science pharmaceutical expo biotech jobs
Originally published online as doi:10.1189/jlb.0707492 on October 24, 2007 Originally published online as doi:10.1189/jlb.0707492 on October 15, 2007

Published online before print October 15, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0707492v1
jlb.0707492v2
83/1/181    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Achilli, C.
Right arrow Articles by Minetti, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Achilli, C.
Right arrow Articles by Minetti, G.
(Journal of Leukocyte Biology. 2008;83:181-189.)
© 2008 by Society for Leukocyte Biology

Neutrophil granulocytes uniquely express, among human blood cells, high levels of Methionine-sulfoxide-reductase enzymes

Cesare Achilli, Annarita Ciana, Antonio Rossi, Cesare Balduini and Giampaolo Minetti1

University of Pavia, Department of Biochemistry, Pavia, Italy

1 Correspondence: University of Pavia, Department of Biochemistry, via Bassi 21, 27100 Pavia, Italy. E-mail: minetti{at}unipv.it


arrow
ABSTRACT
 
L-Methionine (Met), in its free form or when inserted in proteins, is sensitive to oxidation of its thioether group by reactive oxygen species from exogenous or endogenous sources. Two stable diastereomers of Met sulfoxide [Met-(O)] may be formed [Met-S-(O) and Met-R-(O)], but these can be reduced by two classes of Methionine-sulfoxide-reductase (Msr) enzymes: MsrA, which reduces the S, and MsrB, which reduces the R sulfoxide. In this study, we have examined the levels of expression of Msr in human blood cells by enzymatic activity assay, Western blotting, and RT-PCR of purified populations of polymorphonuclear neutrophils and eosinophils, mononuclear cells, platelets, and erythrocytes. Our data indicate that of the blood cells analyzed, neutrophils expressed the highest activity, which was mainly of MsrB type. During degranulation of activated neutrophils, Msr activity was not released but remained confined within the cell, indicating a non-granular localization. Immunoprecipitation and RT-PCR studies indicated the almost complete lack of mitochondrial forms of Msrs in granulocytes. It is thus likely that Msrs are important as antioxidant/repair systems for neutrophils, cells with enormous capacity for the generation of reactive oxidants and hence, susceptible to oxidative damage.

Key Words: oxidation • oxidative burst • selenoprotein • phagocytosis • eosinophils • inflammation


arrow
INTRODUCTION
 
L-Methionine (Met), in its free form or when inserted in polypeptides, is sensitive to oxidation of its thioether group to sulfoxide by reactive oxygen species (ROS) generated from exogenous or endogenous sources. Such oxidation of Met residues in proteins often results in significant changes in the structural and hence functional properties of the protein [1 ]. Upon oxidation to the sulfoxide, the sulfur atom becomes a new stereogenic center, and two stable diastereomers of Met sulfoxide [Met-(O)] exist, with opposite configurations at the sulfur atom: Met-S-(O) and Met-R-(O) [2 , 3 ]. A repair system has evolved from prokaryotes to eukaryotes to cope with Met oxidation in the form of Methionine-sulfoxide-reductase (Msr) enzymes. Early evidence suggested that the Msr activities, recognized in a number of different cell types [4 5 6 ], might in fact act stereospecifically [7 ]. More recent studies have confirmed this concept, and in the last decade, it has been shown that Msr enzymes are ubiquitously expressed in cells and tissues [8 ] and can be grouped in two classes, depending on their stereoselectivity: MsrAs reduce Met-S-(O), and MsrBs reduce Met-R-(O) [9 10 11 12 13 14 15 ].

Msrs thus play a central role in the antioxidant defense of tissues [16 17 18 ]. Although their function is that of a repair system for oxidized Met, they may be considered as part of an antioxidant buffering system of cells, whereby exposed Met residues on proteins may scavenge ROS, and the Met-(O) is reduced back to Met by Msr activity [18 ]. It has also been proposed that the cyclical nature of Met oxidation/reduction may act as a signaling mechanism within cells [19 ]. Evidence exists for Msrs preventing age-associated diseases, such as pulmonary emphysema, rheumatoid arthritis, cataract formation, and Alzheimer and Parkinson diseases. Overexpression of MsrA confers resistance to oxidation and increases the lifespan of PC-12 cells, yeast, MOLT-4 cells, and Drosophila melanogaster (reviewed in refs. [20 , 21 ]). Finally, Msrs can reduce drugs, such as Sulindac, and xenobiotic compounds, such as alkyl-methyl sulfoxides, to the corresponding sulfides [22 , 23 ]. A system based on NADPH, thioredoxin, and thioredoxin-reductase was found to be the source of reducing power for sustaining the activity of some Msrs [24 , 25 ]. Other Msr forms (MsrB2 and MsrB3) cannot use the above-mentioned reducing system but seem able to use thionein and seleno compounds such as selenocystamine [26 , 27 ]. DTT can be used in vitro as the electron donor [25 ].

The expression of Msr in several human tissues has been documented. One gene encodes MsrA in at least three isoforms [28 29 30 ], whereas three different genes encode MsrB1 (cytosolic), MsrB2 (mitochondrial), and MsrB3, the latter existing in two alternatively spliced isoforms: MsrB3A [endoplasmic reticulum (ER)] and MsrB3B (mitochondrial) [15 ]. It is remarkable that it has been shown by Kim and Gladyshev [15 ] that MsrB1, unlike the other known MsrB isozymes, is a selenoprotein (also known as selenoprotein R or selenoprotein X), whose selenocysteine residue is essential for catalysis: Its mutation to a Cys residue decreases the specific activity of the enzyme by almost three orders of magnitude.

Although it has been shown that granulocytes express high Msr activity [6 , 7 , 31 , 32 ], which isoforms of Msr are expressed are unknown, and Msr activity in other blood components has not been determined. The present study, therefore, was designed to provide a detailed investigation of the Msr expression by a combination of enzymatic activity measurements, Western blotting, and RT-PCR, using purified blood cell populations: neutrophils, eosinophils, mononuclear cells, platelets, and erythrocytes.


