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).
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.
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.
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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
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.
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
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
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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.
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.
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-
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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
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