscriptions/etoc.shtml The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright ©2005 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
  2. 2. The Journal of Immunology Apoptotic Pathways Are Inhibited by Leptin Receptor Activation in Neutrophils1 Andreina Bruno, Sebastien Conus, Ines Schmid, and Hans-Uwe Simon2 ´ ` 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. The Journal of Immunology, 2005, 174: 8090 – 8096. Downloaded from www.jimmunol.org on October 19, 2010 N eutrophils are important effector cells of the immune onstrate that leptin delays neutrophil apoptosis in vitro. Analyzing system that represent the body’s first line of defense the intracellular events in these cells revealed that leptin activates against invading microorganisms. Delayed neutrophil both PI3K and MAPK signaling pathways, resulting in the inhi- apoptosis has been associated with several infectious diseases (1) bition of the mitochondrial death pathway. and often associated with overexpression of neutrophil survival factors such as G-CSF, GM-CSF (2), and macrophage migration Materials and Methods inhibitory factor (3). These survival cytokines have been described Reagents to block the mitochondria-dependent death pathway in neutrophils Recombinant human leptin, G-CSF, and goat polyclonal anti-human Bid (3, 4). Accordingly, the expression of members of the Bcl-2 family Ab were purchased from R&D Systems (distributed by Buhlmann). The ¨ appears to be regulated by antiapoptotic cytokines in these anti-human Fas (CH11) and anti-human caspase-8 mAbs were from Lab- cells (5). Force and Cell Signaling Technology (Bioconcept), respectively. Anti- Leptin is a pleiotropic cytokine involved in different biological GAPDH mAb was from Chemicon International. Rabbit polyclonal anti- human Bax and anti-human caspase-3 Abs, as well as anti-cytochrome c systems. It shares structural similarities with some cytokines, in- and anti-human CD15 mAbs were from BD Biosciences. The anti-Cox1 cluding IL-6, IL-11, IL-12, IL-15, as well as with G-CSF, oncosta- mAb and polyclonal anti-second mitochondria-derived activator of caspase tin M, ciliary neurotrophic factor, and leukemia-inhibitory factor (anti-Smac)3 Ab were purchased from Molecular Probes (distributed by (6). Leptin binds to short and long forms of leptin receptors, which Invitrogen Life Technologies) and Stratagene Europe, respectively. The are generated by differential splicing (7). The leptin receptors are goat polyclonal anti-leptin receptor (sc-1834) was from Santa Cruz Bio- technology (Lab-Force). Mouse and rabbit HRP-conjugated secondary Abs expressed in multiple cells and tissues, including kidney, lung, were obtained from Amersham Biosciences. The pharmacological inhibi- adrenal gland, hemopoietic precursor cells, and bone marrow, as tors SB 203580 (inhibitor of p38 kinase) and LY 294002 (inhibitor of well as in neutrophils, monocytes, and T cells (8). The signal PI3K) as well as polymyxin B sulfate were from Calbiochem. All other transduction pathways regulated by leptin are diverse and in- reagents were, unless stated otherwise, from Sigma-Aldrich. clude those characteristic for both cytokine and growth factor re- Cells ceptor signaling (9). Leptin plays a key role in the regulation of body weight, but also Mature peripheral blood neutrophils were purified from healthy normal individuals by Ficoll-Hypaque centrifugation, as previously described (14, exerts other biological functions, which modulate hemopoiesis, an- 15). The resulting cell populations contained 5% contaminating cells. giogenesis, and immune responses (8). Interestingly, leptin pro- Cell purity was assessed by staining with Diff-Quik (Medion) and light duction is increased in patients suffering from sepsis and other microscopy analysis. Written informed consent was obtained from all pa- inflammatory diseases (10, 11). Moreover, leptin mediates both tients and control individuals, and the study was approved by the ethics committee of the Canton Bern. proliferative and antiapoptotic activities in a variety of cell types, including T cells (12) and monocytes (13). In this study, we dem- Cell cultures Human mature neutrophils were cultured at 1 106 per ml in complete Department of Pharmacology, University of Bern, Bern, Switzerland culture medium (RPMI 1640 containing 10% FCS) in the