after release from the bone marrow , mature neutrophils ( polymorphonuclear neutrophils ( pmns ) ) are either rapidly recruited to sites of inflammation , where they represent the immediate defense against bacterial and fungal microorganisms [ 1 , 2 ] , or undergo senescense within hours , which results in apoptosis and rapid clearance from the circulation . without efficient removal of aged neutrophils , the release of reactive oxygen species , proteases , and other cytotoxic enzymes from the granules of pmn during uncontrolled apoptosis and degradation would result in tissue damage . pmns that enter inflammatory sites have a prolonged life - span that allows efficient phagocytosis of bacteria before they become apoptotic and are ultimately eliminated by tissue macrophages . by which mechanism senescent neutrophils are cleared from the circulation in vivo is not completely understood . it is believed that aged pmns acquire functional defects   and are subsequently recognized and phagocytosed by macrophages . however , for a rapid and efficient clearance , additional mechanisms must exist to selectively direct senescent neutrophils from the bloodstream into the tissue where marcophages reside , which is reminiscent of lymphocyte or hematopoietic stem cell homing . the chemokine receptor cxcr4 and its ligand cxcl12 ( stromal cell - derived factor-1 , ( sdf-1 ) ) have emerged as major factors regulating bone marrow homing of circulating hematopoietic stem cells , either mobilized endogenously after cytotoxic damage or infused therapeutically during stem cell transplantation . in both instances , stem cell homing occurs rapidly within hours , which implies that a similar chemokine receptor - based homing - like mechanism could also contribute to neutrophil clearance from the circulation . however , in contrast to hematopoietic stem cells that intrinsingly express high levels of cxcr4 , the expression level of this chemokine receptor is low in neutrophils . indeed , differential regulation of cxcr4 and of the interleukin-8 ( il-8 ) receptor cxcr2 appears to control the release of murine neutrophils and their return to the bone marrow after senescence , as aging is associated with upregulation of cxcr4 and loss of cxcr2 on the cell surface . these and other studies also showed that senescence of neutrophils , a process which is almost solely dependent on time and temperature , takes place not only in vivo , but also during culturing without stimulatory cytokines in vitro .   however , regulatory mechanisms observed in murine neutrophils that involve il-8-mediated effects may be different in the human system . for instance , while human neutrophils express two il-8 receptors ( cxcr1 and cxcr2 ) ,   only the cxcr2 homologue is found in mice . in contrast , other in vitro and in vivo studies rather suggested that downregulation of cxcr4 occured during maturation of neutrophils , which was further augmented by activation [ 1315 ] . however , neutrophils recovered from sites of inflammation as analyzed by suratt et al . [ 13 , 14 ] are different from aged pmn in the bloodstream . thus , the idea that upregulation of cxcr4 is a key mechanism involved in the clearance of aged neutrophils is not generally accepted . we therefore analyzed cell surface and mrna expression as well as function of cxcr4 in human granulocytes undergoing aging in vitro . our results support the concept that upregulation of cxcr4 in senescent granulocytes is an active process , which results in increased responsiveness to sdf-1 , and supports their clearance from the circulation in a homing - like fashion . the myeloid cell lines kg1a and hl-60 were cultured in imdm or rpmi 1640 medium ( seromed - biochrom ) supplemented with 10% fcs . peripheral blood ( pb ) was obtained from normal volunteers after informed consent according to the ethical committee of the university of tbingen ( project no . isolation of mononuclear cells ( mncs ) and polymorphonuclear neutrophils ( pmns ) was performed using dextran sedimentation , ficoll density centrifugation , and nh4cl lysis as described previously . pmns ( purity > 98% ) were resuspended in serum - free medium ( x - vivo 20 , biowhittaker , walkersville , md , usa ) , and incubated at 37c/5% co2 in 6-well plates for up to 18 hours to allow aging . in some experiments , stimulatory cytokines were added : rhg - csf ( recombinant human granulocyte - colony - stimulating factor ) at 100 ng / ml ( amgen , thousand oaks , calif , usa ) , rhtnf - alpha ( recombinant human tumor - necrosis factor - alpha ) at 20 ng / ml , il-1-beta ( recombinant human interleukin-1-beta ) at 20 ng / ml , and rhil-8 ( recombinant