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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-06-1734.
HEMATOPOIESIS
From the Hematology Branch, National Heart, Lung and
Blood Institute, National Institutes of Health, Bethesda, MD.
There is evidence that neutrophil production is a balance between
the proliferative action of granulocyte-colony-stimulating factor
(G-CSF) and a negative feedback from mature neutrophils (the chalone).
Two neutrophil serine proteases have been implicated in granulopoietic
regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth, and elastase mutations cause cyclic and congenital neutropenia. We further studied the action
of the neutrophil serine proteases (proteinase 3, elastase, azurocidin,
and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM
in methylcellulose culture. In serum-free suspension cultures of
CD34+ cells, elastase completely abrogated the
proliferation induced by G-CSF but not that of GM-CSF or stem cell
factor (SCF). The blocking effect of elastase was prevented by
inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride
(PMSF) or heat treatment. When exposed to enzymatically active
elastase, G-CSF, but not GM-CSF or SCF, was rapidly cleaved and
rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF.
(Blood. 2003;101:1752-1758) The generation of neutrophils from pluripotent
progenitors is regulated primarily by granulocyte
macrophage-colony-stimulating factor (GM-CSF) and
granulocyte-colony-stimulating factor (G-CSF).1 However,
at the stage of committed progenitor of neutrophils and monocytes
(granulocyte macrophage-colony-forming unit [CFU-GM]), G-CSF is the
only growth factor required for neutrophil granulopoiesis. G-CSF
stimulates proliferation and differentiation of CFU-GM and is required
for cell survival. Mice deficient for the G-CSF growth factor show a
deficit in absolute neutrophil number,2 and neutropenia is
seen in a canine model by inducing antibody against the G-CSF protein.3 For many years, it has been debated whether the
positive G-CSF-mediated regulation of granulopoiesis requires a
compensatory negative-feedback arm. Various regulatory models involving
the monocyte4 and negative feedback from a granulocyte
chalone have been proposed. The granulocyte chalone (never completely identified) present in serum and granulocytes was believed to specifically, but reversibly, inhibit granulopoiesis.5
Although no definitive candidate molecule causing negative feedback of granulopoiesis has been found, there is evidence that some neutrophil granule proteins inhibit granulopoiesis. Lactoferrin inhibits hematopoiesis by blocking the production of growth factors by monocytes,6 and pro-proteinase 3 inhibits granulopoiesis
by reducing the number of CFU-GMs in S phase.7,8 In 1991 Shellard et al9 showed that a membrane extract from acute
myelogenous leukemia (AML) cells ("CAMAL") could inhibit mouse and
human hematopoiesis. Sequencing revealed that CAMAL contained elastase,
together with cathepsin G, azurocidin, and proteinase 3. Proteolytic
inhibitors were able to reverse this inhibition.9-11 These
findings suggested that some of the neutrophil serine proteases might
act like a chalone. Finally, recent studies12,13 have
shown that mutations in the elastase gene are responsible for
congenital and cyclic neutropenia. These results are of interest
because they raise the possibility that some neutrophil primary granule
proteases may negatively regulate granulopoiesis.
Here we further explore the effect of purified neutrophil serine
protease on neutrophil granulopoiesis in culture. Of the proteases
tested, elastase alone rapidly digested G-CSF (but not other growth
factors) and could directly antagonize proliferation and
maturation-inducing effects of G-CSF in culture. Our results suggest a
role for elastase as a negative regulator of G-CSF-stimulated neutrophil granulopoiesis.
Materials
Cells
Progenitor cell (CFU-GM) assays in methylcellulose BMMCs (3 × 105) were incubated in serum-free Iscove medium with varying concentration of elastase, proteinase 3, cathepsin G, or azurocidin (10 ng/mL-10 µg/mL) for 2 hours.10 Cells were washed once, resuspended in 1 mL serum-free Iscove medium, and added to 4.5 mL Methocult H4230. GM-CSF (5 ng/mL) was then added, and 1-mL cultures were plated in triplicate in 6-well plates and were incubated at 37°C, 5% CO2 for up to 14 days, at which time colonies of more than 20 cells were scored using an inverted microscope. Percentage CFU-GM inhibition was calculated as 100 (mean colonies in elastase-stimulated
wells/mean of colonies in unstimulated cultures × 100).
