Characterization of Calcineurin in Human Neutrophils
INHIBITORY EFFECT OF HYDROGEN PEROXIDE ON ITS ENZYME ACTIVITY
AND ON NF-
B DNA BINDING*
Modesto
Carballo
§,
Gracia
Márquez
§¶,
Manuel
Conde
,
José
Martín-Nieto
,
Javier
Monteseirín**,
José
Conde**,
Elizabeth
Pintado
, and
Francisco
Sobrino
**
From the
Departamento de Bioquímica
Médica y Biología Molecular, Facultad de Medicina,
** Departamento de Medicina, Servicio Regional de Inmunología y
Alergia, Hospital Universitario Virgen Macarena, Universidad de
Sevilla, 41009 Seville and
Departamento de
Biotecnología, Campus Universitario San V. del Raspeig,
Universidad de Alicante, Alicante, Spain
 |
ABSTRACT |
We describe here a specific calcineurin activity
in neutrophil lysates, which is dependent on Ca2+,
inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both
the A and B chains of calcineurin. Neutrophils treated with cyclosporin
A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited
with aminotriazole, exhibited a specific inhibition of calcineurin
activity. However, the addition of reducing agents to neutrophil
extracts partially reversed the inhibition caused by
H2O2. A similar inhibitory effect of
H2O2 on calcineurin activity was observed to
occur in isolated lymphocytes. This is the first demonstration that
redox agents modulate calcineurin activity in a cellular system. In
addition, electrophoretic mobility shift assays revealed that
lipopolysaccharide-induced activation of NF-
B in human neutrophils
is inhibited by cell pretreatment with H2O2 in
a dose-dependent manner. These data indicate that
calcineurin activity regulates the functional activity of
lipopolysaccharide-induced NF-
B/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of
calcineurin activity and that the
H2O2-dependent NF-
B inactivation
in neutrophils occurs in concert with inhibition of calcineurin.
 |
INTRODUCTION |
Calcineurin (CN)1 has recently been established as a
key enzyme in the signal transduction
cascade leading to T cell activation (1-4), and an important regulator
of transcription factors such as NF-AT, NF-
B, and AP-1, which are
involved in the expression of a number of important T cell early genes,
i.e. interleukin-2, tumor necrosis factor-
, and
interleukin-2R (5-8). CN, also known as phosphatase 2B, is a
calcium/calmodulin-dependent serine/threonine phosphatase
(9-11) and is composed of the following two subunits: a 59-kDa
catalytic subunit (CNA), which contains a calmodulin-binding domain and
an autoinhibitory region, and a 19-kDa intrinsic calcium-binding regulatory subunit (CNB) (12-14). Human CN possesses a Fe-Zn active center. The assignment of stoichiometric amounts of Zn2+
and Fe3+ in the CNA center is based on atomic absorption
experiments (15). The same assignments were made for the di-metal site
in the structure of some CNA (16).The central role of CN in T cell
signaling was appreciated by its identification as the target of the
immunosuppressive drugs cyclosporin A (CsA) and FK 506 (1-4). The
phosphatase activity of CN is inhibited by either drug when complexed
to intracellular binding proteins (immunophilins), i.e. CsA
to cyclophilin and FK 506 to the FK 506-binding protein 12 (FKBP12),
respectively. Neither drug nor immunophilin alone bind to or affect the
activity of CN (1). This phosphatase is expressed ubiquitously in
eukaryotic cells. In mammals, CN is most abundant in the brain (17) but has also been detected in T cells (1-4). On the other hand, it is
known that NF-AT-mediated transactivation depends on the CN activity
(18, 19). Other findings suggest that NF-
B activity is also under CN
control (20-22).
