neutrophils play an important role as an effector of inflammation , tissue injury , and host defense against microbial infection .
the lifetime of neutrophils , terminally differentiated blood cells , is relatively short , and they constitutively undergo apoptosis .
apoptotic neutrophils are phagocytosed by macrophages without release of proinflammatory mediators , leading to the limitation of tissue injury and resolution of inflammatory process [ 24 ] . in this context
, it is interesting to note that spontaneous apoptosis of neutrophils is inhibited in patients with sepsis , systemic inflammatory syndrome ( sirs ) , and acute respiratory distress syndrome ( ards ) by the action of various pathogen- and host - derived substances , such as bacterial products ( i.e. , gram - negative lipopolysaccharide ; lps ) , cytokines , and chemokines ( i.e. , il-1 and il-8 ) [ 3 , 58 ]
. the suppressed neutrophil apoptosis results in the prolongation of their life span and causes the uncontrolled release of cytotoxic metabolites and proinflammatory substances ( i.e. , reactive oxygen species and proteases ) , which leads to the amplification of systemic inflammation , tissue injury , and organ failure observed in those disorders [ 9 , 10 ] .
in contrast , neutrophil apoptosis can be accelerated by fas ligand , reactive oxygen species , immune complexes , and bacterial toxins ( such as pseudomonas aeruginosa exotoxin , pyocyanin ) produced at the sites of inflammation and infection [ 1114 ] .
inappropriate induction of neutrophil apoptosis is likely to deplete neutrophil numbers and functions , thereby impairing host defense and favoring bacterial invasion and persistence .
mammalian cells express a number of antimicrobial peptides ( host defense peptides ) that function as effector components in the innate host defense system .
they are found in blood , secretions , epithelial tissues as well as in neutrophil granules and exhibit potent antimicrobial activities against a broad spectrum of invading microorganisms , including both gram - positive and gram - negative bacteria , fungi , and viruses [ 1620 ] . among these peptides , defensins and cathelicidin
are considered as the two major classes of antimicrobial peptides in humans [ 1620 ] .
defensins are characterized by the six - cysteine residues forming three intramolecular disulfide bridges and are divided into - and -defensins based on the distribution of cysteines and the linkages of disulfide bonding [ 16 , 17 , 19 , 20 ] .
human -defensins are found in neutrophils and the paneth cells of the small intestine , whereas human -defensins ( hbds ) are mainly expressed by epithelial tissues . in contrast , cathelicidins are a family of antimicrobial peptides , characterized by the highly conserved cathelin - like prosequence and variable c - terminal sequences that correspond to the mature antibacterial peptides .
about 30 cathelicidin members have been identified from various mammalian species ; however , only one cathelicidin , human cationic antibacterial protein of 18  kda ( hcap18 ) has been found in humans , and its c - terminal mature antimicrobial peptide , called ll-37 , which comprises 37 aminoacid residues ( llgdffrkskekigkefkrivqrikdflrnlvprtes ) , has been identified [ 21 , 22 ] . to date , six different human -defensin molecules have been described [ 16 , 17 , 19 , 20 ] .
human -defensin-1 , -2 , -3 , and -4 are also termed as human neutrophil peptide ( hnp)-1 , -2 , -3 , and -4 , as they are mainly expressed in neutrophils .
hnp-1 , -2 and -3 , which differ only in the first aminoacid , account for 57% of total neutrophil proteins , whereas hnp-4 , with an aminoacid sequence distinct from other hnp sequences , comprises < 1% of total defensins in neutrophils . since no gene that encodes hnp-2 has been identified , it is regarded as a proteolytic product of hnp-1 and/or hnp-3 .
the other two human -defensins , human defensin ( hd)-5 and hd-6 , are constitutively expressed in the paneth cells within the epithelium in the small intestine .
similarly , six hbds ( hbd-1 to -6 ) have been identified in human tissues .
hbd-1 is constitutively expressed in various epithelial tissues , including urogenital and respiratory tracts [ 26 , 27 ] .
