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PHAGOCYTES
From the Laboratoire de Biochimie, Hôpital
de Montfermeil, Montfermeil, France; and the Laboratoire de Biochimie,
INSERM U 408, Laboratoire d'Hématologie et d'Immunologie et
INSERM U 479, and Service de Pneumologie, Hôpital Bichat
Assistance Publique-Hôpitaux de Paris, Paris, France.
Hepatocyte growth factor (HGF), a heparin-binding factor, is
synthesized as a single-chain inactive precursor (pro-HGF), which is
converted by proteolysis to an active heterodimer (mature HGF). HGF has
pleiotropic activities and has been implicated in the regulation of
mitogenesis, motogenesis, and morphogenesis of epithelial and
endothelial cells. As polymorphonuclear neutrophils (PMNs) secrete
numerous cytokines involved in the modulation of local inflammation, we
investigated their ability to produce HGF. We found that HGF was stored
in secretory vesicles and in gelatinase/specific granules. This
intracellular stock was rapidly mobilized by degranulation when
neutrophils were stimulated with phorbol myristate acetate or
N-formylmethionyl-leucyl-phenylalanine. Cycloheximide did not affect
the release of HGF. Moreover, HGF messenger RNA and protein expression
was found in bone marrow myeloid cells, suggesting that HGF synthesis
likely occurs during PMN maturation. In mature circulating PMNs,
intracellular HGF was in the pro-HGF form, whereas the HGF secreted by
degranulation was the mature form. Furthermore, PMNs pretreated with
diisopropyl fluorophosphate only released the pro-HGF form, suggesting
that PMN-derived serine protease(s) are involved in the proteolytic
process. We also obtained evidence that secreted mature HGF binds
PMN-derived glycosaminoglycans (probably heparan sulfate). These
findings suggest that PMNs infiltrating damaged tissues may modulate
local wound healing and repair through the production of HGF, a major
mediator of tissue regeneration.
(Blood. 2002;99:2997-3004) Hepatocyte growth factor (HGF), a heparin-binding
factor, was originally described as a potent mitogen for the growth of
hepatocytes in vitro and was subsequently purified from rat
platelets,1 plasma of a patient with fulminant hepatic
failure,2 and normal human plasma.3 HGF has
mitogenic, motogenic, and morphogenic effects on various
epithelial and endothelial cell types.4,5 Many studies
have demonstrated that embryogenesis, angiogenesis, hematopoiesis, as
well as wound healing and organ regeneration, are critically controlled
by HGF.5-7 HGF is secreted as a 92-kd single-chain pro-HGF
that requires endoproteolytic processing. This processing is mediated
by a serine protease. The HGF activator,8 blood
coagulation factor XII,9 urokinase,10 and
tissue-type plasminogen activator11 are reported to
activate HGF. Processing of HGF results in a bioactive form (mature
HGF) consisting of a disulfide-linked 69-kd The biologic effects of HGF are mediated by the activation of its
high-affinity binding site known as the tyrosine kinase receptor
c-met.13 Besides c-met, HGF possesses
lower-affinity/high-capacity binding sites corresponding to
extracellular matrix molecules (glycosaminoglycans or collagen) or to
cell surface-associated heparan sulfate14,15; this
correspondence creates a molecular reservoir of HGF on the cell
surface, whereas HGF transfer to c-met initiates the cellular
response.16 The broad range of HGF activities on many
physiologic and pathologic processes is reflected by c-met expression
in a variety of organs and cell types. The finding that c-met receptor
expression is induced by inflammatory cytokines17 suggests
that the HGF/c-met system is involved in inflammatory responses to
tissue injury.
Polymorphonuclear neutrophils (PMNs), the predominant tissue
infiltrating cells during acute inflammatory process, participate in
host defenses by generating reactive oxygen species and releasing proteolytic enzymes. Human PMNs can synthesize and secrete many proteins that influence the course of inflammatory responses and contribute to the modulation of local inflammation.18,19
After reports that human liver and bone marrow PMNs contain
immunoreactive HGF,20,21 we investigated the regulation of
HGF production by human blood PMNs, together with the subcellular
localization and molecular form of HGF in these cells.