Suspension cultures of CD34 cells CD34+ cells (105) were plated in triplicate in 24-well plates in 1 mL StemSpan serum-free medium with varying additions of growth factors, with or without neutrophil serine proteases at the concentrations indicated. The plates were then incubated at 37°C, 5% CO2 for 10 days, at which time cells were counted using a Coulter particle counter ZM as follows: 200 µL each well were diluted to 20 mL with isotone, and cells larger than 5.5 µm were counted. To inhibit elastase enzymatic activity, PMSF was preincubated with elastase (20 µg/mL) for 1 hour at 5 mM at 37°C before use. Alternatively elastase (20 µg/mL) was heat inactivated at 95°C for 15 minutes.Proliferation assay CD34+ cells (2.5 × 104) were plated in triplicate in 96-well round-bottom plates in 200 µL StemSpan; serum-free medium was supplemented with various growth factor combinations with or without neutrophil elastase at the concentrations indicated in the figures. Plates were then incubated at 37°C in 5% CO2. Proliferation was assessed by 3H-thymidine incorporation (final concentration 1 µCi (0.037 MBq)/well), added for the final 16 hours of culture. All assays, with or without protein, were carried out in triplicate. Cultures were harvested on fiberglass paper, and incorporation of 3H-thymidine was determined by scintillation counting.Measurement of cell death CD34+ cells (106) were plated in 12-well plates in 2 mL StemSpan serum-free medium with 25 ng/mL of G-CSF, GM-CSF, and SCF, with or without 1 µg/mL neutrophil elastase. Plates were then incubated at 37°C, 5% CO2. Apoptosis was measured daily by flow cytometry using the Annexin V-PE apoptosis detection kit. Daily samples were also examined morphologically: maturation of the myeloid series and apoptotic cell numbers were evaluated on Camco Stain Pak-stained slides. Maturation index was determined by the following formula: (Stab cell number + segmented nuclear cell number)/total number of all myeloid series. Apoptotic cells were morphologically assessed by shrinkage of the cells, condensation and fragmentation of the nuclei, and presence of apoptotic bodies, and the apoptotic cell ratio to total cell number was calculated. Data were expressed as the mean of 3 counts of 1000 cells each in 3 areas of the slide. On day 9, remaining cells were harvested, labeled with monoclonal anti-CD66b-fluorescein isothiocyanate (FITC), and analyzed by flow cytometry.Flow cytometry CD34+ (105) cells were plated in 24-well plates in 1 mL StemSpan serum-free medium with 10 ng/mL G-CSF, GM-CSF, and SCF with or without 1 µg/mL neutrophil elastase. Plates were incubated at 37°C in 5% CO2. On day 7, cells were harvested, labeled with FITC-conjugated anti-CD66b or phycoerythrin (PE)-conjugated anti-CD114 (G-CSF receptor) mouse antibodies for 30 minutes on ice, washed twice, fixed in 1% paraformaldehyde, and analyzed by flow cytometry. Neutrophils (106) were incubated overnight at 37°C, 5% CO2 in 2 mL StemSpan serum-free medium with or without 1 µg/mL neutrophil elastase, directly stained with FITC-conjugated anti-CD66b or PE-conjugated anti-CD114, and analyzed by flow cytometry.Cleavage of G-CSF by elastase Proteolytic digestion assays were performed as previously described14 with the following modification. Lyophilized rhG-CSF, rhGM-CSF, rhSCF, and fibronectin were resuspended in phosphate-buffered saline (PBS) at 100 µg/mL. Aliquots of 20 µL were mixed with 2 µg/mL neutrophil elastase, cathepsin G, 25 µg/mL proteinase 3, or no enzyme and were incubated at 37°C. After 15 minutes the reaction was stopped by the addition of NuPAGE sample buffer (106 mM Tris HCl, 10% glycerol, and 2% lithium dodecyl sulfate) and incubated for 10 minutes at 70°C. Samples were separated by electrophoresis on NuPAGE Bis-Tris gels (4%-12%). Gels were stained using the Gelcode Blue Stain Reagent.Statistical analysis Values shown are the mean ± standard error of the mean (SEM). Significant differences between groups were evaluated using the Student 2-tailed t test.