arrow
MATERIALS AND METHODS
 
Antibodies and chemicals
Mouse monoclonal 1C8 anti-MsrA (ab16742), rabbit polyclonal anti-MsrA (ab16803), rabbit polyclonal anti-MsrB1 (ab16804), and rabbit polyclonal anti COX IV (ab16056) were from Abcam (Cambridge, UK). Mouse monoclonal 2C3 anti-matrix metalloproteinase (MMP)-9 (sc-21733) and goat polyclonal anti-neutrophil elastase (sc-9520) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal SZ2 anti-CD42b (0409) was from Immunotec (Marseille, France). All corresponding secondary antibodies, conjugated to HRP and Immun-Blot polyvinylidene difluoride (PVDF) membranes (0.2 µm), were from Bio-Rad Laboratories (Milan, Italy). Dextran 70, Ficoll-Paque Plus, Protein G Sepharose 4 Fast Flow, and ECL Plus Western blotting detection system were from GE Healthcare (Milan, Italy). Eosinophil Isolation Kit, MACS columns (MS size), and miniMACS separator were purchased from Miltenyi Biotec (Bologna, Italy). Skimmed milk, Zymosan, Percoll, Di-isopropylfluorophosphate (DFP), iodoacetamide, PMA, 1,4-dithioerythritol (DTE), Met-(O), 4-(dimethylamino)azobenzene-4'-sulfonyl (DABS) chloride, acetonitrile for HPLC, and Supelcosil LC-18-T (15 cmx4.6 mm, 3 µm) HPLC column were from Sigma-Aldrich (Milan, Italy). Centrifugal filter devices (Centricon-3, 3000 Da cutoff) were from Millipore (Milan, Italy). Surfact-Amps X-100 (10% Triton X-100 solution) and bicinchoninic acid (BCA) protein assay kit were from Pierce Biotechnology (Rockford, IL, USA). DMEM, FBS, L-glutamine, and penicillin-streptomycin antibiotic mix were from Euroclone (Milan, Italy). Trizol was from Invitrogen (Milan, Italy); High-Capacity cDNA Archive Kit, human kidney cDNA, TaqMan Gene Expression Assay, and TaqMan Universal PCR Master Mix (no AmpErase UNG) were from Applied Biosystems (Milan, Italy); and Taq DNA polymerase (native, plus BSA) was from Fermentas (St.Leon-Rot, Germany).

Isolation and purification of blood cells
Blood was collected from healthy human donors in 0.1 vol 3.8% (w/v) tri-sodium citrate or acid-citrate-dextrose (130 mM citric acid, 152 mM tri-sodium citrate, 112 mM glucose) as anticoagulants. Erythrocytes were recovered by centrifugation and purified further from leukocytes and platelets by filtration through cellulose, as described [33 ]. Platelet-rich plasma was obtained by centrifugation at 130 gmax for 10 min and used for subsequent platelet purification. The volume of the residual erythrocyte-leukocyte-rich sample was restored to the original value with PBS (5 mM sodium phosphate, 154.5 mM NaCl, 4.5 mM KCl, pH 7.4) and then processed for leukocyte purification by following the procedure of Böyum [34 ] with minor modifications [35 ]. The fraction of mononuclear cells at the Ficoll-Hypaque interface was collected, washed twice with PBS, supplemented with 2 mM EDTA and 0.5% (w/v) BSA (PBS-EDTA-BSA), sedimented, and stored at –80°C or used immediately as described below. The granulocyte-rich pellet from the Ficoll-Hypaque gradient was subjected to differential hypotonic lysis to eliminate contaminating erythrocytes [35 ], and the resulting granulocyte fraction was stored at –80°C or used immediately. The platelet-rich plasma obtained above was centrifuged at 700 gmax for 15 min, the supernatant discarded, and the platelet pellet washed with PIPES buffer (20 mM PIPES, 137 mM NaCl, pH 6.5) and finally, resuspended in HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, pH 7.4).

For eosinophil purification, whole blood was enriched preliminarily in eosinophils by a combination of Dextran 70 and Percoll gradient centrifugation, as described previously [36 ], with modifications. In brief, between the Dextran 70 and the Percoll steps, the leukocyte-platelet-rich fraction, as a cell suspension in 1.2% (w/v) Dextran 70, was diluted with 2 vol PBS-EDTA-BSA and centrifuged at 100 g in a fixed-angle rotor. The platelet-rich supernatant was aspirated and discarded. The leukocyte-rich pellet was resuspended directly in Percoll and processed further as described [36 ]. The resulting eosinophil-enriched fraction was finally subjected to immunomagnetic removal of residual contaminating leukocytes and erythrocytes, using the Eosinophil Isolation Kit from Miltenyi Biotec, following the manufacturer’s instructions.

Preparation of cellular extracts
Cellular extracts were obtained by one of two procedures (detergent-based or detergent-free), depending on the nature of the experiments (see below). As well as blood cells, other cell types and tissues were also used to prepare extracts, as follows. Escherichia coli (strain DH5{alpha}) was cultured at 37°C in Luria-Bertani medium without antibiotics and harvested in the exponential growth phase when the culture reached an OD540 of 0.5. Kidney, liver, and heart tissues were explanted from mice (strain 129Sv/Jae, 4–5 months old) killed after i.p. total anesthesia. The organs were washed with ice-cold PBS and then stored at –80°C until use.

Detergent-free lysis
E. coli, granulocytes, and murine organs were resuspended in PBS containing 0.1% DFP (v/v) and homogenized with a manual glass potter in ice. After centrifugation at 30,000 g for 30 min at 4°C, the supernatant was collected and used directly.

Detergent-based lysis
Purified blood cell populations were resuspended (at 108 cells/ml) in PBS containing 1% (v/v) Triton X-100 and 0.1% (v/v) DFP and incubated on ice for 30 min. The lysate was then centrifuged at 18,000 g for 10 min at 4°C. The supernatant was collected and stored frozen at –80°C or used directly.

Protein concentrations in extracts were assayed using the BCA method with BSA as a standard.

Immunoprecipitation
Triton X-100 lysates (described above), corresponding to 6 x 106 cells, were mixed with 5 µg antibody (rabbit polyclonal anti-MsrA or anti-MsrB1), and the final volume was adjusted to 60 µl. The sample was then incubated at 4°C for 2 h, followed by addition of 45 µl protein G Sepharose suspension (reconstituted with 2 vol PBS) and further incubation for 1 h at 4°C with gentle agitation. The slurry was then centrifuged at 18,000 g for 10 min at 4°C, the supernatant was collected, and the protein G Sepharose was washed three times with PBS and resuspended in the same volume of PBS as the supernatant. Aliquots of the precipitate and the supernatant were used for enzymatic analysis and SDS-PAGE/Western blotting. Protein detection by Western blotting was carried out for MsrB1 with the same antibody used for immunoprecipitation and with a mouse monoclonal anti-MsrA for MsrA. In both cases, to avoid interference during immunodetection, from the light chains of the IgGs used for immunoprecipitation, the SDS-PAGE (10–20% acrylamide gradient gels) was conducted under nonreducing conditions: Aliquots of the supernatant and the resuspended precipitate from the immunoprecipitation step were treated with 20 mM (final concentration) iodoacetamide for 20 min at 4°C to alkylate thiol groups in proteins and avoid their possible oxidation with formation of protein aggregates during the following step of dissociation under nonreducing conditions. This was obtained by adding to the iodoacetamide-treated samples, 0.5 vol SDS-PAGE sample buffer without DTT (see below, SDS-PAGE and Western blotting).