presence and Received for publication January 26, 2005. Accepted for publication March 23, 2005. absence of rG-CSF (25 ng/ml), anti-Fas mAb (1 g/ml), and leptin (0.0001–5 M, usually 0.5 M) for the indicated times. In some experi- The costs of publication of this article were defrayed in part by the payment of page ments, we added 15 g/ml polymyxin B. Leptin-blocking experiments charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. were performed with anti-human leptin receptor mAb (80 –320 M). SB 1 203580 and LY 294002 (both at 25 and 50 M) were added 30 min before This work was supported by grants from the Swiss National Science Foundation adding leptin and left in the culture. (31-58916.99 and 31-107526.04) and OPO-Foundation (Zurich). 2 Address correspondence and reprint requests to Dr. Hans-Uwe Simon, Department 3 of Pharmacology, University of Bern, Friedbuhlstrasse 49, CH-3010 Bern, Switzer- ¨ Abbreviations used in this paper: Smac, second mitochondria-derived activator of land. E-mail address: hus@pki.unibe.ch caspase; PS, phosphatidylserine. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
  3. 3. The Journal of Immunology 8091 Determination of cell death and apoptosis strand synthesis was performed using total RNA, oligo(dT) 15 primer (Pro- mega, distributed by Catalys), and Superscript reverse transcriptase Neutrophil death was assessed by uptake of 1 M ethidium bromide and (Invitrogen Life Technologies). Primers for the long isoform of human flow cytometric analysis (FACSCalibur) (16 –18). To determine whether leptin receptor (5 -GAA GAT GTT CCG AAC CCC AAG AAT TG-3 and cell death was apoptosis, DNA fragmentation and redistribution of phos- 5 -CTA GAG AAG CAC TTG GTG ACT GAA C-3 ) (19), the short phatidylserine (PS) were measured (16 –18). 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 Enzymatic caspase-3 assay CA-3 ) (19), and GAPDH (5 -CCC CTT CAT TGA CCT CAA CTA C-3 Caspase-3-like activity was measured using a commercial kit (QuantiZyme and 5 -GAG TCC TTC CAC GAT ACC AAA G-3 ) (20) amplifications caspase-3 cellular activity kit; BIOMOL), according to the manufacturer’s were synthesized (MWG Biotec). The cycling parameters for leptin recep- instructions and as previously described (17, 18). tor 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. Immunoblotting Leptin receptor short isoform (329 bp), leptin receptor long isoform (427 bp), and GAPDH (417 bp) PCR products were separated on 1% agarose Neutrophils (1 106/ml) were cultured for 6 and 9 h, respectively, washed gels and visualized by ethidium bromide staining. with cold PBS supplemented with a protease inhibitor mixture (Sigma- Aldrich), and lysed by using RIPA buffer (50 mM Tris-HCl, 150 mM Confocal laser-scanning microscopy NaCl, 1% Nonidet P-40, 1 mM EGTA, and 0.25% sodium deoxycholate supplemented with a protease inhibitor mixture) with frequent vortexing on Cytospins were prepared from freshly purified neutrophils and neutrophils, ice for 15 min. Equal amounts of cell lysates were mixed with running which were cultured in the presence or absence of leptin, G-CSF, and buffer, boiled, and subjected to gel electrophoresis on 12% NuPage-Gels anti-Fas mAb for 13 h. Cells were fixed and permeabilized, as previously (NOVEX, distributed by Invitrogen Life Technologies). Separated proteins described (3, 17). Immunofluorescent stainings were performed at room were electrotransferred onto polyvinylidene difluoride membranes (Immo- temperature for 1 h using the following primary Abs diluted in PBS plus bilion-P; Millipore). The filters were incubated with blocking solution 3% BSA plus 2% normal goat serum: anti-cytochrome c mAb (1/100), (TBS/0.1% Tween 20, 5% nonfat dry milk) at room temperature for 1 h. polyclonal anti-leptin receptor Ab (1/100), anti-Cox1 mAb (1/500), and The primary Abs anti-Bid (1/1000), anti-Bax (1/1000), anti-caspase-3 polyclonal anti-Smac Ab (1/1500). Incubation with appropriate tetrameth- (1/1000), and anti-caspase-8 (1/1000), respectively, were incubated in ylrhodamine isothiocyanate- and FITC-conjugated secondary Abs was per- Downloaded from www.jimmunol.org on October 19, 2010 blocking solution overnight at 4°C. For loading controls, stripped filters formed in the dark at room temperature for 1 h. The antifading agent were incubated with anti-GAPDH mAb (1/3000). Filters were