Effect of serine proteases on CFU-GM CFU-GMs were cultured with varying concentrations of elastase, proteinase 3, azurocidin, and cathepsin G. In 4 persons tested, colony numbers in the presence of elastase were reduced in a dose-dependent fashion, with the maximum colony inhibition of 32% observed at 10 µg/mL. Colonies grown in the presence of elastase also appeared smaller, and the cells were less refractile. In contrast, no consistent CFU-GM inhibition was observed with other serine proteases (Table 1).
Suspension cultures To further examine the effect of elastase on granulopoiesis, suspension cultures of CD34+ cells stimulated with G-CSF, SCF, and GM-CSF were sequentially analyzed over a 10-day period. Proliferation was measured by nucleated cell count and tritiated thymidine uptake. Apoptosis and maturation index were quantitated morphologically. In control cultures, cell numbers increased from 1 × 105 to 2.76 × 106 ± 0.15 (28-fold increase). Cell counts in the presence of 1000 ng/mL and 500 ng/mL elastase increased only 2.5 ± 0.4- and 9.5 ± 2-fold, respectively. There was a concordantly lower tritiated thymidine uptake in cells cultured with elastase (Figure 1). Although a decrease in counts and proliferation relative to the controls was observed by day 1 of culture, no morphologic differences between control or elastase cultures were observed in the first 3 days. However, on day 4, an increased proportion of apoptotic cells was observed, reaching 58% ± 12% by day 6. Correspondingly, the maturation index ([Stab cell number + segmented nuclear cell number]/total number) of all myeloid series fell dramatically after day 6 (Figures 2-3). On day 9, cells were harvested and examined by flow cytometry. In the untreated culture, more than 35% of the cells were positive for the neutrophil-specific marker CD66b, compared with only 4% of cells cultured in elastase (Figure 2), confirming the elastase-induced neutrophil maturation arrest observed morphologically. Apoptosis was confirmed using annexin V staining; CD34+ cells were cultured with the same cocktail of cytokine in the presence or absence of elastase (1000 ng/mL) and was analyzed daily by flow cytometry. In elastase-treated cultures, apoptosis was already higher by day 2 of culture and reached a maximum on day 5, showing that, in the presence of elastase, neutrophil precursors switched from proliferation and maturation to apoptosis. To determine whether elastase had a nonspecific antiproliferative effect, cultures of B-lymphoblastoid cell lines, K-562 cells, and IL-2-stimulated T cells were incubated for 5 days with or without 1 µg/mL elastase, at which time cells were counted. No effect on cell proliferation was observed in these cultures (data not shown).
Interactions between elastase and growth factors The effect of elastase in cultures stimulated with single growth factors or combinations of growth factors was further explored. SCF and GM-CSF, but not G-CSF, induced proliferation in CD34+ elastase-treated cultures (Figure 4). Untreated cultures combining G-CSF and GM-CSF showed enhanced proliferation over cultures in the presence of GM-CSF alone. Elastase weakly affected the proliferation of GM-CSF-stimulated cultures but reduced the proliferation of G-CSF plus GM-CSF cultures to the level observed with GM-CSF alone. Finally, elastase completely blocked proliferation of cultures grown in the presence of G-CSF alone. The abrogation of G-CSF-stimulated proliferation by elastase was dose dependent and required at least 500 ng/mL elastase (Figure 4; Table 2).
Inhibition of G-CSF-stimulated growth requires enzymatic activity of elastase The inhibitory effect of elastase on G-CSF was potent and could only be modestly overcome by increasing the dose of G-CSF to 200 ng/mL. To determine whether the inhibitory effect of elastase on G-CSF was mediated by enzymatic activity, the effect of blocking enzymatic activity was studied using PMSF, which irreversibly inhibits the serine proteases, or using heat inactivation to 95°C for 15 minutes. PMSF treatment and heat inactivation completely blocked the inhibitory effect of elastase on G-CSF-stimulated proliferation by cell count and also by tritiated thymidine uptake (data not shown), indicating that enzymatically active elastase was required to inhibit G-CSF (Figure 5).