human interleukin-8 ) , at 100 ng / ml ( peprotech inc . , new york , ny , usa ) . following incubation all cells were removed from the flask ( adherent cells were removed using 0.2 g / l edta in balanced saline ) and used for flow cytometric analysis . as a control , granulocytes were kept at 4c . expression of cxcr1 , cxcr2 , and cxcr4 on pmn was analyzed by flow cytometry as described previously . the mean fluorescence intensity was calculated from the fluorescence histograms and expressed in arbitrary units . total rna was isolated using rneasy mini kit ( qiagen , hilden , germany ) , and 1 - 2 g were separated in a 1% agarose formaldehyde gel and transferred onto positively charged nylon membranes by capillary transfer . a 484 bp dna probe for detection of cxcr4 mrna dig - labeling of the pcr - products and the colorimetric detection was performed using the  dig dna labeling and detection kit  ( boehringer mannheim , mannheim , germany ) . freshly isolated pmns were cultured in x - vivo20 medium for 18 hours at 37c or kept at 4c , respectively . after stimulation with 100 ng / ml sdf-1 for the indicated periods of time at 37c , whole cell lysates were prepared by resuspending the pelleted cells in buffer containing 50 mm hepes ( ph 7.5 ) , 10% glycerol , 1% triton x-100 , 1.5 mm mgcl2 , 150 mm nacl , protease inhibitors ( complete mini ; roche , mannheim , germany ) and the phosphatase inhibitors naf ( 100 mm ) , na4p2o7 ( 10 mm ) and activated na3vo4 ( 1 mm ; all from sigma ) . equal amounts of protein were separated on a 10% sds - polyacrylamide gel , and transferred onto nitrocellulose membrane ( schleicher & schuell , dassel , germany ) . the blots were probed with phospho - specific polyclonal antibodies against p44/42 map kinase ( thr202/tyr204 ) , or with the control antibody against the nonphosphorylated form ( cell signaling techn . bands were visualized by ecl staining ( amersham biosciences , freiburg , germany ) and analyzed by densitometry using the scion image for windows software ( scion corporation ; frederick , md , usa ) . the value obtained from the phospho - mapk band was divided by the value of the corresponding total mapk band for each sample ( relative phospho - mapk intensity ) . for all experiments , identical reagents , concentrations and exposure times were used . in vitro analysis of migration across microvascular endothelium was performed as described previously , using the human endothelial cell line bmec-1 grown on 3 m transwell microporous membranes ( transwell , corning - costar , bodenheim , germany ) . rhsdf-1alpha ( recombinant human stromal cell - derived factor-1 , r&d systems , wiesbaden , germany ) and rhil-8 ( recombinant human interleukin-8 , peprotech inc . , new york , ny , usa ) were added to the lower chamber ( underneath the transwell inserts ) at 100 ng / ml . blocking cxcr4 mab ( clone 12g5 , pharmingen ) was added to the cells in the upper chamber at a final concentration of 10 g / ml . results are expressed as the mean    standard error of the mean ( sem ) from at least 3 independent experiments . differences between groups were compared by student 's t - test ( e.g. , chemokine - induced migration of freshly isolated pmn versus control ) or , where applicable ( chemokine receptor expression 0 hours versus 18 hours and migration of aged neutrophils versus cells kept at 4c ) , paired t - test , and considered significant at p  .05 . by flow cytometry , cell surface expression of cxcr4 on human pmn was very weak immediately after isolation from peripheral blood as shown in   figures 1(a ) and 1(d ) . aging for up to 18 hours in vitro ( 37c/5% co2 ) resulted in a significant upregulation of cxcr4 on the cell surface , as reflected by a shift of the mean fluorescence of at least one log step . increased cxcr4 expression was detected starting from 3 hours and reached maximal levels after 1218 hours . as an additional control , pmns from the same blood sample were kept at 4c to inhibit aging . in these cells , an increase in cxcr4 expression was not detected , indicating that upregulation of cxcr4 with age is an active process depending on an intact cell metabolism ( figures 1(a ) and 1(d ) ) . we also incubated freshly isolated human pmn with a panel of stimulatory cytokines for 18 hours . addition of g - csf , il-1 , tnf , or il-8 to the cells did not influence the observed increase of cxcr4 on the cell surface ( figures 1(b ) and 1(d ) ) . as il-8/cxcr2 were proposed to be involved in murine neutrophil homeostasis , we included expression analysis of the two human cxc receptors recognizing il-8 ( cxcr1 and cxcr2 ) in our experiments . in contrast to cxcr4 , expression of both receptors was relatively high in freshly isolated cells with unchanged cxcr1 expression during aging for 18 hours , whereas cxcr2 was even significantly reduced on the cell surface , as shown in figures 1(c ) and 1(d ) . as changes of the cell surface expression of chemokine receptors may depend on posttranslational effects such as receptor internalisation and recycling , we used northern blot for quantitative mrna analysis and could demonstrate increased cxcr4 mrna expression in pmn aged in vitro ( 18 hours ) compared to freshly isolated cells , as shown in figure 1(e ) . these results indicate that both increased mrna transcription and consecutive protein synthesis contribute to upregulation of cxcr4 expression on the cell surface in pmn undergoing senescence rather than only re - expression of stored and/or recycled cxcr4 receptor . as shown in figure 2(a ) , transendothelial migration of freshly isolated pmn toward sdf-1 in vitro was approximately twofold increased compared to spontaneous migration and statistically significant . the migratory effect of sdf-1 was completely blocked by the cxcr4 monoclonal antibody 12g5 , indicating that migration toward sdf-1 was exclusively mediated by cxcr4 . in accordance with the observed high expression of cxcr1 and cxcr2 in freshly prepared pmn , migration toward il-8 furthermore , we were interested to know if the observed changes in cxcr4 and cxcr2 surface expression during aging ( figure 1(d ) ) were reflected in functional tests . indeed , migration toward sdf-1 was much more pronounced in aged pmn ( about tenfold increase compared to spontaneous migration , figure 2(b ) ) than in freshly isolated pmn ( twofold increase , figure 2(a ) ) . sdf-1-induced migration of aged pmn was reduced to the baseline level in the presence of the blocking cxcr4 antibody . in contrast to pmn undergoing senescence , sdf-1-induced migration of cells kept at 4c for the same time period was only marginally increased ( figure 2(b ) ) . however , the migratory response of aged pmn to il-8 was significantly reduced after aging compared to cells kept at 4c , but still considerably greater than spontanous migration . indeed , il-8-induced transendothelial migration of aged neutrophils in vitro was even slightly smaller than that induced by sdf-1 . without stimulation , only weak basal levels of phosphorylated p44/42 map - kinase / erk1/2 were detected in aged pmn and cells kept at 4c by western blot analysis ( figure 2(c ) ) . however , in both conditions , strong phosphorylation was observed within one minute after stimulation with 100 ng / ml sdf-1 . phosphorylation of cells kept at 4c was transient and returned to the baseline level after 2.5 minutes . the response was significantly stronger ( figure 2(d ) ) and somewhat more sustained ( still detectable at 2.5 minutes ) in senescent neutrophils as compared to cells which were kept at 4c to prevent aging . in this study , we observed basal levels of the chemokine receptor cxcr4 on the cell surface of native human peripheral blood neutrophils as well as basal mrna expression , which were associated with a weak , but significant migratory response to sdf-1 . this is in contrast to previous studies that could not detect cxcr4 in pmn , neither any effect of sdf-1 ,   but may depend on the sensitivity of the methods employed . after aging in vitro , both an increased mrna and surface expression of cxcr4 were found , which was associated with more efficient migration across bone marrow endothelium and increased phosphorylation of p44/42 map kinase ( erk1/2 ) in response to sdf-1 . in postmitotic pmn , erk1/2 signaling is not related to cell proliferation , but rather plays a   role in the regulation chemotaxis   [ 19 , 20 ] . this is supported by our finding that the map kinase phosphorylation in response to sdf-1 was only transient and returned to baseline levels after a few minutes . the effects observed were not due to selection of a subpopulation with increased cxcr4 expression during culturing in vitro , as neutrophils with a similar high cxcr4 level were virtually absent at the beginning of the culture period . in contrast to a previous study by martin et al .   