hbd-2 , which was originally isolated from lesional scale of psoriatic skin , is mainly found in skin and respiratory as well as gastrointestinal tracts and is upregulated by various stimuli ( e.g. , lps and cytokines ) [ 28 , 29 ] . the third -defensin , hbd-3 , which was also isolated from human lesional psoriatic scale , is detected in both epithelial and nonepithelial tissues , and its expression is inducible upon stimulation with bacteria and cytokines [ 30 , 31 ] .
hbd-4 , which was initially identified by screening of human genomic sequences , is upregulated in epithelial cells by stimulation with bacteria and cytokines [ 32 , 33 ] .
however , the antimicrobial and other cellular activities have not yet been determined for hbd-5 and hbd-6 , the newly discovered hbds based on the human genome database .
in addition to their antimicrobial properties , hnps , hbds , and ll-37 have the potential to stimulate various host cell types to induce cytokine and chemokine production as well as to chemoattract immune and inflammatory cells [ 19 , 35 ] . in this context ,
hnps , hbds , and ll-37 possess the ability to chemoattract neutrophils , monocyte , t cells , and immature dendritic cells [ 3639 ] . given that hnps , hbds , and ll-37
are multifunctional molecules as host defense peptides and act on neutrophils as chemoattractants [ 36 , 38 , 39 ] , we hypothesized that they may have a potential to modulate the lifetime ( apoptosis ) of neutrophils .
here , we provided the evidence that hnp-1 , hbd-3 , and ll-37 can potently suppress neutrophil apoptosis , possibly via the actions on the distinct receptors ; the p2y6 nucleotide receptor , the cc chemokine receptor ( ccr ) 6 , and the low - affinity formyl - peptide receptor , formyl - peptide receptor - like 1 ( fprl1)/the nucleotide receptor p2x7 , respectively [ 2325 ] .
before looking at the actions of ll-37 , we determined the spontaneous apoptosis of neutrophils .
when neutrophils were incubated alone for 18  h at 37c , they exhibited characteristic features of apoptosis , such as chromatin condensation , formation of rounded nuclear profiles , cell shrinking , and presence of cytoplasmic vacuolization ( figure 1(b ) ) , compared to resting cells incubated for 18  h at 4c ( figure 1(a ) ) .
alternatively , neutrophil apoptosis was evaluated by flow cytometry using fitc - annexin v and propidium iodide staining .
incubation of neutrophils alone for 18  h at 37c substantially induced apoptosis defined as annexin v positive but propidium iodide negative ( figure 1(e ) ) , compared to resting cells incubated for 18  h at 4c ( figure 1(d ) ) .
evaluation of neutrophil apoptosis based on the morphological changes revealed that > 50% of neutrophils underwent apoptosis after incubation alone for 18  h ( figure 2(a ) ) .
interestingly , spontaneous apoptosis of neutrophils was inhibited by incubation with ll-37 ( figures 1(c ) , 1(f ) , and 2(a ) ) .
next , we evaluated the activation of caspase 3 , a key executor for apoptosis .
consistent with the changes in the number of apoptotic cells , caspase 3 activity was increased after 18  h of incubation ( figure 2(b ) ) , and the activity was reduced by lps - stimulation . of importance , ll-37 dose dependently suppressed the activation of caspase 3 . to clarify the mechanism for the action of ll-37 , we investigated the signaling molecules by western blot analysis .
first , we looked at the effect of ll-37 on the phosphorylation of erk , a member of mitogen - activated kinase family .
ll-37 ( 1  g / ml ) stimulation strikingly enhanced the phosphorylation of erk-1/-2 ( data not shown ) .
further , we evaluated the effect of ll-37 on the expression of bcl - xl , an antiapoptotic protein .
ll-37 ( 1  g / ml ) markedly induced the expression of bcl - xl ( data not shown ) .
it has been demonstrated that ll-37 uses fprl1 as a receptor to chemoattract neutrophils , monocytes and t cells .
in addition , ll-37 is reported to promote the processing and release of il-1 from monocytes via the activation of p2x7 receptor .