Interaction of elastase with G-CSF and its receptor To determine whether elastase blocked the effect of G-CSF by its receptor or by direct interaction with the growth factor, G-CSF receptor expression on neutrophils and CD34+ cultivated cells was measured by flow cytometry in the presence and absence of elastase. Although CD66b expression was lost on elastase-exposed CD34+ cultures, no reduction in the surface expression of the G-CSF receptor was seen, nor was it seen on CD66b+ blood neutrophils exposed to elastase (Figure 6). To study the enzymatic inactivation of G-CSF by elastase, G-CSF, SCF, and GM-CSF were incubated for 15 minutes with 2µg/mL elastase and were analyzed by gel electrophoresis. G-CSF alone was rapidly cleaved by elastase. When the 3 growth factors were first mixed and treated with elastase, only G-CSF was cleaved by elastase. In contrast, proteinase 3 did not cleave G-CSF, and cathepsin G only induced minimal cleavage of G-CSF. Fibronectin, used as a positive control, was cut by the 3 proteases. However, because it has lower affinity for fibronectin, the concentration of proteinase 3 was increased to 25 µg/mL (Figure 7).
Here we show that neutrophil elastase blocks the ability of G-CSF to provide proliferative, maturational, and survival signals to neutrophils and their precursors. First, elastase inhibited normal CFU-GM growth, with the result that colonies that form do not attain the size of control colonies and appear to senesce before day 10. The mechanism of colony inhibition is unclear. It is possible that elastase digests G-CSF produced by BMMCs during preincubation in the absence of growth factor or in the culture in methylcellulose bound to the membrane of the BMMCs.15 To further study the effect of elastase on granulopoiesis, suspension cultures of CD34 cells in the presence of abundant growth factors were studied. Elastase blocked the generation of mature CD66b+ neutrophils, increasing the apoptotic score of maturing neutrophil progenitors but only minimally affecting undifferentiated cells. Apoptosis of developing neutrophils was confirmed by annexin V flow cytometric analysis. Because annexin V is an early marker of apoptosis, the maximum apoptosis was seen on day 5, preceding the morphologic changes seen on day 6. To determine which growth factor was antagonized by elastase, we cultured CD34 cells in the presence of G-CSF, GM-CSF, or SCF. CD34 cells proliferated with GM-CSF or SCF alone, but proliferation under the influence of G-CSF was almost completely blocked by elastase. The elastase preparation used was more than 95% pure, but it is derived from neutrophil extracts and could contain other granule proteins. However, the other neutrophil serine protease granules (proteinase 3, cathepsin G, and azurocidin) did not inhibit colony growth and showed no comparable effect on proliferation in suspension culture. The blocking effect on G-CSF-stimulated proliferation was thus attributable to elastase. The possibility that elastase had a nonspecific effect on proliferating cells was also considered. However, elastase did not inhibit proliferation in leukemic cell lines, B cells, or T cells. Furthermore, identical results in repeated experiments using another commercial source of elastase (Calbiochem, La Jolla, CA) were found (data not shown). Finally, elastase appeared to counteract primarily the proliferative effect of G-CSF but not GM-CSF or SCF. Inactivation of elastase with PMSF or heat treatment showed that the blocking effect on G-CSF required enzymatic activity. This raised the possibility that elastase might act by cleaving G-CSF or its receptor. The G-CSF receptor contains a fibronectin 3 domain in the extracellular region near the cytoplasmic junction.16 Elastase is known to cleave fibronectin.17 The possibility was considered that the G-CSF receptor could be enzymatically clipped from the cell surface by elastase, thus depriving the cell of a mechanism for G-CSF signaling. This did not appear to be the case; using antibodies specific for the G-CSF receptor, neither elastase-treated CD34 cells nor neutrophils lost surface G-CSF receptors. Because the epitope recognized by the antibody is unknown, the possibility that elastase cuts above this epitope cannot be excluded. Rather, the main antagonistic action of elastase appeared to be caused by enzymatic digestion of G-CSF (but not SCF or GM-CSF). By extrapolation from the in vitro experiment, a single molecule of elastase appears to be capable of rapidly digesting more than 70 molecules of G-CSF. The rapid digestion of G-CSF was not observed with the other serine