Purification of blood PMNs and bone marrow myeloid cells
Immunocytochemical staining of HGF
Subcellular fractionation of blood PMNs Specific and azurophilic granules were purified as previously described.23 Briefly, purified neutrophils (100 × 106) in 5 mL ice-cold relaxation buffer (100 mM KCl, 3 mM NaCl, 1 mM Na2ATP, 3.5 mM MgCl2, 10 mM PIPES, pH 7.2) supplemented with EGTA and antiproteases were pressurized with N2 for 20 minutes at 450 psi with constant stirring in a nitrogen bomb. The cavitate was then collected dropwise into EGTA, pH 7.4, sufficient for a final concentration of 1.25 mM. Nuclei and unbroken cells were pelleted by centrifugation of the cavitate at 400g for 15 minutes. The supernatant was decanted, loaded on top of a 2-layer Percoll gradient (1.05/1.12 g/mL), precooled to 4°C, and centrifuged at 4°C for 30 minutes at 40 000g. Three visible bands were identified: a lower band containing azurophil granules, an intermediate band containing specific and gelatinase granules, and an upper band containing plasma membranes and secretory vesicles. The cytosol remained above the upper band. The different fractions were collected, and the purity of specific and azurophilic granule fractions was assessed by enzyme-linked immunosorbent assay (ELISA) measurement of their respective markers (lactoferrin and myeloperoxidase) in each fraction and in the total cavitate.Degranulation experiments and purification of PMN-derived HGF Pure PMNs (107/mL) were resuspended in Hanks balanced salt solution (HBSS with Ca++/Mg2+; Life Technologies, Cergy Pontoise, France). Part of the cells (unstimulated control PMNs) were kept for 20 minutes in medium alone, on ice, or at 37°C; the other part was preincubated at 37°C for 20 minutes with the following degranulating agents: lipopolysaccharide at 1 µg/mL (LPS from Escherichia coli 055:B5; Sigma, St Louis, MO), 10 6 M and 108 M
N-formylmethionyl-leucyl-phenylalanine (fMLP; Sigma), or phorbol myristate acetate at 100 ng/mL (PMA; Sigma). In some experiments, to
ensure total degranulation, PMNs were preincubated with 5 µg/mL cytochalasin B (Sigma) for 5 minutes, then with 106 M
fMLP for 15 minutes at 37°C. In another set of experiments, to
inhibit serine proteases, neutrophils were preincubated with 2 mM
diisopropyl fluorophosphate (DFP; Sigma) for 15 minutes at 4°C and
then treated as indicated above with cytochalasin B and fMLP. All tubes
were then centrifuged, and the cell-free supernatants were stored at
 80°C until HGF quantitation and Western blot analysis.
For HGF purification, the cell-free supernatant of PMNs stimulated with cytochalasin B + fMLP was applied to a Hi-Trap heparin affinity column (Amersham Pharmacia Biotech); the starting washing buffer was 0.02 M Tris, 0.2 M NaCl, pH 7.5, and the eluting buffer was 0.02 M Tris, 2 M NaCl, pH 7.5. Each harvested fraction (1 mL) was dialyzed against distilled water and assayed for HGF with an ELISA method. Blood PMN culture Pure PMNs (107/mL) were cultured for 24 hours at 37°C with 5% CO2 in 24-well tissue-culture plates (Costar, Cambridge, MA). The culture medium was RPMI 1640 (Sigma) supplemented with 2 mM glutamine and antibiotics. To avoid contaminating HGF and/or protease activities,24 all culture media were free of fetal calf serum. Neutrophils were cultured with or without the following stimulating agents: LPS at 1 µg/mL, fMLP at 106 M, PMA at 100 ng/mL, and interleukin-1
(IL-1) at 10 ng/mL (Immugenex, Los Angeles, CA). To evaluate de novo
HGF synthesis, PMNs (107/mL) were preincubated with or
without 10 µg/mL cycloheximide (Sigma) for 30 minutes at 37°C and
further incubated with the stimulating agents for 24 hours. Cell-free
PMN culture supernatants were then collected and stored at 80°C
until HGF assay.