thus , we determined the involvement of fprl1 and p2x7 in the ll-37-induced suppression of neutrophil apoptosis by using fprl1 antagonist and p2x7 inhibitors . as shown in figure 3 ,
antagonistic agents for fprl1 ( wrw , trp - arg - trp - trp - trp - trp - conh2 )   and p2x7 ( oxidized atp and kn-93 ) [ 4345 ] significantly reversed the ll-37-induced suppression of neutrophil apoptosis . similarly , fprl1 antagonist ( wrw ) and p2x7 inhibitors ( oxidized atp and kn-93 ) obviously attenuated the ll-37-induced inhibition of caspase 3 activity ( data not shown ) .
these observations apparently suggest that fprl1 and p2x7 are involved in the ll-37-induced suppression of neutrophil apoptosis .
next , to further determine the involvement of fprl1 and p2x7 in the suppression of neutrophil apoptosis , neutrophils were directly incubated with the fprl1 and p2x7 agonists . as shown in figure 4 ,
agonistic agents for fprl1 ( wkymvm , trp - lys - tyr - met - val - d - met - conh2 and mmk-1 , lesifrsllfrvm ) , and p2x7 ( bz - atp , benzoylbenzoyl - atp ) [ 43 , 44 ] dose dependently suppressed neutrophil apoptosis .
notably , the combinations of fprl1- and p2x7-agonists cooperatively suppressed neutrophil apoptosis as well as the activation of caspase 3 ( data not shown ) . these observations
most likely indicate that the activation of fprl1 and p2x7 in concert acts to induce the suppression of neutrophil apoptosis .
interestingly , hbd-3 dose dependently inhibited the neutrophil apoptosis ( figure 5 ) . of note
, neither hbd-1 , hbd-2 nor hbd-4 significantly influenced neutrophil apoptosis at the concentrations examined .
similarly , hbd-3 but not hbd-1 , hbd-2 and hbd-4 significantly suppressed the activation of caspase 3 ( data not shown ) .
further , we evaluated the expression of apoptosis - associated proteins , truncated bid ( a proapoptotic protein ) and bcl - xl ( an antiapoptotic protein ) .
of note , consistent with its suppressive action on neutrophil apoptosis , hbd-3 downregulated truncated bid , whereas it upregulated bcl - xl ( figure 6 ) .
the dissipation of mitochondrial electrochemical potential gradient is known as an early event of apoptosis .
thus , we investigated the effect of hbd-3 on the mitochondrial membrane potential change in apoptotic neutrophils
. assessment of the mitochondrial membrane potential change with a cationic lipophilic dye jc-1   revealed that the percentage of cells with intact mitochondrial membrane potential ( enhanced red but diminished green fluorescent cells ) decreased , whereas that with disrupted mitochondrial membrane potential ( enhanced green but diminished red fluorescent cells ) increased after incubation at 37c for 18  h ( figure 7 ) .
importantly , hbd-3 suppressed the mitochondrial membrane potential change ; hbd-3 ( 1 ~ 10  g / ml ) dose dependently decreased neutrophil number with disrupted mitochondrial membrane potential , but increased that with intact mitochondrial membrane potential even after incubation at 37c for 18  h. although ccr6 is known as a specific receptor for ccl20/mip-3 , hbd-3 also utilizes ccr6 to stimulate chemotaxis for monocytes and ccr6-transfected human embryonic kidney ( hek ) 293 cells .
thus , we determined the participation of ccr6 in the hbd-3-induced suppression of neutrophil apoptosis .
when neutrophils were incubated with hbd-3 in the presence of anti - ccr6 mab or control igg , anti - ccr6 mab but not control igg significantly reversed the hbd-3-induced suppression of neutrophil apoptosis ( figure 8) . to further clarify the involvement of ccr6 in the suppression of neutrophil apoptosis
mip-3 ( 0.01 ~ 1  g / ml ) dose dependently suppressed neutrophil apoptosis , and mip-3-induced suppression of neutrophil apoptosis was obviously attenuated by anti - ccr6 mab but not control igg .
these observations suggest that ccr6 is involved in not only the mip-3-induced but also the hbd-3-induced suppression of neutrophil apoptosis .
interestingly , hnp-1 ( 5 ~ 40  g / ml ) dose dependently suppressed neutrophil apoptosis ( figure 9 ) .
however , hnp-2 , hnp-3 and hd-5 ( data not shown ) did not significantly influence the neutrophil apoptosis at the concentrations examined .