proteases tested, suggesting a specific elastase-G-CSF interaction. How might these in vitro observations relate to the action of G-CSF in vivo? Normally serum neutrophil elastase levels are in the order of 200 ng/mL, but much of it is complexed with serpins and therefore is inactive. However, elastase levels correlate directly with the number of peripheral blood neutrophils,18 and 106 neutrophils can release up to 2 µg/mL elastase.19 Furthermore, changes in elastase accompany the administration of G-CSF. After a single subcutaneous dose of G-CSF, elastase levels rise, both in the blood20 and in the bone marrow.14 Thus the concentration of 500 ng/mL, effective in our cultures, could easily be reached in G-CSF-stimulated bone marrow. The potent inactivation of G-CSF by elastase could explain its rapid disappearance from the circulation seen after parenteral administration. Interestingly, a large body of data defines an inverse relationship between neutrophil count and G-CSF level.21-23 It has been suggested that the presence of G-CSF receptors on neutrophils is responsible for this relationship, but because neutrophil counts and elastase levels also correlate, this correlation could alternatively be reinterpreted as a direct effect of elastase on G-CSF. It is therefore possible that elastase release by neutrophils represents a negative feedback loop of granulopoietic regulation. The fact that mice deficient in the elastase gene have normal neutrophil counts24,25 could be explained by redundancy in the system controlling granulopoiesis. Recent studies have shown that granulopoiesis is negatively regulated by pro-proteinase 3 and pro-azurocidin (but not pro-elastase, pro-cathepsin G, mature proteinase 3, or azurocidin).7,8 Because these neutrophil serine protease proforms are only produced in primary granules at the promyelocyte stage, they may represent a primary granule feedback path, distinct from an elastase-G-CSF feedback from mature neutrophils. Elastase is normally retained within the neutrophil granule until the
cell degranulates or dies in the tissue compartment. Although large
amounts of elastase must be generated daily from the normal process of
neutrophil senescence, the events occur in tissues peripheral to the
marrow. Furthermore, elastase is normally inactivated by circulating
Recently, neutrophil serine proteases have been implicated in the process of neutrophil mobilization.14 In mice, exogenously administered G-CSF increases intramedullary elastase and cathepsin G, which in turn cleaves stromal cell vascular cellular adhesion molecule-1 (VCAM-1), reducing cellular adhesiveness and mobilizing marrow cells into the circulation. Again, by inactivating G-CSF, elastase may modulate mobilization. Recent findings implicate elastase mutations in the pathophysiology of
cyclic neutropenia and congenital neutropenia. However, the pattern of
mutation in the neutrophil elastase gene differs in the 2 disorders,26 suggesting that the mutated enzyme acts by a
different mechanism in each disease. Mutated elastase complexes poorly
with Finally, elastase levels are very high in certain myeloid leukemias.18,29,30 The question arises whether an inhibitory effect of elastase on normal granulopoiesis contributes to the dominance of a G-CSF-independent leukemic clone or, alternatively, whether elastase release by leukemic cells could contribute to the maturation arrest of a G-CSF-sensitive leukemic blast cell. In conclusion, our study points toward an important role for elastase in antagonizing G-CSF, which blocks granulopoiesis in vitro and may modulate the actions of the growth factor in vivo. Further studies will be necessary to define the significance of elastase in the regulation of neutrophil production and G-CSF function in vivo in normal and pathologic states.
We thank Dr Magnusson Magnus for critical reading of this manuscript. We also thank Dr Philippe Martiat, Dr Philippe Lewalle, and Dr Redouane Rouas for their help.
Submitted June 11, 2002; accepted October 7, 2002.
Prepublished online as Blood First Edition Paper, October 17, 2002; DOI 10.1182/blood-2002-06-1734.
Supported by grants from MEDIC Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: John Barrett, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bldg 10, Rm 7C103, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: barrettj{at}nih.gov.
1. Boxer L, Dale DC. Neutropenia: causes and consequences. Semin Hematol. 2002;39:75-81[CrossRef][Medline] [Order article via Infotrieve].
2.
Lieschke GJ, Grail D, Hodgson G, et al.
Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.
Blood.