Activation of granulocytes
Granulocytes were resuspended in incubation buffer (9.6 mM sodium phosphate, pH 7.4, 138 mM NaCl, 2.7 mM KCl, 0.6 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose) at 25 x 106 cells/ml and preincubated at 37°C for 10 min. The suspensions were then treated with 0.8 µM PMA or 5 mg/ml opsonized Zymosan (final concentrations) and incubated for 30 min at 37°C, after which they were cooled on ice, supplemented with 0.1% (v/v) DFP (final concentration), and centrifuged. The incubation medium (supernatant) was collected and concentrated (to allow subsequent enzymatic analysis and Western blotting) to approximately one-fifth the original volume by centrifugation in a Centricon-3 ultrafiltration device, and the pelleted cells were resuspended in the Triton X-100-containing lysis buffer described above to a volume equal to one-fifth that of the original suspension. Opsonized Zymosan was obtained [37 ] by boiling Zymosan powder suspended in PBS for 20 min, followed by three washes with PBS. Finally, 1 vol human serum was added to a 20 mg/ml suspension of Zymosan in PBS, and the mixture was incubated for 60 min at 37°C, then washed three times with the incubation buffer, and resuspended in the same buffer.

Determination of Msr activity
The diastereoisomers of Met-(O) were separated as described [38 , 39 ] and obtained in optically pure forms. DABS-Met-R-(O) and DABS-Met-S-(O) were prepared as described [7 ]. MsrA- and MsrB-type enzymatic activities were assayed separately using the DABS derivatives of the two diastereoisomers of Met-(O) as substrates. The reaction mixture (usually 50 µl) contained 5 mM sodium phosphate (pH 7.4), 154.5 mM NaCl, 4.5 mM KCl, 20 mM DTE, 500–600 µM substrate, and 30–60 µg total protein from cell extract. The reaction was carried out at 37°C for 30 min and stopped by dilution 1:10 with a solution containing equal parts of sodium acetate (pH 4.25) and ethanol. The reaction product, DABS-Met, was analyzed by reverse-phase HPLC, as described [7 ]. The specific activity of samples was reported as pmoles DABS-Met produced per minute per mg protein.

SDS-PAGE and Western blotting
Cell extracts were mixed with Laemmli’s 3x SDS-PAGE sample buffer [50 mM Tris/HCl, pH 6.8, 5% SDS (w/v), 5 mM EDTA, 200 mM DTT, 35% sucrose (w/v), 0.01% bromophenol blue], boiled for 5 min, separated by SDS-PAGE on 5–15% or 10–20% acrylamide gradient gels [40 ], and electrotransferred to a 0.2 µm PVDF membrane [41 ], which was blocked with 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 5% skimmed milk (w/v). After incubation with a primary antibody (monoclonal anti-MsrA, polyclonal anti-MsrA, and polyclonal anti-MsrB, diluted 1:2000; polyclonal anti-neutrophil elastase and monoclonal anti-MMP-9, diluted 1:200; anti-CD42b, diluted 1:1000) and the corresponding secondary antibody, the proteins of interest were revealed with the ECL detection system.

RNA extraction and reverse transcription
RNA was extracted from total granulocytes, purified eosinophils, and mononuclear cells using Trizol reagent, according to the supplier’s instructions. In some experiments, RNA was extracted from human embryo kidney (HEK) 293-T cells grown in DMEM, supplemented with 10% FBS, L-glutamine, and penicillin-streptomycin antibiotic mix at 37°C in the presence of 5% CO2, and harvested at ~90% confluence. First-strand cDNA was synthesized using 2–10 µg total RNA using the High Capacity cDNA Archive Kit, according to the manufacturer’s recommendations.

RT-PCR
Analysis of human MsrB3A and MsrB3B transcripts was performed using cDNA from granulocytes and mononuclear, HEK 293-T, and human kidney cells (the latter commercially available). The primers used, which give rise to 2 amplicons 578 bp and 707 bp for MsrB3A and MsrB3B, respectively, were the following [42 ]: 5'-ATGAGCCCGCGGCGGACC-3' (forward), 5'-TTAGAGCTCCGCTTTGTCTG-3' (reverse). After preheating at 94°C for 4 min, reactions were cycled (x38) as follows: denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 1.5 min. The PCR products were separated on 1.5% agarose gels and then stained with ethidium bromide.

Quantitative (q)RT-PCR
The expression levels of MsrA, MsrB1, MsrB2, and GAPDH were evaluated using qRT-PCR based on the TaqMan Gene Expression Assay (Assay ID: Hs00737165_m1 for MsrA; Hs00249482_m1 for MsrB1; Hs00255292_m1 for MsrB2; and Hs99999905_m1 for GAPDH), according to the manufacturer’s instruction. Standard curves were constructed by serial dilution (from 1 to 10–3 in tenfold steps) of cDNA samples for the different Msrs. Direct quantitation of levels of expression of Msrs in different cell types was impossible, as a result of intrinsic difficulties in normalizing the expression levels for each gene to the housekeeping gene chosen for these experiments (GAPDH). Therefore, the data were analyzed as follows: Within a single, multiwell plate, three calibration curves were generated, each point in triplicate, with serial dilutions of human kidney cDNA for MsrA, MsrB1, and MsrB2. In the same plate, a triplicate sample of cDNA from a given cell type was run, whose relative amount of transcript was interpolated with the calibration curve for each gene. The gene with the highest relative transcription level within a given plate/cell type was taken as 100%, and the transcript levels of the other two genes were given as a percent of that (see also Discussion).


arrow
RESULTS
 
Granulocytes display levels of Msr activity comparable with those present in highly expressing cells
Levels of MsrA and MsrB activity of human granulocytes were measured using the separate S and R diastereomers of Met-(O) derivatized with DABS chloride and DTE as an electron donor, according to the methods developed previously in our lab [7 ]. Msr activities were compared with those measured in other tissues that have been shown previously to express high levels of the enzymes, such as kidney, heart, and liver [5 , 6 , 32 , 43 ]. Murine organs were used in this study, as they were more readily available than human tissues. Lysates of E. coli were also analyzed under the same conditions. As shown in Figure 1 , MsrB-type specific activity was greater than MsrA-type activity in all tissue extracts, except in E. coli, where the situation was reversed, which had greater MsrA activity and an overall lower total Msr activity. MsrB specific activity in granulocytes was comparable with that of highly expressing tissues, and MsrA activity was decidedly lower than measured in liver and kidney. In particular, the MsrB specific activity level for granulocytes is ~25 times higher than the MsrA levels, and this ratio decreases to fivefold and fourfold for liver and kidney, respectively. This clearly indicates that MsrB is the predominant Msr activity in granulocytes.