washed in Slowfade (Molecular Probes) was added, and the cells were covered by TBS/0.1% Tween 20 for 30 min and incubated with the appropriate HRP- coverslips. conjugated secondary Ab (1/3000) at room temperature for 1 h. After an- Immunofluorescent stainings were also performed on 5- m-thick para- other washing step, filters were developed by an ECL technique (ECL kit; formaldehyde-fixed paraffin-embedded tissue sections from appendicitis Amersham), according to the manufacturer’s instructions. and ulcerative colitis patients, as previously described (21). Immunostain- ings were performed at 4°C overnight using goat polyclonal anti-leptin RT-PCR receptor (1/100) together with anti-CD15 mAb (1/20) in blocking buffer. Incubation with secondary Abs and mounting was performed, as described Total RNA was isolated from neutrophils using TRIzol solution (Invitro- above. All slides were analyzed by confocal laser-scanning microscopy gen Life Technologies), according to the manufacturer’s instructions. First- (LSM 510; Zeiss) equipped with Ar and HeNe lasers. FIGURE 1. Neutrophils express leptin receptors. A, RT-PCR. Freshly purified neutrophils express detect- able 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 ex- press leptin surface receptors (ring-like staining pat- tern). Control Abs were used as negative controls. The bars represent 10 m. Lower left corner, Magnification of one cell. C, Immunofluorescence. Leptin surface re- ceptors were readily detected on neutrophils (open ar- rows), which infiltrated in appendicitis and ulcerative colitis tissues. Neutrophils were detected using an anti- CD15 mAb. The bars represent 10 m. Control Abs were used as negative controls and demonstrated no sig- nificant fluorescence signals (data not shown).
  4. 4. 8092 LEPTIN IS A NEUTROPHIL SURVIVAL FACTOR Statistical analysis no further effect (Fig. 2B). The EC50 of leptin used for these ex- Statistical analysis was performed by using the Mann-Whitney U test. If no periments was 0.1 M. Optimal concentrations of leptin and original data are provided, the figures show mean levels SD. A proba- G-CSF had similar antideath effects on neutrophils (Fig. 2C). Anti- bility value of 0.05 was considered statistically significant. Fas stimulation induced neutrophil death in this system, as ex- pected (3, 16, 17). To demonstrate specificity of leptin actions, an Results anti-leptin receptor mAb was used. This mAb dose dependently Leptin receptor expression by neutrophils inhibited the survival effect of leptin, but not of G-CSF (Fig. 2D). We measured the expression of leptin receptor at mRNA and pro- The anti-leptin receptor mAb had no effect on neutrophil viability tein levels in freshly isolated and G-CSF-stimulated (5-h stimula- when used in the absence of cytokine stimulation. Moreover, a tion) blood neutrophils from normal donors. In agreement with control mAb had no effect and leptin effects were not blocked previously published work (22), we observed that freshly isolated when optimal concentrations of polymyxin B were added, exclud- neutrophils express mRNA for the short, but not the long form of ing any potential nonspecific effects via LPS (data not shown). leptin receptor (Fig. 1A). G-CSF stimulation of neutrophils ap- We next investigated whether the antideath effect mediated by peared to increase the mRNA expression of the short form of the leptin was due to inhibition of apoptosis. Leptin reduced redistri- leptin receptor, but the long form was again not detectable. Freshly bution of PS, a characteristic feature of apoptotic neutrophils (16 – purified PBMC and PHA-activated PBMC (5-h stimulation) 18), with the same efficacy as G-CSF did (Fig. 3A). In contrast, served as controls. Fresh PBMC expressed both short and long anti-Fas stimulation accelerated neutrophil apoptosis in this in leptin receptors, but PHA activation appeared to decrease the lev- vitro system. We also analyzed DNA fragmentation, another hall- els of the long form (Fig. 1A). To determine whether the expres- mark of apoptotic cells. Again, leptin and G-CSF significantly sion of mRNA correlates with protein expression, we performed blocked apoptosis, whereas anti-Fas stimulation resulted in in- immunofluorescence analysis on freshly purified blood neutro- creased DNA fragmentation (Fig. 3B). In conclusion, leptin exerts