1994;84:1737-1746 3. Hammond WP, Csiba E, Canin A, et al. Chronic neutropenia: a new canine model induced by human granulocyte colony-stimulating factor. J Clin Invest. 1991;87:704-710[Medline] [Order article via Infotrieve]. 4. Heissig B, Pasternak G, Horner S, Schwerdtfeger R, Rossol S, Hehlmann R. CD14+ peripheral blood mononuclear cells from chronic myeloid leukemia and normal donors are inhibitory to short- and long-term cultured colony-forming cells. Leuk Res. 2000;24:217-231[CrossRef][Medline] [Order article via Infotrieve]. 5. Rytomaa T. Role of chalone in granulopoiesis. Br J Haematol. 1973;24:141-146[Medline] [Order article via Infotrieve].
6.
Broxmeyer HE, Smithyman A, Eger RR, Meyers PA, de Sousa M.
Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production.
J Exp Med.
1978;148:1052-1067
7.
Skold S, Rosberg B, Gullberg U, Olofsson T.
A secreted proform of neutrophil proteinase 3 regulates the proliferation of granulopoietic progenitor cells.
Blood.
1999;93:849-856 8. Skold S, Zeberg L, Gullberg U, Olofsson T. Functional dissociation between proforms and mature forms of proteinase 3, azurocidin, and granzyme B in regulation of granulopoiesis. Exp Hematol. 2002;30:689-696[CrossRef][Medline] [Order article via Infotrieve]. 9. Shellard JE, Leitch HA, Logan PM, McMaster WR, Levy JG. Purification of an in vitro inhibitor of normal myelopoiesis using a monoclonal antibody directed to a common antigen of myelogenous leukemia (CAMAL). Exp Hematol. 1991;19:136-142[Medline] [Order article via Infotrieve]. 10. Leitch HA, Yip ST, Jamieson CH, Levy JG. Inhibition of murine hematopoiesis by CAMAL, an inhibitor of human hematopoiesis. Leukemia. 1993;7:717-724[Medline] [Order article via Infotrieve]. 11. Leitch HA, Levy JG. Reversal of CAMAL-mediated alterations of normal and leukemic in vitro myelopoiesis using inhibitors of proteolytic activity. Leukemia. 1994;8:605-611[Medline] [Order article via Infotrieve]. 12. Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat Genet. 1999;23:433-436[CrossRef][Medline] [Order article via Infotrieve].
13.
Dale DC, Person RE, Bolyard AA, et al.
Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia.
Blood.
2000;96:2317-2322
14.
Levesque JP, Takamatsu Y, Nilsson SK, Haylock DN, Simmons PJ.
Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor.
Blood.
2001;98:1289-1297 15. Koller MR, Bradley MS, Palsson BO. Growth factor consumption and production in perfusion cultures of human bone marrow correlate with specific cell production. Exp Hematol. 1995;23:1275-1283[Medline] [Order article via Infotrieve].
16.
Larsen A, Davis T, Curtis BM, et al.
Expression cloning of a human granulocyte-colony-stimulating factor receptor: a structural mosaic of hematopoietin receptor, immunoglobulin, and fibronectin domains.
J Exp Med.
1990;172:1559-1570
17.
McDonald JA, Kelley DG.
Degradation of fibronectin by human leukocyte elastase: release of biologically active fragments.
J Biol Chem.
1980;255:8848-8858 18. Nagamatsu Y, Yamamoto J, Fukuda A, Ohta M, Tsuda Y, Okada Y. Determination of leukocyte elastase concentration in plasma and serum by a simple method using a specific synthetic substrate. Haemostasis. 1991;21:338-345[Medline] [Order article via Infotrieve].
19.
Renesto P, Chignard M.
Enhancement of cathepsin G-induced platelet activation by leukocyte elastase: consequence for the neutrophil-mediated platelet activation.
Blood.
1993;82:139-144
20.
Falanga A, Marchetti M, Evangelista V, et al.
Neutrophil activation and hemostatic changes in healthy donors receiving granulocyte colony-stimulating factor.
Blood.
1999;93:2506-2514
21.
Layton JE, Hockman H, Sheridan WP, Morstyn G.
Evidence for a novel in vivo control mechanism of granulopoiesis: mature cell-related control of a regulatory growth factor.
Blood.
1989;74:1303-1307
22.