similarly , hnp-1 but not hnp-2 , hnp-3 , and hd-5 significantly suppressed the activation of caspase-3 ( data not shown ) .
moreover , consistent with its suppressive action on neutrophil apoptosis , hnp-1 downregulated truncated bid , whereas it upregulated bcl - xl ( data not shown ) .
importantly , we confirmed that hnp-1 suppressed the mitochondrial membrane potential change ; hnp-1 ( 40  g / ml ) increased neutrophil number with intact mitochondrial membrane potential , but decreased that with disrupted mitochondrial membrane potential even after incubation at 37c for 18  h ( data not shown ) . although p2y6 is known as a purinergic receptor for udp , it has been reported that hnps utilizes p2y6 signaling to stimulate human lung epithelial cells to produce il-8 .
thus , we determined the involvement of p2y6 in the hnp-1-induced suppression of neutrophil apoptosis . to determine the involvement of p2y6 in the suppression of neutrophil apoptosis , neutrophils were directly incubated with a p2y6 agonist udp , and its effect on neutrophil apoptosis was evaluated .
udp ( 30 ~ 3000  m ) dose dependently suppressed neutrophil apoptosis ( data not shown ) .
of importance , mrs2578 significantly reversed the hnp-1-induced as well as udp - induced suppression of neutrophil apoptosis ( figure 10 ) .
these observations suggest that p2y6 is involved in not only the udp - induced but also the hnp-1-induced suppression of neutrophil apoptosis .
we demonstrated that the human antimicrobial peptides , ll-37 , hbd-3 , and hnp-1 can suppress neutrophil apoptosis .
thus , we determined whether ll-37 , hbd-3 , and hnp-1 could cooperatively suppress the neutrophil apoptosis by incubating neutrophils with the combinations of ll-37 , hbd-3 , and hnp-1 .
as shown in figure 11 , the low concentrations of ll-37 ( 0.1  g / ml ) , hbd-3 ( 1  g / ml ) , and hnp-1 ( 10  g / ml ) slightly reduced the neutrophil apoptosis .
of note , the combinations of 2 peptides cooperatively decreased apoptosis , and the combination of 3 peptides further reduced apoptosis .
these observations clearly indicate that ll-37 , hbd-3 , and hnp-1 can act in concert on neutrophils to suppress the apoptosis .
host defense peptides ( - and -defensins ; cathelicidins ) , as the effectors in the innate host defense system , exhibit antimicrobial activities against a broad spectrum of microbes , including both gram - positive and gram - negative bacteria , fungi , and viruses .
in addition to their antimicrobial properties , hnps , hbds , and ll-37 have the potential to stimulate various host cell types to induce cytokine and chemokine production [ 19 , 35 ] .
furthermore , hnps , hbds , and ll-37 possess the ability to chemoattract neutrophils , monocyte , t cells , and immature dendritic cells [ 3639 ] . in this paper
, we demonstrated that hnp-1 , hbd-3 , and ll-37 can potently suppress neutrophil apoptosis among the peptides examined . during the process of apoptosis , truncated bid ( a proapoptotic protein )
is cleaved from bid and translocates to mitochondria to perturb the mitochondrial functions , thereby disrupting mitochondrial membrane potential and promoting cytochrome c release , which results in the activation of effector caspases and finally induces apoptosis [ 40 , 53 ] .
in contrast , bcl - xl acts as an antiapoptotic protein to preserve mitochondrial integrity , thereby suppressing the activation of caspase cascade and apoptosis [ 40 , 54 ] .
consistent with their antiapoptotic actions , hnp-1 , hbd-3 , and ll-37 downregulated truncated bid and upregulated bcl - xl .
furthermore , they inhibited the dissipation of mitochondrial membrane potential and activation of caspase 3 , one of the death proteases functioning as the central executioners of apoptosis .