phils. Neutrophils demonstrated a ring-like staining pattern, sug- antiapoptotic activities on neutrophils in vitro. Downloaded from www.jimmunol.org on October 19, 2010 gesting leptin receptor surface expression (Fig. 1B). To demon- strate leptin receptor expression on neutrophils under in vivo conditions, we analyzed neutrophils in tissue sections of patients Leptin inhibits caspase activation as well as Bid and Bax with acute appendicitis and ulcerative colitis by a double immu- cleavage nofluorescence technique. Infiltrating neutrophils were identified us- Caspase-3 is a critical effector caspase in neutrophil apoptosis (3, ing an anti-CD15 mAb. Neutrophils expressed leptin receptors, dem- 17). Moreover, there is evidence that neutrophil apoptosis is asso- onstrated by a ring-like staining pattern and its colocalization with ciated with the activation of caspase-8 (3, 17), although the mech- CD15, consistent with its expression on the cell surface (Fig. 1C). anism of activation of this initiator caspase in neutrophils is un- clear. In agreement with these earlier findings, spontaneous Leptin delays neutrophil apoptosis apoptosis was associated with both caspase-3 and caspase-8 cleav- Because leptin delivers antiapoptotic signals in T cells (12) and age. In both cases, culturing of the cells revealed in the appearance monocytes (13), we investigated whether leptin delays apoptosis of of the apparent active enzymes (17-kDa fragment of caspase-3 and neutrophils that spontaneously occurs following culturing these 18-kDa form of caspase-8) (Fig. 4). Caspase cleavage was accel- cells (1). Leptin delayed spontaneous neutrophil death in a dose- erated in anti-Fas-stimulated neutrophils. In contrast, leptin and and time-dependent manner (Fig. 2A). Maximal inhibition of neu- G-CSF prevented the occurrence of the active forms of both trophil death was reached with 0.5 M; higher concentrations had caspase-3 and caspase-8. 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 M. Higher leptin concentrations did not further increase this effect. The EC50 for leptin was 0.1 M. C, Leptin (0.5 M, n 6) and G-CSF (25 ng/ml, n 6) had very similar efficacy regarding main- taining neutrophil survival. Anti-Fas mAb treatment (1 g/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 M), but not of G-CSF (25 ng/ml). This panel shows data from a 24-h neutrophil culture and is repre- sentative of six independent experiments. , p 0.05; , p 0.01.
  5. 5. The Journal of Immunology 8093 FIGURE 3. Leptin delays neutrophil apoptosis in vitro. A, Leptin (0.5 M) reduced PS redistribution in neutrophil membranes (8-h cultures, n 3). Right, Rep- resentative examples of flow cytometric analysis. The numbers indicate the percentage of apoptotic and ne- crotic cells, respectively. B, Leptin reduced the forma- tion of hypoploid DNA in neutrophils (20-h cultures, n 3). Right, Representative examples of flow cyto- metric analysis. , p 0.05. Downloaded from www.jimmunol.org on October 19, 2010 Leptin and G-CSF also suppressed caspase-3-like DEVDase ac- 4), in agreement with the observation that active 18-kDa caspase-8 tivity in neutrophils (Fig. 5), supporting the assumption that is not generated under these conditions. Bax, a target of calpain-1 caspase-3 activation is blocked in the presence of these cytokines. in neutrophils (17), was also not cleaved in the presence of leptin Moreover, Bid cleavage was prevented by leptin and G-CSF (Fig. or G-CSF (Fig. 4). Leptin inhibits the mitochondrial release of cytochrome c and Smac Mitochondria have been implicated in the regulation of neutrophil apoptosis (3–5, 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 immu- nostaining and microscopic analysis. Both cytochrome c and Smac 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 FIGURE 5. Leptin blocks enzymatic caspase-3-like activity in cultured anti-CD95 mAb-treated neutrophils. The filters were reprobed with an anti- neutrophils. Leptin (0.5 M) and G-CSF (25 ng/ml) had equal efficacy. In GAPDH mAb to ensure equal loading of the gels. Results of 6- and 9-h contrast, Fas receptor activation resulted in an increase of caspase-3-like cultures are shown. The immunoblots are representative of three indepen- activity compared with cells cultured in medium. Results of 25-h cultures dent experiments. (n 3) are shown. , p 0.05.