Stute N, Santana VM, Rodman JH, Schell MJ, Ihle JN, Evans WE.
Pharmacokinetics of subcutaneous recombinant human granulocyte-colony-stimulating factor in children.
Blood.
1992;79:2849-2854 23. Ericson SG, Gao H, Gericke GH, Lewis LD. The role of polymorphonuclear neutrophils in clearance of granulocyte-colony-stimulating factor (G-CSF) in vivo and in vitro. Exp Hematol. 1997;25:1313-1325[Medline] [Order article via Infotrieve]. 24. Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity. 2000;12:201-210[CrossRef][Medline] [Order article via Infotrieve]. 25. Belaaouai A, McCarthy R, Baumann M, et al. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med. 1998;5:615-618. 26. Dale DC, Bolyard AA, Aprikyan A. Cyclic neutropenia. Semin Hematol. 2002;39:89-94[CrossRef][Medline] [Order article via Infotrieve].
27.
Li FQ, Horwitz M.
Characterization of mutant neutrophil elastase in severe congenital neutropenia.
J Biol Chem.
2001;276:14230-14241
28.
Ancliff PJ, Gale RE, Watts MJ, et al.
Paternal mosaicism proves the pathogenic nature of mutations in neutrophil elastase in severe congenital neutropenia.
Blood.
2002;100:707-709
29.
Hiller E, Jochum M.
Plasma levels of human granulocytic elastase
30.
Saito M, Asakura H, Uotani C, Jokaji H, Kumabashiri I, Matsuda T.
Quantitative estimation of elastase-
© 2003 by The American Society of Hematology.
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  J. Skokowa, J. P. Fobiwe, L. Dan, B. K. Thakur, and K. Welte Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1 Blood, October 1, 2009; 114(14): 3044 - 3051. [Abstract] [Full Text] [PDF]
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D. S. Grenda, M. Murakami, J. Ghatak, J. Xia, L. A. Boxer, D. Dale, M. C. Dinauer, and D. C. Link Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis Blood, December 15, 2007; 110(13): 4179 - 4187. [Abstract] [Full Text] [PDF]
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 L. Padron-Barthe, C. Lepretre, E. Martin, M.-F. Counis, and A. Torriglia Conformational Modification of Serpins Transforms Leukocyte Elastase Inhibitor into an Endonuclease Involved in Apoptosis Mol. Cell. Biol., June 1, 2007; 27(11): 4028 - 4036. [Abstract] [Full Text] [PDF]
|
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|
|
|
|
M. Donini, S. Fontana, G. Savoldi, W. Vermi, L. Tassone, F. Gentili, E. Zenaro, D. Ferrari, L. D. Notarangelo, F. Porta, et al. G-CSF treatment of severe congenital neutropenia reverses neutropenia but does not correct the underlying functional deficiency of the neutrophil in defending against microorganisms Blood, June 1, 2007; 109(11): 4716 - 4723. [Abstract] [Full Text] [PDF]
|
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|
|
M. S. Horwitz, Z. Duan, B. Korkmaz, H.-H. Lee, M. E. Mealiffe, and S. J. Salipante Neutrophil elastase in cyclic and severe congenital neutropenia Blood, March 1, 2007; 109(5): 1817 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Kollner, B. Sodeik, S. Schreek, H. Heyn, N. von Neuhoff, M. Germeshausen, C. Zeidler, M. Kruger, B. Schlegelberger, K. Welte, et al. Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response Blood, July 15, 2006; 108(2): 493 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tavor, I. Petit, S. Porozov, P. Goichberg, A. Avigdor, S. Sagiv, A. Nagler, E. Naparstek, and T. Lapidot Motility, proliferation, and egress to the circulation of human AML cells are elastase dependent in NOD/SCID chimeric mice Blood, September 15, 2005; 106(6): 2120 - 2127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Porter, H. Yang, S. Yavagal, G. C. Preza, O. Murillo, H. Lima, S. Greene, L. Mahoozi, M. Klein-Patel, G. Diamond, et al. Distinct Defensin Profiles in Neisseria gonorrhoeae and Chlamydia trachomatis Urethritis Reveal Novel Epithelial Cell-Neutrophil Interactions Infect. Immun., August 1, 2005; 73(8): 4823 - 4833. [Abstract] [Full Text] [PDF]