Figure 1
View larger version (32K):
[in this window]
[in a new window]

  		Figure 1. Enzymatic assays of MsrA- and MsrB-type activity in various cell types. The reaction mixture was prepared as described in Materials and Methods. The cell extract was added at concentrations varying from 0.2 to 1.0 mg/ml per reaction tube. The analysis was performed in five different cell extracts from different cell preparations. The error bars represent the SD.

Immunoprecipitation of MsrA and MsrB forms from granulocytes
To ascertain whether the Msr activities detected by the enzymatic assay were indeed a result of Msr protein expression, immunoprecipitation experiments were carried out using two commercially available antibodies against MsrA and MsrB1 (Fig. 2A ; antibodies against other forms, such as MsrB2, MsrB3A, and MsrB3B were not available or not working in our hands). As shown in Figure 2 , B and C, MsrA and MsrB, respectively, were immunoprecipitated by the respective antibodies and subsequently detected as enzymatic activity (graphs) and as protein band after immunodetection (Western blots). Nonrelevant antibodies used as a control were ineffective in immunoprecipitating the enzymes. It can be seen from these data that the sum of the activities in the immunoprecipitates and soluble fractions does not equal the total original activity before immunoprecipitation or the activity in the soluble fraction of the samples treated with the nonrelevant antibody. It is hypothesized that this was a result of a partial inhibition of the enzyme after the binding of the antibody. In fact, the antibodies, at the concentrations used in the immunoprecipitation experiments, inhibited MsrA and MsrB activities by ~40% and 60%, respectively (Fig. 2D) . On this basis, the Msr activities measured in the immunoprecipitated fractions of the experiments described above are underestimated. By applying a correction factor to account for this inhibition, it can be calculated that the immunoprecipitated MsrA activity amounts to 97% of total MsrA, and MsrB1 activity is 82% of total MsrB activity.


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		Figure 2. Immunoprecipitation of MsrA and MsrB1 from human granulocytes. Rabbit polyclonal anti-MsrA and anti-MsrB1 antibodies used for immunoprecipitation recognize the respective proteins in unfractionated granulocyte extracts (A). The same antibodies were used to immunoprecipitate the respective proteins as described in Materials and Methods. The immunoprecipitated (P) and soluble (S) materials were assayed for the presence of MsrA- and MsrB-type enzymatic activity using the method described (B and C, histograms), and compared with the activity in the total cell lysate (T); n = 5 for MsrA, and n = 7 for MsrB. The equivalent of 5 x 105 cells was loaded in each lane (B and C, Western blots). (D) The MsrA and MsrB enzymatic activity is inhibited by the presence of the antibody. The enzymatic reactions were conducted exactly as those used for the immunoprecipitation experiments in the presence or absence of protein G (PG)-Sepharose. Results shown are from three independent experiments. Error bars represent the SD.

Most of the blood cell Msr activity is expressed in neutrophils
The granulocytic fraction of circulating leukocytes contains approximately 5 x 103 cells/µl blood, of which ~95% are neutrophils, 5% eosinophils, and 0.02% basophils [44 ]. Apart from the basophils, whose concentration is three orders of magnitude lower than that of neutrophils, eosinophils could contribute significantly to the Msr activity measured in the whole granulocyte populations obtained routinely. To study the levels of activity of eosinophils, a leukocyte suspension was enriched in eosinophils by density separation on Percoll gradients [36 ], followed by an immunomagnetic depletion step, which allows the removal of the following cell types: B cells, T cells, NK cells, neutrophils, dendritic cells, erythroid cells, and monocytes (see Materials and Methods), thus providing a pure population of eosinophils. During this procedure, other cell populations (platelets, mononuclear cells) were obtained and used to analyze Msr activity and expression. The purity of the populations of platelets, mononuclear cells, erythrocytes, and eosinophils was evaluated preliminarily by measuring the levels of contamination by neutrophils by immunodetection of the neutrophil-specific marker MMP-9. The contamination was found to be less than 0.5% (Fig. 3A ). Similarly, the purity of the granulocyte and mononuclear cell populations with regard to platelet contamination was evaluated by immunodetection of CD42b, a specific marker of platelets. This contamination was found to be less than 1% (Fig. 3B) . Levels of MsrA and MsrB enzymatic activity were then measured in whole lysates of the purified cell populations. As shown in Figure 4A and 4B , eosinophils were found to express MsrA and MsrB activities at levels corresponding, respectively, to ~30% and 18% of the specific activity in the total granulocyte population. As eosinophils represent only ~5% of the total granulocytes, the Msr activity measured in the total granulocyte population can therefore be considered to reflect essentially the activity of neutrophils. MsrA activity in mononuclear cells, platelets, and erythrocytes was extremely low and barely above background noise (Fig. 4A) . MsrB activity in the mononuclear cell fraction was similar to that of eosinophils, and it was virtually undetectable in platelets and erythrocytes (Fig. 4B) .


Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		Figure 3. Assessment of the purity of the blood cell preparations. After purification of the various cell populations, as described in Materials and Methods, the degree of contamination by neutrophils in the preparations of platelets, erythrocytes, mononuclear cells, and eosinophils (A) or the contamination by platelets in the preparations of granulocytes and mononuclear cells (B) was evaluated, respectively, by the presence of the neutrophil-specific marker MMP-9 or the platelet-specific marker CD42b. To this purpose, a calibration curve was constructed by loading SDS-PAGE gels with increasing quantities of neutrophil lysates (from 0.1 to 10 µg total protein) or platelet lysates (from 0.5 to 10 µg), from which to compare the levels of MMP-9 and CD42b, possibly present in lysates of the other cell populations. The latter was loaded in higher quantities (20 µg total protein) to ensure detection of even trace amounts of contamination. The blots shown are representative of at least three independent experiments.


Figure 4
View larger version (42K):
[in this window]
[in a new window]

  		Figure 4. Msr activity in various blood cell populations. The levels of MsrA-type and MsrB-type activities were measured, as described in Materials and Methods, in total granulocytes (n=4), eosinophils (n=3), mononuclear cells (n=2), platelets (n=3), and erythrocytes (n=3), as shown in the histograms in panels A and B, respectively, where the error bars represent the SD. For mononuclear cells, where the mean of two results is shown, they varied by less than ±5%. MsrA and MsrB1 proteins were immunodetected in the same cell populations as shown in the representative blots. SDS-PAGE gels (5–15% acrylamide gradient) were loaded with 5 µg/lane or 10 µg/lane total protein in A and B, respectively. Erythrocyte samples could not be probed because of the high amounts of hemoglobin giving rise to artifacts in the chemiluminescence detection system.