HGF, lactoferrin, and myeloperoxidase assays HGF was quantified by using a commercial ELISA kit (Quantikine; R&D Systems) with a detection limit of 40 pg/mL. As indicated by the manufacturer, both pro-HGF and mature HGF were recognized by this assay. Lactoferrin and myeloperoxidase were quantified by using ELISA kits (Oxis International, Portland, OR; detection limits 1 ng/mL), following the manufacturer's instructions.Western blot analysis Purified subcellular fractions of PMN granules, supernatants from cytochalasin B + fMLP-stimulated PMNs, or recombinant human HGF (rh-HGF, a mixture of pro-HGF and mature HGF according to the manufacturer; R&D Systems) were added to 2× Laemmli sample buffer, with or without 2-mercaptoethanol as reducing agent. Proteins were then resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA), the proteins were immunoblotted. Nonspecific sites were blocked by overnight immersion in 10% nonfat dried milk, and the membranes were then probed with a mixture (1:1) of 2 monoclonal anti-HGF antibodies (0.5 µg/mL) both recognizing the chain (MAB694 clone 24 516.1 and MAB294 clone 24 612.111; R&D
Systems) or, in one set of experiments, with a monoclonal antiheparan
sulfate antibody (5 µg/mL) (Seikagaku, Tokyo, Japan).25
The immunoblots were developed by using an enhanced chemiluminescence
method (Amersham Pharmacia Biotech) following the manufacturer's
instructions. In selected experiments, before Western blot analysis,
supernatants from cytochalasin B + fMLP-stimulated PMNs were
incubated in 2 M NaCl, 0.02 M Tris, pH 7.5 for 30 minutes at 37°C to
disrupt ionic interactions.26 To check their specificity,
the anti-HGF antibodies (5 µg) were preincubated with or without
rh-HGF (1 µg) for 1 hour at room temperature before probing the
membranes. The intensities of HGF bands (pro-HGF and chain)
decreased when the primary antibodies were preincubated with
rh-HGF.
Treatment of pro-HGF from specific granules with serine proteases Aliquots of purified specific granules (containing 1 ng pro-HGF as determined by Western blot analysis and ELISA) were incubated at 37°C for 10 and 60 minutes with either 0.1 to 100 IU urokinase-type plasminogen activator (uPA)/ng pro-HGF in 20 mM phosphate buffer pH 7.4,11 or with 1 to 100 pmol elastase/ng pro-HGF in HBSS pH 7.5,27 or with 1 to 100 pmol cathepsin G/ng pro-HGF in HBSS pH 7.5.27 Elastase and cathepsin G were kindly provided by M. Chignard from Institut Pasteur-INSERM 285 (Paris, France); uPA was from Hoechst (Paris La Defense, France). After incubation, samples were immediately added to 2× Laemmli sample buffer with 2-mercaptoethanol as reducing agent and analyzed by Western blotting as detailed above.Reverse transcriptase-polymerase chain reaction Purified blood PMNs and bone marrow cells prepared as described above were harvested and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for HGF messenger RNA (mRNA) expression. In selected experiments, highly purified PMNs were incubated for 1 hour in 2 mL culture medium with or without 1 µg/mL LPS, 100 ng/mL PMA, 106M fMLP, or 10 ng/mL IL-1 as stimulating agents.
Total cellular RNA was isolated with Trizol (Life Technologies)
according to the manufacturer's instructions. Total RNA (4 µg) was
reverse transcribed in reverse transcriptase reaction buffer (10 mM
MgCl2, 50 mM KCl, 50 mM Tris-HCl pH 8.3) with 25 µg/mL
oligo-dT primer, 1 mM each deoxynucleotide triphosphate (Life
Technologies), 1 U/µL Rnasin, and 0.5 U/µL AMV reverse
transcriptase (Promega, Madison, WI). Samples were incubated for 5 minutes at 65°C and then at 42°C for 60 minutes. A specific pair of
primers designed for HGF (Life Technologies) was then added to each
complementary DNA reaction mix; the sense primer was
5'-TCCCCATCGCCATCCCC-3' and the antisense primer was
5'-CACCATGGCCTCGGCTGG-3'.28 Amplification was performed in
a solution containing 2 mM MgCl2, 1× Taq polymerase buffer, 0.2 mM each deoxynucleotide triphosphate, 5% dimethyl sulfoxide (vol/vol), 25 U/mL Taq polymerase AmpliTaq Gold (Perkin Elmer, Foster City, CA), and 0.4 µmol/L specific primer, in an automated thermal cycler (Uno II; Biometra, Maidstone, United Kingdom)
at 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 40 seconds. Amplification was stopped after 35 cycles. The MRC-5 human
embryonic lung fibroblast line served as a positive control for HGF
gene expression,29 and water was used instead of RNA as a
negative control of RT-PCR. The housekeeping gene porphobilinogen
deaminase (PBGD) was amplified in each sample to confirm the integrity
of RNA and the efficiency of complementary DNA synthesis. The expected
PCR products of 749 (HGF) and 338 (PBGD) base pairs were detected by
electrophoresis in 2% agarose gel containing ethidium bromide, along
with molecular weight standards (Roche Diagnostics, Mannheim, Germany).