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 H. Fujiwara, J. J. Melenhorst, F. El Ouriaghli, S. Kajigaya, M. Grube, G. Sconocchia, K. Rezvani, D. A. Price, N. F. Hensel, D. C. Douek, et al. In vitro Induction of Myeloid Leukemia-Specific CD4 and CD8 T Cells by CD40 Ligand - Activated B Cells Gene Modified to Express Primary Granule Proteins Clin. Cancer Res., June 15, 2005; 11(12): 4495 - 4503. [Abstract] [Full Text] [PDF]
|
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P. Massullo, L. J. Druhan, B. A. Bunnell, M. G. Hunter, J. M. Robinson, C. B. Marsh, and B. R. Avalos Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes Blood, May 1, 2005; 105(9): 3397 - 3404. [Abstract] [Full Text] [PDF]
|
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 I. G. Winkler, J. Hendy, P. Coughlin, A. Horvath, and J.-P. Levesque Serine protease inhibitors serpina1 and serpina3 are down-regulated in bone marrow during hematopoietic progenitor mobilization J. Exp. Med., April 4, 2005; 201(7): 1077 - 1088. [Abstract] [Full Text] [PDF]
|
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L. J. Druhan, J. Ai, P. Massullo, T. Kindwall-Keller, M. A. Ranalli, and B. R. Avalos Novel mechanism of G-CSF refractoriness in patients with severe congenital neutropenia Blood, January 15, 2005; 105(2): 584 - 591. [Abstract] [Full Text] [PDF]
|
|
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|
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 O. Levy Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes J. Leukoc. Biol., November 1, 2004; 76(5): 909 - 925. [Abstract] [Full Text] [PDF]
|
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G. Carlsson, A. A. G. Aprikyan, R. Tehranchi, D. C. Dale, A. Porwit, E. Hellstrom-Lindberg, J. Palmblad, J.-I. Henter, and B. Fadeel Kostmann syndrome: severe congenital neutropenia associated with defective expression of Bcl-2, constitutive mitochondrial release of cytochrome c, and excessive apoptosis of myeloid progenitor cells Blood, May 1, 2004; 103(9): 3355 - 3361. [Abstract] [Full Text] [PDF]
|
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H. Fujiwara, F. El Ouriaghli, M. Grube, D. A. Price, K. Rezvani, E. Gostick, G. Sconocchia, J. Melenhorst, N. Hensel, D. C. Douek, et al. Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase Blood, April 15, 2004; 103(8): 3076 - 3083. [Abstract] [Full Text] [PDF]
|
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C. R. D. Carter, K. M. Whitmore, and R. Thorpe The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation J. Leukoc. Biol., March 1, 2004; 75(3): 515 - 522. [Abstract] [Full Text] [PDF]
|
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T. Papayannopoulou Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization Blood, March 1, 2004; 103(5): 1580 - 1585. [Abstract] [Full Text] [PDF]
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F. E. Ouriaghli, E. Sloand, L. Mainwaring, H. Fujiwara, K. Keyvanfar, J. J. Melenhorst, K. Rezvani, G. Sconocchia, S. Solomon, N. Hensel, et al. Clonal dominance of chronic myelogenous leukemia is associated with diminished sensitivity to the antiproliferative effects of neutrophil elastase Blood, November 15, 2003; 102(10): 3786 - 3792. [Abstract] [Full Text] [PDF]
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B. Jones, S. Adams, G. T. Miller, M. I. Jesson, T. Watanabe, and B. P. Wallner Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies Blood, September 1, 2003; 102(5): 1641 - 1648. [Abstract] [Full Text] [PDF]
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 M. H. Cottler-Fox, T. Lapidot, I. Petit, O. Kollet, J. F. DiPersio, D. Link, and S. Devine Stem Cell Mobilization Hematology, January 1, 2003; 2003(1): 419 - 437. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
,
control; and 
, elastase.
, GM-CSF; and 
, SCF. Data are representative of 3 independent experiments. (C) Loss of synergy between GM-CSF and G-CSF
in elastase-treated cultures. Data presented are the mean (± SEM) of
triplicates. 
, control GM-CSF + G-CSF; 
-1 antitrypsin and