who analyzed trafficking of aged pmn in vivo in a mouse model , our results indicate that upregulation of cxcr4 during aging in human pmn is not solely due to translocation of intracellular cxcr4 to the cell surface , but also to new receptor synthesis . previously it has been thought that senescence of neutrophils is associated with a general loss of cellular functions [ 6 , 21 ] . however , our results confirm the notion that human pmn undergoing senescence can upregulate specific mrna transcription and even acquire functions such as sdf-1/cxcr4-mediated chemotaxis and signaling . the effect of sdf-1 was specifically mediated by cxcr4 , since it was abrogated by a functionally blocking cxcr4 antibody . aging of pmn is associated with controlled apoptosis , as suggested by weinmann et al . . according to the results of this study , factors that mediate neutrophil migration such as adhesion molecules are also involved in the regulation of apoptosis . it is therefore conceivable that increased expression of cxcr4 in senescent pmn represents a further event in the regulatory network that controls their apoptosis and neutrophil clearance from the circulation . taken together , our results demonstrate that during aging , human pmns acquire a phenotype that supports migration to areas of high sdf-1 concentration . although particularly bone marrow homing of both hematopoietic stem cells   and murine granulocytes   has been shown to depend on sdf-1/cxcr4 , production of sdf-1 is by no means confined to the bone marrow . for instance , in acute lymphoblastic leukemia , high expression of cxcr4 generally correlates with the ability of the leukemic cells to infiltrate extramedullary sites . therefore , sdf-1/cxcr4 could also be involved in pmn sequestration in organs such as the liver and spleen . other mechanisms like adhesion molecules may additionally determine to which extent the clearance of senescent pmn takes place in the liver , spleen , or bone marrow . indeed , the adhesion molecule p - selectin expressed in hepatic sinusoids supported phagocytosis of pmn by kupffer cells in an animal model of inflammation caused by endotoxinemia . interestingly , vascular selectins are also involved in hematopoietic stem cell homing , which is not solely dependent on sdf-1/cxcr4 . thus , increased expression and function of cxcr4 is only one of many factors that ensure targeted direction of senecent pmn to their individual site of sequestration . in the liver and in the spleen , macrophages appear to play a more direct role in the clearance of neutrophils . this might be explained by the specific microenvironment , particularly by the open microcirculation of the spleen . in the bone marrow , transendothelial migration and extravasation must have occurred before pmn are phagocytosed by macrophages in the tissue . sdf-1 not only may be important as a chemoattractant , but also may directly contribute to the apoptosis of senescent granulocytes , as suggested by lum et al . . in this study , engagement of cxcr4 in granulocytes resulted in the increase of tnf - related apoptosis - inducing ligand ( trail ) and trail receptors , which eventually supported apoptosis of these cells . from this study , it can be concluded that once senescent pmns have reached their site of sequestration , high concentration of sdf-1 released by stromal cells may support the process of apoptosis , which then allows nearby macrophages to immediately phagocytose these cells . high initial expression of the il-8 receptor cxcr2 followed by downmodulation during aging also resembles observations in mice , and may indicate that cxcr2 is similarly involved in the release of human neutrophils from the bone marrow . furthermore , cxcr2 is mainly responsible for the il-8-induced inflammatory neutrophil attachment and chemotaxis , , suggesting that the loss of cxcr2 in circulating senescent neutophils also results in impaired recruitment to sites of inflammation . in contrast , expression of cxcr1 remained unchanged which may be important for neutrophils after extravasation . this idea is supported by the previous suggestion that cxcr1 expressed on human neutrophils may require higher concentrations of il-8 for chemotaxis and , therefore , may be involved in neutrophil activities closer to the site of injury and not in the early attachment and extravasation events . in addition , also sdf-1 is produced at sites of tissue damage , which is mediated by upregulation of the hypoxia - inducible factor-1 ( hif-1 ) . high expression of cxcr4 could therefore contribute to the attraction of pmn to inflammatory areas after extravasation , even when the process of aging has already been initiated . based on our findings and the previous study in mice , we propose a model of cxc chemokine receptor modulation during aging ( figure 3 ) that allows circulating , aged granulocytes to home to the bone marrow ( or other sites of sequestration ) , dependent on cxcr4 and its ligand sdf-1 , while after extravasation cxcr1 and cxcr4 sustain attraction of neutrophils to areas of inflammation and tissue damage , where il-8 and sdf-1 are released .