In neutrophils, only indirect evidence has been presented on the
occurrence of the phosphatase CN. The treatment of these cells with
inhibitors of CN (e.g. CsA and FK 506) inhibited the neutrophils chemokinesis on vitronectin matrix (23, 24). Furthermore, intracellular calcium and CN regulate neutrophil motility on
vitronectin through a receptor identified by antibodies against the
integrins
v and
3 (25, 26). The first purpose of the
present work was to assess the presence of CN in neutrophils using as a
substrate a specific peptide corresponding to the phosphorylation site
of the RII subunit of cyclic AMP-dependent protein kinase.
Additionally, reactive oxygen intermediates (ROI) have been implicated
in mediating signal transduction by a variety of stimuli in lymphoid
cells, and transcription factors seem to be responsible for the
inducible expression of a number of genes in response to oxidative
stress (27, 28). In this context, the addition of
H2O2 to the culture medium has been shown to
activate NF-
B (29). Hydroxyl radicals produced from
H2O2 cannot function as diffusible
intracellular messengers, since they can react with the nearest
molecule in a nonspecific fashion. A more suitable ROI messenger would
be the less reactive H2O2. However, arguments against ROI involvement in NF-
B activation have been published (30-32), and despite the fact that phorbol 12-myristate
13-acetate-dependent NF-
B stimulation is cancelled by
antioxidants, it has been recently shown that phorbol 12-myristate
13-acetate does not increase intracellular ROI (33). Peroxide-mediated
stimulation of NF-
B appears to be cell line-specific, since
N-acetylcysteine, an antioxidant, elicited up-regulation of
NF-
B binding activity in monocyte-derived macrophages (34).
Moreover, NF-
B is not the only nuclear factor whose activity is
altered by H2O2. In a fashion opposite to that observed for NF-
B, NF-AT has been shown to be actively suppressed by
H2O2 in Jurkat T cells (35). Recently, the
development of stabilized peroxovanadium compounds has provided the
opportunity to more fully characterize the action of oxidants within
the cell (36). Treatment of lymphocytes with sodium
oxodiperoxo(1,10-phenanthroline)vanadate (V), pV(phen), results in a
large increase in intracellular oxidation, which correlates with a
strong induction of cellular tyrosine phosphorylation and activation of
kinases. The same authors (37) have documented that antioxidant
treatment does not prevent the activation of NF-
B by pV(phen).
Therefore, we have also used pV(phen) as another tool to study the
effect of oxidative stress on calcineurin activity.
In summary, previous data suggest that, first, CN modulates the DNA
binding activity of essential transcription factors (e.g. NF-AT and NF-
B), and second, ROI regulates positively or negatively those transcription factors. However, a link between both signals, that
is CN and ROI, is lacking. In this paper we address this question and
the implications of the ROI as universal messenger to activate
NF-
B.
 |
EXPERIMENTAL PROCEDURES |
Cell and Reagents--
Neutrophils were isolated from fresh
heparinized blood of healthy human donors by dextran sedimentation,
followed by Ficoll-Paque gradient centrifugation and hypotonic lysis of
residual erythrocytes as indicated (38). Neutrophils were washed twice
in Hanks' balanced salt solution (HBSS), suspended at a density of
1 × 107 cells/ml in HBSS supplemented with 0.1 mg/ml
BSA, and maintained at 37 °C in an atmosphere of 5% CO2
and 95% O2 for 1-2 h. Peripheral blood lymphocytes were
obtained from heparinized venous blood of normal volunteers by
Ficoll-Paque centrifugation.
Dextran T-500 was obtained from Pharmacia Biotech (Barcelona, Spain).