  6. 6. 8094 LEPTIN IS A NEUTROPHIL SURVIVAL FACTOR were present in mitochondria of freshly purified neutrophils, as a subgroup of cells. Both leptin and G-CSF preserved the punctate indicated by the colocalization with the mitochondrial marker pro- pattern in the majority of the cells, whereas anti-Fas treatment tein Cox1 (23). In these cells, a punctate staining pattern was ob- revealed a diffuse pattern of both cytochrome c and Smac in almost served (Fig. 6A). Culturing of neutrophils for 13 h demonstrated all cells. No difference was observed when cytochrome c and Smac evidence for cytochrome c and Smac release ( diffuse pattern) in stainings were compared. A statistical analysis of these experi- ments is given in Fig. 6B. Leptin and G-CSF had the same efficacy to block the transition into a diffuse cytosolic staining pattern as- sociated with neutrophil apoptosis. 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 prein- cubated 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 par- tially blocked by each inhibitor, but leptin was still able to signif- icantly increase neutrophil viability under these conditions. How- ever, 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 Downloaded from www.jimmunol.org on October 19, 2010 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. Discussion 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 func- tional is a matter of debate. For instance, the effect of leptin on CD11b expression of neutrophils appeared to be indirect via monocyte-derived TNF- (22). In contrast, it was reported that FIGURE 6. Leptin prevents the mitochondrial release of cytochrome c FIGURE 7. Pharmacological inhibition of p38 MAPK and PI3K blocks and Smac. Freshly isolated neutrophils and neutrophils from 13-h cultures antideath effects of leptin in cultured neutrophils. Neutrophils were cul- were investigated by confocal microscopy. A, Cytochrome c and Smac tured in the presence (f) and absence ( ) of leptin (0.5 M) for 24 h (n colocalized with each other and with Cox1, demonstrating that they are 3). The indicated inhibitors were used at 25 M and preincubated for 30 mitochondrial proteins in freshly isolated neutrophils. Leptin preserved the min before leptin stimulation. Both inhibitors accelerated spontaneous neu- punctate pattern in cultured neutrophils, whereas we observed diffuse stain- trophil death, but did only partially prevent the leptin-mediated antideath ing in the absence of cytokine support or after anti-Fas mAb treatment. No effect. Only combined treatment with SB 203580 and LY 294002 abolished detectable staining was observed using control Abs (data not shown). Re- the effect of leptin in this system. Same results were observed when the sults are representative of three independent experiments. B, Quantitative inhibitors were used at 50 M and if neutrophils were cultured for 48 h and statistical analysis of the experiments shown in A. , p 0.05. (data not shown). , p 0.05.
  7. 7. The Journal of Immunology 8095 reactive oxygen production is the consequence of direct stimula- the high concentrations of leptin that are required for the antiapop- tion of neutrophils by leptin (25) and that leptin could induce the totic action on neutrophils in vitro occur under in vivo conditions. phagocytic activity in leptin-deficient purified mouse neutrophils Leptin serum levels up to 400 ng/ml were reported in children with (26). In this study, we investigated apoptotic pathways in neutro- chronic renal failure (40). In addition, in obese subjects treated phils after stimulation with leptin. The following new findings are with leptin, serum levels of 700 ng/ml were measured (41). Even reported: 1) Leptin receptors are expressed on neutrophils under in higher leptin serum levels might have been reached (based on our vivo conditions. 2) Leptin activates directly neutrophils and delays own calculations 0.1 M) when mice were treated with 2 g/g spontaneous apoptosis of these cells. 3) Leptin appears to exert its body weight leptin to improve wound healing (42). These levels antiapoptotic function by inhibiting proapoptotic events proximal are in the range in which significant antiapoptotic effects on neu- to mitochondria. 4) The antiapoptotic function of leptin is medi- trophils are observed under in vitro conditions. Moreover, it should ated via PI3K and p38 MAPK signaling pathways. be noted that recombinant leptin, which has been used in this Our study adds additional information on the potential role of study, is known to have a lower potency than native leptin, perhaps leptin in regulating immune responses, which are often associated due to differences in glycosylation (43). In addition, it is possible with elevated levels of leptin. Besides sepsis (10, 11), leptin has that higher leptin concentrations are present in inflamed tissues also been implicated in the pathogenesis of autoimmune diseases. that contain leptin-producing adipocytes. Therefore, leptin may