Msrs have a cytosolic, nongranular localization in granulocytes
As the cell lysates used in the immunoprecipitation experiments were prepared with the use of a nonionic detergent to ensure complete solubilization of the subcellular compartments (including the azurophil, specific, and gelatinase granules), the possibility existed that the Msr activities measured in these experiments were due to previously undetected Msr forms present in one or more types of cytosolic granules. To explore this possibility, neutrophils were activated with PMA or opsonized Zymosan to induce differential activation: maximal activation of the respiratory burst and degranulation but limited phagocytic activity, triggered by PMA; maximal phagocytic response with degranulation localized to the phagosome compartment and weaker respiratory burst induced by Zymosan [44 ]. The effectiveness of these activation processes was verified by measuring the release into the medium of MMP-9, a marker of specific and gelatinase granules, and of elastase, as a marker of azurophil granules (Fig. 5A ). The presence of MsrA and MsrB1 released into the extracellular medium and within the cells was then assayed by Western blotting with the corresponding antibodies, and the enzymatic activity was also measured in the two compartments after PMA or Zymosan treatment. As shown in Figure 5B and 5C , the two Msr forms were retained entirely within the activated cells by immunodetection and by enzymatic activity under conditions where the granule content was released partially (Zymosan) or completely (PMA) into the extracellular space, thus confirming a nongranular localization of MsrA and MsrB activities in neutrophils. The MsrB activity measured in the residual cells after activation with Zymosan appeared to be decreased, compared with control, unstimulated activity (see Discussion).


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		Figure 5. Intracellular and extracellular MsrA and MsrB during granulocyte activation. Granulocytes (25x106 cells/ml) were treated with PMA or Zymosan, as described in Materials and Methods. S, cell-free supernatant; C, cell pellet recovered after incubation. (A) Granulocyte activation was confirmed by measuring the levels of the granule proteins MMP-9 and elastase released into the extracellular medium. (B and C) The levels of MsrA-type and MsrB-type of activity (histograms), measured as described in Materials and Methods, and the immunodetection of MsrA and MsrB1 (Western blot) are shown, respectively. SDS-PAGE gels (10–20% acrylamide gradients) were loaded with an equivalent of 106 cells/lane. The activation with PMA was conducted in four independent experiments. The error bars represent the SD. Activation with Zymosan was performed in two experiments, whose results varied within a margin of ±10%.

RT-PCR of Msr transcripts in blood cells
The presence of MsrA, MsrB1, and MsrB2 transcripts was evaluated by qRT-PCR in cDNA obtained from total granulocytes, purified eosinophils, and human kidney. Initial attempts to normalize the results with respect to the classical housekeeping gene GAPDH to allow direct comparison of the levels of expression of the various Msrs in the different cell types were frustrated by the inconsistency of results, likely a result of the widely varying levels of expression of GAPDH in such diverse cell types as proliferating epithelial kidney cells and terminally differentiated granulocytes. Therefore, the data were analyzed relative to the most highly expressed Msr in the same cell type. As shown in Figure 6A 6B 6C , the most highly expressed Msr in total granulocytes and eosinophils was (cytosolic) MsrB1, and the transcript for (mitochondrial) MsrB2 was the most abundant form in human kidney, followed by MsrB1 and MsrA. This finding, again, is consistent with the prevalence of cytosolic forms of Msr in granulocytes and the lack of mitochondrial isoforms. The analysis by non-qRT-PCR for MsrB3A and MsrB3B confirmed this idea, as the only mRNA detected in granulocytes was that of MsrB3A (ER), and that of MsrB3B (mitochondrial) was completely undetectable. Conversely, both transcripts were present in human kidney at comparable levels (Fig. 6D) . Mononuclear cells expressed transcripts for MsrB3A and trace amounts of transcripts for MsrB3B. Both transcripts for MsrB3 isoforms were also found in HEK 293 cells, used here as a positive control, as shown previously [42 ].


Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		Figure 6. RT-PCR of Msr in various cell types. The relative levels of transcripts for MsrA, MsrB1, and MsrB2 were evaluated by qRT-PCR for granulocytes (A), eosinophils (B), and human kidney (C). No direct comparison between the levels of transcript of a given gene in different cell types is possible (see Materials and Methods and Discussion). Each histogram shows the mean and SD of three independent experiments, except for eosinophils, for which two experiments were conducted, whose results varied within ±20%. (D) The products of a RT-PCR reaction conducted, using cDNA from the cell types indicated, with primers allowing the simultaneous detection of the two isoforms of MsrB3 (MsrB3A and MsrB3B), are shown.


arrow
DISCUSSION
 
In this work, we report that neutrophils, unlike eosinophils, mononuclear cells, platelets, and erythrocytes, express high levels of Msr activity, similar to those found in other cell types already characterized for high expression of these enzymes. We show here, for the first time, that eosinophils, another polymorphonuclear phagocyte endowed with a functional NADPH-oxidase system capable of producing even higher amounts of ROS than neutrophils [45 ], express Msr at much lower levels than neutrophils. MsrB with a cytosolic localization is the major expressed form in neutrophils, and the mitochondrial forms are not expressed or else are only present at very low levels. In fact, mitochondria in neutrophils are of low number and appear to perform little, if any, oxidative phosphorylation, their role mainly restricted to modulation of the intrinsic pathway of apoptosis [46 , 47 ]. We could detect only minor amounts of mRNA for MsrB2, compared with the levels of the transcript for the cytosolic form MsrB1 (Fig. 6) . Whether these levels are directly proportional to the presence of the protein MsrB2 could not be determined, as a result of the unavailability of anti-MsrB2 antibodies. However, the immunoprecipitation experiments showed that more than 80% of MsrB activity could be precipitated with the anti-MsrB1 antibody used. Another clear indication that nonmitochondrial forms of Msr are expressed in neutrophils comes from the separate analysis of the levels of mRNA for the two alternatively spliced isoforms of MsrB3: Only the transcript corresponding to MsrB3A, the form that targets to the ER, is present in neutrophils, and MsrB3B, the mitochondrial form, is absent. The residual MsrB activity, which is nonimmunoprecipitable by anti-MsrB1 in lysates of neutrophils, could be therefore due to MsrB3A or possibly to residual MsrB2. As it was shown previously in studies with the recombinant enzyme that a peculiar feature of MsrB2 is its inhibition by substrate [15 ], we ran additional enzymatic assays in samples from the immunoprecipitation experiments with granulocyte lysates at different substrate concentrations, trying to unmask possible MsrB2-type activity. On the basis of the results obtained, we could rule out a significant presence of such activity (not shown). The HEK 293 cells were used in this study as a positive control for MsrB3 transcripts, as previously proposed [42 ]; we were also able to determine, for the first time, both transcripts of MsrB3 in the human kidney.