Ficoll-Paque, HBSS, and RPMI 1640 were obtained from BioWhittaker
(Verviers, Belgium). CsA and FK 506 were kindly provided by Dr. S. F. Borel (Sandoz Ltd., Basel, Switzerland) and Fujisawa GmbH
(München, Germany), respectively. Chemicals were of analytical grade from Merck (Darmstadt, Germany). Dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) were obtained from Boehringer Mannheim (Barcelona, Spain). Bovine serum albumin (BSA), okadaic acid,
trifluoroperazine, pyrrolidine dithiocarbamate (PDTC), hydrogen peroxide (30% v/v), 3-amino-1,2,4-triazole (aminotriazole, AMT), soybean trypsin inhibitor, leupeptin, aprotinin, Nonidet P-40, and goat
anti-rabbit IgG conjugated to horseradish peroxidase were purchased
from Sigma (Madrid, Spain). Rabbit anti-bovine calcineurin IgG was
kindly provided by C. B. Klee. The synthetic peptide used as a
substrate for calcineurin was purchased from Peninsula Laboratories
(Bellmont, CA). pV(phen) was synthesized as described previously (37).
[
-32P]ATP was obtained from NEN Life Science Products.
2-Mercaptoethanol, SDS, acrylamide,
N,N'-methylene-bisacrylamide, Coomassie Brilliant Blue
R-250, and blotting nitrocellulose membranes were purchased from
Bio-Rad. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and
diisopropyl fluorophosphate (DFP) were purchased from Serva (Madrid,
Spain), and 4-iodophenol was from Aldrich (Madrid, Spain). Molecular
weight standards (Rainbow markers) was obtained from Amersham Corp.
(London, UK); Sephadex G-25 was from Pharmacia (Barcelona, Spain); and
double-stranded oligonucleotide probe (5'-AGTTGAG GGGACTTTCC CAGGC-3')
containing NF-
B sites was from Boehringer Mannheim Gmbh (Mannheim,
Germany). 2',7'-Dichlorodihydro-fluorescein diacetate
(H2DCFDA) was purchased from Molecular Probes (Leiden, The Netherlands).
Crude Neutrophils Extract--
Untreated neutrophils (1 × 108 cells/ml) were lysed for 30 min on ice in 500 µl of
buffer A (50 mM Tris, pH 8.0, 0.5% Triton X-100, 150 mM NaCl, 50 µg/ml PMSF, 50 µg/ml soybean trypsin
inhibitor, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and
disrupted by sonication. The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant fluid (crude
extract) was separated from low molecular weight material by passage
through a 0.5 × 10-cm Sephadex G-25 column equilibrated with 50 mM Tris-Cl, pH 7.5, 0.1 mM EDTA, 0.1%
-mercaptoethanol, and a mixture of protease inhibitors as above (39). Fractions containing most of the A280 nm
material eluting with the void volume of the column were pooled and
used to measure CN activity.
Cell Treatments and Lysis--
Immunosuppressive agents were
dissolved in dimethyl sulfoxide (Me2SO) at a concentration
1000-fold higher than that used for cell treatments. Neutrophils
(7 × 106 cells/ml) were suspended in 1 ml of HBSS
supplemented with 0.1 mg/ml BSA in microcentrifuge tubes, 1 µl of
Me2SO or CsA or FK 506 was added, and the cells were
incubated at 37 °C for 2 h. For experiments with
H2O2 and other stimuli, neutrophils were incubated at 37 °C. The incubation times and concentration of agents
are indicated in the figure legends. After incubation, the cells were
washed once with 1 ml of HBSS on ice and lysed in 60 µl of buffer B
(50 mM Tris, pH 7.5, 0.1 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 50 µg/ml
PMSF, 50 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and
10 µg/ml aprotinin) and disrupted by sonication. Cell debris was
removed by centrifugation at 4 °C for 10 min at 12,000 × g, and supernatant was used as the source of CN.