in- For instance, in a mouse model of multiple sclerosis, it was found deed represent a neutrophil survival factor at sites of inflammation, that leptin levels were particularly high before and at the start of but probably not in the circulation. clinical disease (27). In contrast, leptin-deficient mice did not de- Besides neutrophils, leptin has been shown to exhibit antiapop- velop this disease (28), and starvation, which reduced leptin levels, totic activity in T cells (12), monocytes (13), and neuroblastoma inhibited the progression of clinical symptoms (27). Neutrophils cells (44). In contrast, leptin has been reported to induce apoptosis play an important pathogenic role in both infectious and autoim- in human bone marrow stromal cells (45). The reason for these mune diseases. Therefore, the question as to whether leptin acti- contrasting responses in different cell types is unclear, but differ- Downloaded from www.jimmunol.org on October 19, 2010 vates neutrophils, and how the functional response(s) of such an ences in the expression patterns of leptin receptors and associated interaction might be, seems to be of great interest. Because leptin signaling molecules may play a role. Despite these uncertainties, it delayed spontaneous neutrophil apoptosis, it is possible that leptin seems that leptin, like other hormones, regulates apoptosis, and contributes to neutrophil accumulation at inflammatory sites (2), that cells of the immune system are targets of leptin. Further work and, perhaps, it may then also stimulate the release of proinflam- is required to understand how the control of apoptosis by leptin matory mediators from these cells (1). influences innate and/or adaptive immunity. We confirm in this study previously published work, demon- strating that neutrophils express the short form of leptin receptors Acknowledgments (22). Both forms have identical extracellular and transmembrane We thank E. Kozlowski (Department of Pharmacology, University of domains, but differ in their intracellular domains (7). Although Bern) for excellent technical assistance, as well as Drs. D. Simon (Depart- initial studies suggested that the short form is unable to deliver ment of Dermatology, University of Bern) and M. Neef (Department of signals into the cells (7), it is now clear that this is not the case. Clinical Pharmacology, University of Bern) for providing blood samples. Despite lacking the STAT3 docking site, the short leptin receptor is still able to bind and activate Jak2, which subsequently activates Disclosures the MAPK pathway (29). The data reported in this manuscript The authors have no financial conflict of interest. suggest that besides the MAPK pathway, PI3K is also activated in leptin-stimulated neutrophils. Both MAPK (30, 31) and PI3K (32, References 33) activation have previously been shown to mediate neutrophil 1. Simon, H.-U. 2003. Neutrophil apoptosis pathways and their modifications in inflammation. Immunol. Rev. 193: 101–110. antiapoptosis. 2. Dibbert, B., M. Weber, W. H. Nikolaizik, P. Vogt, M. H. Schoni, K. Blaser, and ¨ The leptin-initiated signaling cascades somehow block sponta- H.-U. Simon. 1999. Cytokine-mediated Bax deficiency and consequent delayed neous neutrophil apoptosis before mitochondria release their pro- neutrophil apoptosis: a general mechanism to accumulate effector cells in inflam- mation. Proc. Natl. Acad. Sci. USA 96: 13330 –13335. apoptotic factors. This is indicated by the observation that leptin 3. Baumann, R., C. Casaulta, D. Simon, S. Conus, S. Yousefi, and H.-U. Simon. delays cleavage of caspase-8, Bid, and Bax. Cleaved Bid (34, 35) 2003. Macrophage migration inhibitory factor delays apoptosis in neutrophils by and Bax (36) have previously been shown to be highly efficient in inhibiting the mitochondria-dependent death pathway. FASEB J. 17: 2221–2230. 4. Maianski, N. A., F. P. J. Mul, J. D. van Buul, D. Roos, and T. W. Kuijpers. 2002. proapoptotic mitochondrial triggering. The delay of Bid and Bax Granulocyte colony-stimulating factor inhibits the mitochondria-dependent acti- cleavage by leptin was associated with inhibition of the release of vation of caspase-3 in neutrophils. Blood 99: 672– 679. 5. Moulding, D. A., C. Akgul, M. Derouet, M. R. H. White, and S. W. Edwards. cytochrome c into the cytosol. Cytochrome c forms a complex with 2001. BCL-2 family expression in human neutrophils during delayed and accel- Apaf-1 and caspase-9 that, in the presence of dATP, leads to the erated apoptosis. J. Leukocyte Biol. 70: 783–792. activation of caspase-3 (37). Smac is another proapoptotic factor 6. Faggioni, R., K. R. Feingold, and C. Grunfeld. 2001. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J. 15: released from mitochondria that inactivates members of the inhib- 2565–2571. itors of apoptosis protein family (38, 39). Leptin and G-CSF in- 7. Lee, G. H., R. Proenca, J. M. Montez, K. M. Carroll, J. G. 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