A quantitative real-time PCR method, based on the TaqMan system, was adopted in this study. This technique allows for the quantification of transcripts for a gene of interest relative to that of a housekeeping gene in a given cell type, thus permitting direct comparison of absolute levels of transcripts in different tissues. However, this quantitation is based on the assumption that levels of the housekeeping gene do not vary across samples, a situation that may not always be valid. The housekeeping gene initially chosen for our quantitative study was one that is commonly adopted in other studies—GAPDH—but was found to be too differently expressed in granulocytes and kidney cells to be usable as a normalizing control. There are serious concerns that GAPDH, as well as other common housekeeping genes, can be used as a valid reference in qRT-PCR, as the levels of its transcripts were shown to vary over as much as four to five orders of magnitude in normal human colon epithelium from different individuals [48 ]. Therefore, our results did not permit direct comparison of the Msr levels in the various cell types examined. However, they were still informative of the relative levels of transcripts of MsrA, MsrB1, and MsrB2 within a given cell type. Together, with the qualitative RT-PCR approach to the study of MsrB3, these results clearly indicated the net prevalence of non-mitochondrial forms of Msr in granulocytes.

Neutrophils are polymorphonuclear phagocytes involved in the innate immune response, which represents the first line of defense against infecting microorganisms. Neutrophils usually encounter the pathogens outside the circulatory system. Before this contact is made, a number of highly controlled and hierarchically organized events have to take place, such as the sensing by the neutrophil of chemoattractants, crossing the endothelial barrier, and the migration in the subendothelial space. In parallel with these steps, the neutrophils change their morphology, rearrange their cytoskeleton, and become "primed" through the expression of new surface receptors necessary for phagocytosis and NADPH-oxidase activity, so that they can recognize, phagocytose, and then kill the pathogens by first including them in the phagosome, followed by the production of superoxide ions. These are converted rapidly into various other oxidant species, such as hydrogen peroxide, hydroxyl ions, hypochlorite, and even gaseous chlorine, which are generated within the phagosome in parallel with the discharge into the phagosome of the cytoplasmic granules. The latter contains several proteolytic and hydrolytic enzymes that contribute to the neutralization of the pathogen.

Neutrophils contain multiple enzymatic and nonenzymatic systems for their protection against oxidative insults and repair of oxidative damage [49 ]. In view of the results presented here, Msr enzymes should now be considered an important addition to this list of antioxidants. The production of oxidants by the neutrophils is under strict control and does not appear to take place, even at low levels, in non-activated, circulating cells. It has thus been proposed that the complex antioxidant systems in neutrophils must have evolved to counteract auto-oxidation of the cell itself, a deleterious event that has been shown to occur during the oxidative burst [37 , 49 , 50 ].

Being that the neutrophils are terminally differentiated, it is unlikely that the protection of the machinery for cell replication is of high priority. It is conceivable that the antioxidants serve to maintain a functional intracellular environment while the cell is actively engaged in the phagocytosis/killing of pathogens and live until "the job is done," as previously suggested [49 ]. This would imply that Msrs, whose peculiar function is that of scavengers for ROS [18 ] and of a repair system, must be able to restore Met residues in the proteins of neutrophils during the most intense phase of the oxidative burst and/or in the immediate aftermath, when the functionality of the machinery of neutrophils is still needed to complete phagocytosis and neutralization of the pathogen.

In this respect, it is somewhat surprising that eosinophils, another professional phagocyte capable of higher production of oxidants than the neutrophil, express relatively low levels of Msr activity. There is, however, an important difference in the function of these cells in that eosinophils, which specialize in the killing of parasites without engulfing them in a phagosome, release the ROS and granule contents in the extracellular space rather than into the phagosome vacuole, as neutrophils do. This release of oxidants probably restricts the levels of oxidants produced intracellularly, and charged oxidants (e.g., superoxide) are unlikely to diffuse back into the cytoplasm to cause intracellular damage. In contrast, it has been shown that phagocytizing neutrophils are sensitive to oxidation of their own proteins [37 , 49 , 50 ]. It is interesting to note that MsrB activity appears decreased in Zymosan-activated neutrophils (Fig. 5C) . This could be related to auto-oxidation of MsrB1, when the reactive oxidant species are retained in close vicinity of the cell structures, as occurs when a phagosome is generated, instead of released in the extracellular medium, where they can rapidly diffuse away.

Nevertheless, the discrepancy in the levels of Msr expression in neutrophils and eosinophils remains and raises the question whether the powerful antioxidant machinery of neutrophils serves the sole scope of protection of the cell’s components from self-inflicted oxidation. It would be interesting, for instance, to verify if Msr activity plays any role in the function of resting, non-activated neutrophils. It is interesting that although the activity of the NADPH-oxidase of resting neutrophils is considered to be negligible, low levels of activity (perhaps below the level of detection of many assays) can be detected in the absence of the formation of phagolysosomes. In these cases, the NADPH-oxidase appears to undergo activation when still in the granules, without translocation to the plasma membrane, and superoxide and other oxidants may be found within the cytoplasm, rather than the phagosome [51 52 53 ].

It has also been proposed that the cell can use ROS for signaling purposes [54 ]. There is increasing evidence that the redox balance plays a key role in the regulation of apoptosis [55 ], an important process for the regulation of neutrophil function. If, in neutrophils, oxidants can be produced independently of the phagosome [51 52 53 ] and are present within the cytoplasm, where they can accelerate the apoptotic process [56 ], then antioxidants and hence Msr activities may play an anti-apoptotic role. It is therefore conceivable that Msrs are expressed at high levels to be functional at late stages of the cell’s lifespan: in the circulatory system, as a peripheral blood neutrophil, and in the extravasation to the sites of inflammation.

In this respect, it is noteworthy that a recent study, using microarray technology to analyze the transcriptional profiles of human peripheral blood neutrophils and their bone marrow precursors (promyelocytes, myelocytes, and bone marrow neutrophils; see ref. [57 ] and supplementary material therein), found transcripts for MsrB1 (identified in that study as selenoprotein X1) in the transcriptome of neutrophils. Selenoprotein X (or selenoprotein R) was first identified in silico [58 ] and later shown to be a Msr activity and renamed MsrB1 [12 ]. Selenoprotein X appears to be up-regulated throughout terminal granulopoiesis, showing an abrupt increase in expression from the promyelocyte to the myelocyte stage [57 ]. It is interesting that this pattern of transcription is superimposable to that of the components of the NADPH-oxidase: {alpha} and β subunits of the membrane-associated heterodimeric cytochrome b-245 (p22phox and p91phox, respectively) and the cytosolic components p47phox (neutrophil cytosolic factor 1), p67phox (neutrophil cytosolic factor 2), and p40phox (neutrophil cytosolic factor 4) [57 ], and the transcripts for other antioxidant enzymes, such as catalase, glutathione peroxidase, various peroxiredoxins, and superoxide dismutase 1 were all down-regulated starting from the myelocyte stage [57 ].