PMNs contain an intracellular pool of pro-HGF As shown in Figure 1, immunocytochemistry revealed the presence of intracellular HGF in unstimulated blood PMNs. To determine the precise localization of HGF, HGF, lactoferrin, and myeloperoxidase were determined in subcellular fractions. HGF was mainly located in the gelatinase/specific granules fraction and, to a lesser extent, the plasma membrane fraction (Table 1); the distribution of HGF (ELISA) matched that of lactoferrin but not that of myeloperoxidase. Western blot analysis confirmed the subcellular localization of HGF (Figure 2). We observed a distinct 92-kd band in the gelatinase/specific granules fraction and a weak band in the plasma membrane fraction, both migrating at the level of pro-HGF. HGF was not detected in azurophilic granules or in cytosol fractions. As Western blot analysis was conducted in reducing conditions, these results indicate that HGF was present in PMNs as the pro-HGF single-chain form.
PMNs release HGF by degranulation As shown in Figure 3, PMN stimulated with 1 µg/mL LPS, 108 and 106
M fMLP, the combination of cytochalasin B + 106 M
fMLP, or 100 ng/mL PMA for 20 minutes at 37°C released a large amount
of HGF (730 ± 120, 741 ± 142, 1 234 ± 165, 2 322 ± 141, and 2 202 ± 246 pg/mL/107 PMN,
respectively) in comparison with unstimulated control cells maintained
at 37°C (261 ± 30 pg/mL/107 PMNs) or at 4°C
(< 40 pg/mL).
To investigate the ability of PMNs to synthesize HGF de novo,
purified blood PMNs were stimulated, in the presence or absence of
cycloheximide, with LPS, PMA, fMLP, or IL-1, which induce cytokine synthesis in PMNs.30 After 24 hours, unstimulated control
PMNs cultured in medium alone showed spontaneous HGF release that was not affected by the presence of cycloheximide (Table
2). Each stimulating agent slightly
increased HGF concentrations in PMN supernatants as compared with
unstimulated PMNs. However, whatever the stimulus, cycloheximide
treatment did not significantly modify the amounts of HGF detected
after 24 hours of stimulation, suggesting that the HGF released in the
culture supernatants was mainly derived from degranulation induced by
LPS, PMA, fMLP, or IL-1. Similar results were obtained when PMNs were
cultured with tumor necrosis factor
In agreement with the absence of active HGF synthesis, a very low
level of HGF mRNA was detected in unstimulated mature PMNs (Figure
4), and none of the stimulating agents
(LPS, PMA, fMLP, or IL-1) modified the HGF mRNA level compared with
unstimulated PMNs (densitometric analysis, in comparison with the
housekeeping gene PBGD; data not shown).
Expression of HGF in human bone marrow myeloid cells As most of the proteins stored in PMN granules are actively synthesized during granulocyte maturation, we investigated whether HGF protein and mRNA could be detected in bone marrow myeloid cells. We first performed immunocytochemical analysis of normal human bone marrow smears (n = 3). We found strong positive staining in myelocytes, metamyelocytes, band, and segmented cells in all samples. Weak to moderate staining was found in promyelocytes, whereas lymphocytes, monocytes, and megakaryocytes were negative for HGF (Figure 5).