Calcineurin Phosphatase Assay--
CN phosphatase activity was
measured using an assay adapted from Hubbard and Klee (40), basically
as described (22). Neutrophils (7 × 106 cells/ml)
were incubated for 2 h at 37 °C in the presence or absence of
drugs as indicated in the text. Reaction mixtures containing 2 µM 32P-labeled phosphopeptide, 500 nM okadaic acid (added to inhibit PP-1A and PP-2A type
phosphatase activities), and 20 µl of cell lysate (about 80 µg of
protein) were incubated in a total volume of 60 µl of assay buffer C
(20 mM Tris, pH 8.0, 100 mM NaCl, 6 mM MgCl2, 0.5 mM dithiothreitol,
and 0.1 mM CaCl2 or 5 mM EGTA, as
indicated in the figures) for 15 min at 30 °C. After this time, reactions were terminated by the addition of 0.5 ml of 100 mM potassium phosphate buffer, pH 7.0, containing 5%
trichloroacetic acid. The reaction mixture was passed through a
500-µl column of activated Dowex cation-exchange resin, and free
inorganic phosphate was quantitated in the eluate by scintillation
counting. It was verified that at 15 min of incubation time the assay
was linear. Assays were performed in triplicate, and the counts/min
measured in blank assay lacking cell lysate were subtracted. Data are
expressed as the number of picomoles of 32PO4
released in 15 min per mg of protein.
32P-Labeled Phosphopeptide--
The synthetic
peptide
(Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu),
corresponding to a segment of the RII subunit of
cAMP-dependent kinase (41), was phosphorylated on the
unique serine residue by the catalytic subunit of
cAMP-dependent protein kinase using
[
-32P]ATP, essentially as described (40), and used as
phosphatase substrate. The specific activity of fresh preparations of
32P-labeled phosphopeptide was about 500 µCi/µmol peptide.
Western Blot Analysis--
Cells (7 × 106
cells/ml) were lysed for 30 min on ice in 100 µl of buffer A (see
above). Lysates were clarified by centrifugation at 4 °C for 2 min
at 12,000 × g. Protein concentrations in the lysates
were determined by the Bradford method (42), using BSA as a standard.
For the Western blot analysis of CN subunits A and B, neutrophil
lysates were subjected to 12.5% SDS-PAGE followed by electroblotting
onto nitrocellulose using the Bio-Rad Mini-blotting apparatus. Filters
were blocked for 1 h in TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.5) containing 3% BSA. Furthermore, they
were rinsed twice with TBS containing 0.1% Tween 20 (TBST), and they were incubated overnight with rabbit anti-bovine calcineurin IgG diluted 1:1000 in TBST. After three washes in TBST, the filters were
incubated for 90 min with horseradish peroxidase-conjugated goat
anti-rabbit IgG (1:5000, final dilution). Filters were then washed
twice with TBS and then twice with TBST for 10 min each time. The bound
secondary antibody was detected by enhanced chemiluminescence (43).
Briefly, the membranes were incubated for 1 min in 10 ml of fresh
luminescent reagent solution, composed of 10 mM Tris-HCl, pH 8.5, 2.25 mM luminol, 0.015% (v/v)
H2O2, and 0.45 mM 4-iodophenol, the
latter acting as an enhancer of the chemiluminescence reaction (44).
These concentrations of luminol, H2O2, and
4-iodophenol were determined to be optimal for maximum light
production.2 Luminol and
4-iodophenol were freshly prepared in 10 ml of 10 mM
Tris-HCl, pH 8.5. Luminol was previously dissolved in 50 µl of 1 M NaOH. The use of Me2SO as solvent should be
avoided since in alkaline/Me2SO conditions, luminol
autoxidizes with emission of intense luminescence (45). After 1 min of
incubation, the membranes were placed on paper filter, covered with
Saran Wrap, and exposed to x-ray films (X-Omat, Eastman Kodak Co.) in
the dark for 1-5 min.