The data in this paper confirm that the cytosolic MsrB1 is expressed in neutrophils, and from its timing of expression during granulopoiesis, Msr activities in the circulating neutrophil are not vestigial forms but may play important roles in regulating neutrophil function in inflammation.


arrow
ACKNOWLEDGEMENTS
 
This work was supported with funds from the "Ministero dell’Istruzione, Università e Ricerca Scientifica," Italy (PRIN 2006). Part of this work was generated within the CellPROM project, funded by the European Community as contract no. NMP4-CT-2004-500039 under the 6th Framework Program for Research and Technological Development in the thematic area of "nanotechnologies and nano-sciences, knowledge-based multifunctional materials and new production processes and devices." We thank Prof. Herbert Weissbach and Prof. Steven Edwards for their critical reading of the manuscript.

Received July 26, 2007; revised September 3, 2007; accepted September 22, 2007.


arrow
REFERENCES
 
    1
  1. Vogt, W. (1995) Oxidation of methionyl residues in proteins: tools, targets, and reversal Free Radic. Biol. Med. 18,93-105[CrossRef][Medline]
    2. 2
  2. Christensen, B. W., Kjær, A. (1965) The absolute configuration of methionine sulphoxide Chem. Commun. (Camb.). 11,225-226
    3. 3
  3. Bentley, R. (2005) Role of sulfur chirality in the chemical processes of biology Chem. Soc. Rev. 34,609-624[CrossRef][Medline]
    4. 4
  4. Ejiri, S. I., Weissbach, H., Brot, N. (1979) Reduction of methionine sulfoxide to methionine by Escherichia coli J. Bacteriol. 139,161-164[Abstract/Free Full Text]
    5. 5
  5. Brot, N., Weissbach, L., Werth, J., Weissbach, H. (1981) Enzymatic reduction of protein-bound methionine sulfoxide Proc. Natl. Acad. Sci. USA 78,2155-2158[Abstract/Free Full Text]
    6. 6
  6. Fliss, H., Vasanthakumar, G., Schiffmann, E., Weissbach, H., Brot, N. (1982) Enzymatic reduction of oxidized chemotactic peptide N-formyl-L-methionyl-sulfoxide-L-leucyl-L-phenylalanine Biochem. Biophys. Res. Commun. 109,194-201[CrossRef][Medline]
    7. 7
  7. Minetti, G., Balduini, C., Brovelli, A. (1994) Reduction of DABS-L-methionine-dl-sulfoxide by protein methionine sulfoxide reductase from polymorphonuclear leukocytes: stereospecificity towards the l-sulfoxide Ital. J. Biochem. 43,273-283[Medline]
    8. 8
  8. Delaye, L., Becerra, A., Orgel, L., Lazcano, A. (2007) Molecular evolution of peptide methionine sulfoxide reductases (MsrA and MsrB): on the early development of a mechanism that protects against oxidative damage J. Mol. Evol. 64,15-32[CrossRef][Medline]
    9. 9
  9. Sharov, V. S., Ferrington, D. A., Squier, T. C., Schöneich, C. (1999) Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase FEBS Lett. 455,247-250[CrossRef][Medline]
    10. 10
  10. Moskovitz, J., Poston, J. M., Berlett, B. S., Nosworthy, N. J., Szczepanowski, R., Stadtman, E. R. (2000) Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity J. Biol. Chem. 275,14167-14172[Abstract/Free Full Text]
    11. 11
  11. Grimaud, R., Ezraty, B., Mitchell, J. K., Lafitte, D., Briand, C., Derrick, P. J., Barras, F. (2001) Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase J. Biol. Chem. 276,48915-48920[Abstract/Free Full Text]
    12. 12
  12. Kryukov, G. V., Kumar, R. A., Koc, A., Sun, Z., Gladyshev, V. N. (2002) Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase Proc. Natl. Acad. Sci. USA 99,4245-4250[Abstract/Free Full Text]
    13. 13
  13. Moskovitz, J., Singh, V. K., Requena, J., Wilkinson, B. J., Jayaswal, R. K., Stadtman, E. R. (2002) Purification and characterization of methionine sulfoxide reductases from mouse and Staphylococcus aureus and their substrate stereospecificity Biochem. Biophys. Res. Commun. 290,62-65[CrossRef][Medline]
    14. 14
  14. Etienne, F., Spector, D., Brot, N., Weissbach, H. (2003) A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide Biochem. Biophys. Res. Commun. 300,378-382[CrossRef][Medline]
    15. 15
  15. Kim, H. Y., Gladyshev, V. N. (2004) Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases Mol. Biol. Cell 15,1055-1064[Abstract/Free Full Text]
    16. 16
  16. Moskovitz, J., Berlett, B. S., Poston, J. M., Stadtman, E. R. (1999) Methionine sulfoxide reductase in antioxidant defense Methods Enzymol. 300,239-244[Medline]
    17. 17
  17. Friguet, B. (2006) Oxidized protein degradation and repair in ageing and oxidative stress FEBS Lett. 580,2910-2916[CrossRef][Medline]
    18. 18
  18. Stadtman, E. R., Moskovitz, J., Berlett, B. S., Levine, R. L. (2002) Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism Mol. Cell. Biochem. 234–235,3-9
    19. 19
  19. Hoshi, T., Heinemann, S. (2001) Regulation of cell function by methionine oxidation and reduction J. Physiol. 531,1-11[Abstract/Free Full Text]
    20. 20
  20. Stadtman, E. R., Van Remmen, H., Richardson, A., Wehr, N. B., Levine, R. L. (2005) Methionine oxidation and aging Biochim. Biophys. Acta 1703,135-140[Medline]
    21. 21
  21. Koc, A., Gladyshev, V. N. (2007) Methionine sulfoxide reduction and the aging process Ann. N. Y. Acad. Sci. 1100,383-386[CrossRef][Medline]
    22. 22
  22. Etienne, F., Resnick, L., Sagher, D., Brot, N., Weissbach, H. (2003) Reduction of Sulindac to its active metabolite, sulindac sulfide: assay and role of the methionine sulfoxide reductase system Biochem. Biophys. Res. Commun. 312,1005-1010[CrossRef][Medline]
    23. 23
  23. Moskovitz, J., Weissbach, H., Brot, N. (1996) Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins Proc. Natl. Acad. Sci. USA 93,2095-2099[Abstract/Free Full Text]
    24. 24
  24. Gonzalez Porque, P., Baldesten, A., Reichard, P. (1970) The involvement of the thioredoxin system in the reduction of methionine sulfoxide and sulfate J. Biol. Chem. 245,2371-2374[Abstract/Free Full Text]
    25. 25
  25. Ejiri, S. I., Weissbach, H., Brot, N. (1980) The purification of methionine sulfoxide reductase from Escherichia coli Anal. Biochem. 102,393-398[CrossRef][Medline]
    26. 26
  26. Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N., Weissbach, H. (2006) Thionein can serve as a reducing agent for the methionine sulfoxide reductases Proc. Natl. Acad. Sci. USA 103,8656-8661[Abstract/Free Full Text]