Mononuclear cell-depleted bone marrow samples and circulating purified PMNs were subjected to RT-PCR in the same run. HGF mRNA expression was found in bone marrow samples but was only weakly positive in mature circulating PMNs (Figure 4). By using densitometric analysis in comparison with the housekeeping gene PBGD, we found that the relative intensity was higher in bone marrow cells than in mature circulating PMNs (data not shown). Taken together, these results show that HGF mRNA and protein are present in neutrophil precursors and suggest that HGF synthesis occurs during PMN maturation. PMNs process pro-HGF to mature HGF in vitro Western blot analysis in reducing conditions was used to characterize the HGF form released by PMNs into the extracellular medium. As shown in Figure 6, HGF released by cytochalasin B + fMLP-stimulated PMNs was mainly the mature form (presence of the 69-kd chain). As the processing of
pro-HGF to mature HGF involves serine proteases, the same degranulating
experiment was conducted with an additional preincubation step of 15 minutes with 2 mM DFP, used to irreversibly inhibit neutrophil serine
proteases. HGF secreted by DFP-pretreated PMNs was exclusively in the
pro-HGF form, as shown by the sole detection of the 92-kd band by
Western blotting in reducing conditions (Figure 6). These results
indicated that the pro-HGF contained in PMNs was processed to mature
HGF by neutrophil serine protease(s) during degranulation. We, thus, investigated the effects of elastase and cathepsin G, which are the
main PMN serine proteases; uPA, which is present in specific granules31 and processes pro-HGF,11 was also
tested. As shown in Figure 7, none of
these agents were able to catalyze the conversion of pro-HGF from
specific granules, whatever the incubation period (from 10 to 60 minutes) or concentration (elastase and cathepsin G, from 1 to 100 pmol/ng pro-HGF; uPA, from 0.1 to 100 IU/ng pro-HGF).
Secreted HGF binds PMN-derived heparan sulfate As HGF is a heparin-binding factor,32 we investigated whether HGF secreted by PMNs could link to glycosaminoglycans. First, in an attempt to purify PMN-derived HGF, the cell-free supernatant of cytochalasin B + fMLP-stimulated PMNs was submitted to heparin affinity chromatography. Contrary to control rh-HGF, HGF secreted by PMNs was not retained on the heparin column (ELISA; data not shown), suggesting that HGF binding sites for heparin were not available. Second, contrary to HGF purified from specific granules, Western blot analysis in nonreducing conditions of HGF secreted by PMNs failed to yield a specific HGF band (Figure 8). This latter result suggests that HGF epitopes were inaccessible to anti-HGF antibodies because of the presence of masking substances. As a high salt concentration is commonly used to release HGF from polyanionic substances such as glycosaminoglycans,26 the supernatant of cytochalasin B + fMLP-stimulated PMNs was then incubated with 2 M NaCl. Western blot analysis revealed a 92-kd band in nonreducing conditions (Figure 8). This finding suggested that HGF secreted by PMNs binds glycosaminoglycans. As heparan sulfate is known to interact with HGF,32 we performed Western blot analysis of PMN-derived HGF with antiheparan sulfate monoclonal antibodies.25 A specific 69-kd band migrating at the level of the HGF chain was revealed, whereas no band was found with
rh-HGF (Figure 9). Taken together, our
results suggest that HGF secreted by PMNs binds a PMN-derived
heparinlike substance (likely heparan sulfate).