it has been shown that the activation of erk , a member of mitogen - activated kinase family , generates the survival signals via the upregulation of antiapoptotic proteins of bcl-2 family ( such as bcl - xl ) to prolong the life span of cells [ 3 , 55 ] . since we confirmed that hnp-1 and hbd3 as well as ll-37 induced the phosphorylation of erk-1/-2 in neutrophils , it is feasible to assume that the stimulation of neutrophils with hnp-1 , hbd-3 , and ll-37 induces the phosphorylation of erk-1/-2 and the subsequent upregulation of antiapoptotic protein bcl - xl and downregulation of proapoptotic protein truncated bid , which inhibits the mitochondrial membrane potential change and caspase 3 activity , thereby suppressing neutrophil apoptosis ( figure 12 ) .
cationic antimicrobial peptides ( such as defensins and cathelicidins ) kill the invaded microorganisms by perturbing their membranes ; the action of those peptides is not receptor - mediated but involves a less specific interaction with microbial membrane components , since the peptides target cell surface anionic lipids such as phosphatidyl glycerol and cardiolipin that are abundant in microorganisms .
in contrast , the mammalian cell membrane is mainly composed of electrically neutral phospholipids such as phosphatidylcholine and sphingomyelin , for which the affinity of antimicrobial peptides is generally low .
recently , antimicrobial peptides have been shown to act on the cell surface receptors to modulate various host cell functions ( i.e. , proinflammatory mediator production ; immune and inflammatory cell activation ) .
for instance , hnps induce il-8 production from a549 lung epithelial cells via the action on p2y6 nucleotide receptor , and hbd-3 utilizes a chemokine receptor ccr6 to chemoattract monocytes and ccr6-transfected hek 293 cells .
moreover , ll-37 functions as a chemoattractant for neutrophils , monocytes , and t cells via the interactions with fprl1 , a low - affinity formyl - peptide receptor   and promotes the processing and release of il-1 from monocytes via the activation of p2x7 nucleotide receptor . using antagonistic and agonistic agents and a neutralizing antibody , we demonstrated that p2y6 , ccr6 , and fprl1/p2x7 are likely involved in the hnp-1- , hbd-3 , and ll-37-induced suppression of neutrophil apoptosis , respectively . to date , it is proposed that ll-37 and hbd-3 can directly bind to and activate fprl1 and ccr6 [ 24 , 46 ] . however , the exact mechanisms by which ll-37 and hnps signal through the p2y7 and p2y6 receptors remain to be elucidated [ 41 , 51 ] . the current study demonstrated that among -defensins , hnp-1 but not hnp-2 , hnp-3 and hd-5 suppresses the apoptosis of neutrophils via the p2y6 signaling . of interest , aminoacid sequences of hnp-1 ,
-2 , and -3 differ only in the n - terminal end , whereas hd-5 is distinct from other hnps .
thus , it can be postulated that the difference in the n - terminal sequences of hnp-1 ~ -3 can be recognized by the p2y6 signaling to induce the suppression of neutrophil apoptosis . supporting this sequence - specific actions of hnps
, it has been reported that hnp-1 most potently exhibits the chemotactic activity for monocytes among hnp-1 ~ -3 .
hnps are synthesized in neutrophils , whereas hbds are mainly produced in epithelial tissues , including respiratory and urogenital tracts and skin [ 16 , 17 , 19 , 20 ] .
furthermore , ll-37 is expressed in keratinocytes and lung epithelial cells as well as neutrophils [ 19 , 57 ] .
interestingly , the expression of these antimicrobial peptides is locally induced at the sites of inflammation and infection within epithelial cells , and invading neutrophils represent an additional source for the peptides [ 57 , 58 ] . in this context
, it has been reported that the concentrations of hnps , hbds , and ll-37 are increased up to 40  g / ml in bronchoalveolar and nasal fluids from patients with inflammation and infection [ 5860 ] .
importantly , we demonstrated that ll-37 , hbd-3 , and hnp-1 can suppress neutrophil apoptosis in vitro at the concentrations ( 0.01 ~ 40  g / ml ) comparable to those at the sites of inflammation and infection , and that ll-37 , hbd-3 , and hnp-1 can cooperatively suppress neutrophil apoptosis in vitro .
thus , it can be speculated that antimicrobial peptides ll-37 , hbd-3 , and hnp-1 , in concert modulate neutrophil apoptosis in vivo in the local milieu at the sites of inflammation or infection by utilizing different receptors ( e.g. , fprl1 , p2x7 , ccr6 , and p2y6 ) .