    27. 27
  27. Sagher, D., Brunell, D., Brot, N., Vallee, B. L., Weissbach, H. (2006) Selenocompounds can serve as oxidoreductants with the methionine sulfoxide reductase enzymes J. Biol. Chem. 281,31184-31187[Abstract/Free Full Text]
    28. 28
  28. Kim, H. Y., Gladyshev, V. N. (2005) Role of structural and functional elements of mouse methionine-S-sulfoxide reductase in its subcellular distribution Biochemistry 44,8059-8067[CrossRef][Medline]
    29. 29
  29. Kim, H. Y., Gladyshev, V. N. (2006) Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases BMC Mol. Biol. 7,11[CrossRef][Medline]
    30. 30
  30. Lee, J. W., Gordiyenko, N. V., Marchetti, M., Tserentsoodol, N., Sagher, D., Alam, S., Weissbach, H., Kantorow, M., Rodriguez, I. R. (2006) Gene structure, localization and role in oxidative stress of methionine sulfoxide reductase A (MSRA) in the monkey retina Exp. Eye Res. 82,816-827[CrossRef][Medline]
    31. 31
  31. Brot, N., Fliss, H., Coleman, T., Weissbach, H. (1984) Enzymatic reduction of methionine sulfoxide residues in proteins and peptides Methods Enzymol. 107,352-360[Medline]
    32. 32
  32. Swaim, M. W., Pizzo, S. V. (1988) Methionine sulfoxide and the oxidative regulation of plasma proteinase inhibitors J. Leukoc. Biol. 43,365-379[Abstract]
    33. 33
  33. Beutler, E., West, C., Blume, K. G. (1976) The removal of leukocytes and platelets from whole blood J. Lab. Clin. Med. 88,328-333[Medline]
    34. 34
  34. Böyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g Scand. J. Clin. Lab. Invest. Suppl. 97,77-89[Medline]
    35. 35
  35. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Horecker, B. L. (1985) Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase Proc. Natl. Acad. Sci. USA 82,6435-6439[Abstract/Free Full Text]
    36. 36
  36. Cramer, R., Dri, P., Zabucchi, G., Patriarca, P. (1992) A simple and rapid method for isolation of eosinophilic granulocytes from human blood J. Leukoc. Biol. 52,331-336[Abstract]
    37. 37
  37. Voetman, A. A., Weening, R. S., Hamers, M. N., Meerhof, L. J., Bot, A. A., Roos, D. (1981) Phagocytosing human neutrophils inactivate their own granular enzymes J. Clin. Invest. 67,1541-1549[Medline]
    38. 38
  38. Lavine, T. F. (1947) The formation, resolution, and optical properties of the diastereoisomeric sulfoxides derived from L-methionine J. Biol. Chem. 169,477-491[Free Full Text]
    39. 39
  39. Greenstein, J. P., Winitz, M. (1984) Chemistry of the Amino Acids vol. 3,2125-2155 Krieger Malabar, FL, USA.
    40. 40
  40. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227,680-685[CrossRef][Medline]
    41. 41
  41. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc. Natl. Acad. Sci. USA 76,4350-4354[Abstract/Free Full Text]
    42. 42
  42. Kim, H. Y., Gladyshev, V. N. (2004) Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase Biochem. Biophys. Res. Commun. 320,1277-1283[CrossRef][Medline]
    43. 43
  43. Petropoulos, I., Mary, J., Perichon, M., Friguet, B. (2001) Rat peptide methionine sulphoxide reductase: cloning of the cDNA, and down-regulation of gene expression and enzyme activity during aging Biochem. J. 355,819-825[CrossRef][Medline]
    44. 44
  44. Edwards, S. W. (1994) Biochemistry and Physiology of the Neutrophil Cambridge University Press Cambridge, UK.
    45. 45
  45. DeChatelet, L. R., Shirley, P. S., McPhail, L. C., Huntley, C. C., Muss, H. B., Bass, D. A. (1977) Oxidative metabolism of the human eosinophil Blood 50,525-535[Abstract/Free Full Text]
    46. 46
  46. Fossati, G., Moulding, D. A., Spiller, D. G., Moots, R. J., White, M. R., Edwards, S. W. (2003) The mitochondrial network of human neutrophils: role in chemotaxis, phagocytosis, respiratory burst activation, and commitment to apoptosis J. Immunol. 170,1964-1972[Abstract/Free Full Text]
    47. 47
  47. Maianski, N. A., Geissler, J., Srinivasula, S. M., Alnemri, E. S., Roos, D., Kuijpers, T. W. (2004) Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis Cell Death Differ. 11,143-153[CrossRef][Medline]
    48. 48
  48. Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays J. Mol. Endocrinol. 25,169-193[Abstract]
    49. 49
  49. Splettstoesser, W. D., Schuff-Werner, P. (2002) Oxidative stress in phagocytes—"the enemy within" Microsc. Res. Tech. 57,441-455[CrossRef][Medline]
    50. 50
  50. Fliss, H., Weissbach, H., Brot, N. (1983) Oxidation of methionine residues in proteins of activated human neutrophils Proc. Natl. Acad. Sci. USA 80,7160-7164[Abstract/Free Full Text]
    51. 51
  51. Lundqvist, H., Follin, P., Khalfan, L., Dahlgren, C. (1996) Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told J. Leukoc. Biol. 59,270-279[Abstract]
    52. 52
  52. Vaissiere, C., Le Cabec, V., Maridonneau-Parini, I. (1999) NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils J. Leukoc. Biol. 65,629-634[Abstract]
    53. 53
  53. Karlsson, A., Nixon, J. B., McPhail, L. C. (2000) Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase J. Leukoc. Biol. 67,396-404[Abstract]
    54. 54
  54. Lander, H. M. (1997) An essential role for free radicals and derived species in signal transduction FASEB J. 11,118-124[Abstract]
    55. 55
  55. Danial, N. N., Korsmeyer, S. J. (2004) Cell death: critical control points Cell 116,205-219[CrossRef][Medline]
    56. 56
  56. Lundqvist-Gustafsson, H., Bengtsson, T. (1999) Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils J. Leukoc. Biol. 65,196-204[Abstract]
    57. 57
  57. Theilgaard-Monch, K., Jacobsen, L. C., Borup, R., Rasmussen, T., Bjerregaard, M. D., Nielsen, F. C., Cowland, J. B., Borregaard, N. (2005) The transcriptional program of terminal granulocytic differentiation Blood 105,1785-1796[Abstract/Free Full Text]
    58. 58
  58. Lescure, A., Gautheret, D., Carbon, P., Krol, A. (1999) Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif J. Biol. Chem. 274,38147-38154[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0707492v1
jlb.0707492v2
83/1/181    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Achilli, C.
Right arrow Articles by Minetti, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Achilli, C.
Right arrow Articles by Minetti, G.