PMNs synthesize inflammatory cytokines and growth factors,30 but there are few reports of preformed intracellular stocks of either cytokines18 or growth factors.33 Our results demonstrate that both secretory vesicles and gelatinase/specific granules of human blood PMNs contain a mobilizable stock of pro-HGF that is proteolytically processed to mature HGF by neutrophil serine protease(s) during degranulation. Furthermore, this mature HGF bound neutrophil-derived polyanionic substances (likely heparan sulfate). The bulk of HGF was found in the gelatinase/specific granules fraction
and colocalized with the lactoferrin-containing fraction. The
efficiency of fractionation was confirmed by measuring the reference
markers of specific and azurophil granules (lactoferrin and
myeloperoxidase, respectively) in each fraction. Interestingly, vascular endothelial growth factor, another heparin-binding factor, has
also been found in specific granules.33,34 Intracellular HGF was also present in the plasma membrane and/or secretory vesicles, constituting a readily mobilizable store. This localization
demonstrated by the results in Table 1 and in Figure 1 was confirmed by
the pattern of HGF release obtained with 10 In addition to releasing numerous preformed mediators into the extracellular medium, PMNs can synthesize cytokines de novo in certain conditions.30 For instance, we have previously demonstrated that PMNs can release oncostatin M through a 2-step mechanism involving the release of a preformed stock followed by de novo protein synthesis.18 In the present study we found no evidence of active HGF synthesis by human blood PMNs, as cycloheximide did not affect HGF levels after 24 hours of stimulation. Therefore, our results show that acute HGF release by mature circulating PMNs does not depend on ongoing or activated synthesis but rather on the release of HGF stored in PMNs. Most of the proteins stored in PMN granules are actively synthesized during granulocyte maturation; indeed, we found that HGF mRNA was expressed by bone marrow cells at a higher level than in circulating PMNs. The timing of granule formation is well documented; specific granules along with their protein contents are mainly formed at the myelocyte and metamyelocyte stages.37,38 As expected, our immunocytochemistry experiments showed that HGF+ cells are essentially myelocytes, metamyelocytes, and segmented cells. Weak to moderate staining was found in promyelocytes. This latter result could be explained by a continuum during precursor maturation.38 Inaba et al39 have shown that the human promyelocytic leukemia cell line HL-60 produces HGF and that dibutyryl cAMP (a granulocyte inducer) stimulates HGF release. In addition, Majka et al40 have shown that highly purified human CD34+ cells express HGF transcripts and produce HGF. Altogether, our results suggest that the bulk of HGF synthesis occurs during PMN maturation, along with most other granule constituents. As shown by Western blot analysis in reducing conditions, HGF was
present as the pro-HGF form in both gelatinase/specific granules and
plasma membrane fractions; this mobilizable intracellular pool of HGF
was released as the mature disulfide-linked heterodimer into the
extracellular medium, as shown by the presence of the 69-kd In addition to its specific signaling receptor c-met, HGF interacts
with glycosaminoglycans, especially heparan sulfate.32 We
infer that HGF secreted by PMNs into the extracellular medium binds
PMN-derived heparan sulfate. Indeed, PMNs contain sulfated glycosaminoglycans (eg, heparan sulfate) in their azurophil
granules,44 which may bind to HGF during the degranulating
process. Antiheparan sulfate antibodies revealed the existence of a
69-kd band comigrating with the HGF These results have functional implications for the role of PMNs in wound repair. First, the existence of a mobilizable stock of HGF would allow PMNs to respond rapidly to changes in the extracellular environment. PMNs represent the majority of infiltrating cells in injured tissues, at least in the early phases, and may, therefore, be an important source of HGF, a critical factor for tissue repair and regeneration. Indeed, HGF acts as a mitogen and morphogen for various epithelial cells and has been shown to play an essential part in liver45 and lung7 regeneration after damage. HGF may exert its protective action by stimulating proliferation and angiogenesis46 and by inhibiting apoptosis.47 Various effects of HGF on peripheral blood cells have been described: HGF primes neutrophil oxidative response,48 triggers PMN transmigration,49 and stimulates T-cell adhesion and migration,50 as well as monocyte cytokine production (eg, IL-6) and monocyte motility and invasiveness.51,52 In monocytes, c-met expression is up-regulated in conditions mimicking inflammation (stimulation with IL-1 or LPS),52,53 making these cells accessible to the functional effects of HGF. Therefore, the autocrine/paracrine effects of HGF may play a role in the regulation of inflammatory process. Finally, PMN-derived metalloproteases (eg, MMP-8 and MMP-9) can mobilize growth factors, including HGF, tethered to extracellular matrix proteoglycans and collagen,14,15 further supporting the role of PMNs in tissue repair. In conclusion, our findings point to a new role of PMNs in tissue repair, through the release of HGF.
We thank A. Barnier, F. Hochedez, S. Laribe, V. Leçon-Malas, and J. Moreau for expert technical assistance and C. Poüs and J. B. Stern for helpful discussions.
Submitted January 10, 2001; accepted December 3, 2001.
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: Monique Dehoux, Laboratoire de Biochimie and INSERM U408, Hôpital Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France; e-mail: monique.dehoux{at}bch.ap-hop-paris.fr.
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Abstract
Introduction