furthermore , hnps , and ll-37 are expected to exert their actions on neutrophils in a paracrine / autocrine fashion , as they are stored in the azurophil and specific granules of neutrophils , respectively , and extracellularly released from activated neutrophils [ 61 , 62 ] . in this context
, it should be noted that antiapoptotic genes are upregulated , and proapoptotic genes are downregulated in vivo in neutrophils that challenged with inflammatory / immunomodulatory molecules ( including ll-37 , hbds , and hnps ) , and transmigrated to the inflammatory skin lesions .
clinical studies have indicated that spontaneous neutrophil apoptosis is inhibited in patients with severe inflammation ( e.g. , sepsis , sirs and ards ) by the actions of various bacterial products , cytokines and chemokines , detected in these disorders [ 3 , 58 ] . activated neutrophils with prolonged survival cause the amplification of inflammation and tissue injury via the uncontrolled release of cytotoxic metabolites and proinflammatory substances [ 9 , 10 ] .
from this point of view , antimicrobial peptides ( ll-37 , hbd-3 , and hnp-1 ) may exert a harmful effect during inflammation by suppressing apoptosis and prolonging the lifespan ( survival ) of neutrophils , which could lead to the augmented inflammatory reactions .
in contrast , physiological process of neutrophil apoptosis can be subverted by bacterial pathogens during infections .
inappropriate or premature apoptosis of neutrophils could deplete cell numbers and functions , impairing host defense and favoring bacterial persistence in infections . in this context
, it has been reported that neutrophil apoptosis is accelerated and neutrophil - mediated host defense is impaired in vivo during infection with pseudomonas aeruginosa by the action of pyocyanin , a predominant phenazine exotoxin . considering their antiapoptotic action ,
antimicrobial peptides ( ll-37 , hbd-3 , and hnp-1 ) can exert an advantageous effect on host defense against bacterial infections by prolonging the lifespan of neutrophils , major phagocytes engaged in the killing of invading bacteria .
hnps , hbds , and ll-37 are originally identified as antimicrobial peptides , which participate in the innate immune system , capable of protecting host from invasive microbial infections [ 16 , 17 ] , and now regarded as multifunctional molecules that link the innate immune response to the adaptive immune system by exerting various immunomodulatory actions , such as cytokine and chemokine production , and immune and inflammatory cell migration [ 19 , 35 ] . in this paper
, we have demonstrated an additional function of hnps , hbds , and ll-37 to prolong the lifespan of neutrophils via the actions on the distinct receptors .
this finding provides a novel insight into the role of antimicrobial peptides in the regulation of neutrophil lifespan ( apoptosis ) as well as the innate and adaptive host defense systems .
 peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense . 
 human antimicrobial peptides , -defensins ( human neutrophil peptides , hnps ) , human -defensins ( hbds ) , and cathelicidin ( ll-37 ) not only exhibit potent bactericidal activities against gram - negative and gram - positive bacteria , but also function as immunomodulatory molecules by inducing cytokine and chemokine production , and inflammatory and immune cell activation . 
 neutrophil is a critical effector cell in host defense against microbial infection , and its lifespan is regulated by various pathogen- and host - derived substances . here 
 , we provided the evidence that hnp-1 , hbd-3 , and ll-37 can not only destroy bacteria but also potently modulate ( suppress ) neutrophil apoptosis , accompanied with the phosphorylation of erk-1/-2 , the downregulation of tbid ( an proapoptotic protein ) and upregulation of bcl - xl ( an antiapoptotic protein ) , and the inhibition of mitochondrial membrane potential change and caspase 3 activity , possibly via the actions on the distinct receptors , the p2y6 nucleotide receptor , the chemokine receptor ccr6 , and the low - affinity formyl - peptide receptor fprl1/the nucleotide receptor p2x7 , respectively . 
 suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion . 
1. Introduction
2. Modulation of Neutrophil Apoptosis by LL-37
3. Modulation of Neutrophil Apoptosis by hBDs
4. Modulation of Neutrophil Apoptosis by 
5. Perspectives