Electrophoretic Mobility Shift Assays (EMSA)--
Human
neutrophils whose viability exceeded 98% after 3 h in culture, as
determined by trypan blue exclusion, were resuspended at a density of
5 × 106 cells/ml in RPMI 1640 medium supplemented
with 10% fetal calf serum. The cells were then incubated at 37 °C
under a 5% CO2 atmosphere for 3 h, with occasional
shaking in the absence or presence of 1 µg/ml LPS, 1 µg/ml CsA, or
H2O2 at the concentrations indicated in each
experiment. Then nuclear extracts were prepared basically as described
by McDonald et al. (46). With this purpose, cell suspensions
were transferred into pre-cooled tubes containing an equivalent volume
of ice-cold RPMI 1640 supplemented with DFP (2 mM, final
concentration). After centrifugation at 4,000 × g for
1 min at 4 °C, the cells were resuspended in 100 µl of ice-cold relaxation buffer, consisting of 10 mM HEPES, pH 7.3, 30 mM KCl, 3 mM NaCl, 3.5 mM
MgCl2, 1.25 mM EGTA, and 0.5 mM
DTT, supplemented with an antiprotease mixture composed of 2 mM DFP, 1 mM PMSF, 10 mM
iodoacetamide, 1 mM benzamidine, and 10 µg/ml each of
aprotinin, leupeptin, and captopril. Cells were disrupted by short
sonication (1 s), and the lysates were spun at 3,000 × g for 10 min at 4 °C to pellet unbroken cells and intact
nuclei. The pellets were resuspended in 100 µl of ice-cold relaxation
buffer, again subjected to short (1 s) sonication, and respun as above
to pellet intact nuclei. These were resuspended in 200 µl of
relaxation buffer, and after new centrifugation, the nuclear pellet was
resuspended in 25 µl of ice-cold relaxation buffer, additionally
containing 10% (v/v) glycerol and 380 mM NaCl. Following a
20-min incubation on ice with occasional mixing, the samples were spun
at 13,000 × g for 15 min at 4 °C, and the resulting
supernatants were stored at
70 °C. EMSA was performed by using as
a probe the double-stranded 22-base pair NF-
B consensus
oligonucleotide indicated above, which was labeled with DIG using the
labeling kit from Boehringer Mannheim. The nuclear extracts (5 µg of
protein) were assayed for
B binding activity using the DIG gel shift
assay kit (Boehringer Mannheim, GmbH). The reactions were performed in
15 µl of binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1%
(v/v) Nonidet P-40, 6% glycerol) and allowed to proceed for 20 min at
room temperature. For competition assays, binding reactions were
performed in the presence of the unlabeled oligonucleotide (100-fold
molar excess) for 20 min at room temperature. Supershift assays
including anti-p50 and anti-p65 were carried out as described previously (46). The samples were finally electrophoresed on 5%
polyacrylamide native gels at 4 °C in 0.25× TBE.
 |
RESULTS |
Calcineurin Is Present in Neutrophil Lysates--
CN is a well
characterized phosphatase that plays an important role in T cell
activation pathways (1-4). In this work we have characterized the
presence of CN activity and immunoreactive CN protein in human
neutrophils. In crude neutrophil lysates, specific CN activity was
observed, and a linear appearance of product during the assay was
obtained in the range of 20-75 µg of lysate proteins (Fig.
1A). The time course of CN
activity is shown in Fig. 1B. Clearly, 32P
release increased linearly along assay time until 15 min, and then a
slow activity was found. Fig. 1 (inset) illustrates an immunoblotting analysis of CN expression in lymphocytes (lane 1) and different amounts of human neutrophil lysates (lanes
2-4). We used an antiserum that recognizes both the A (59 kDa)
and B (19 kDa) chains of CN, confirming that both subunits are
expressed in human neutrophils. As shown, neutrophil CNB migrates as a
16-kDa band in SDS-polyacrylamide gels, and it is detected along with a
predominant CNA band migrating at 59 kDa. A third band, detected at 57 kDa in lymphocytes extracts, is probably a proteolytic fragment of CNA
generated during preparation of the cell lysates. In some preparations
of neutrophil lysates, a similar band of 55-57 kDa was also found
(data not shown). Next experiments were addressed to analyze the
regulation of CN activity in crude neutrophil lysates. Fig.
2 illustrates that the dephosphorylation
of the CN-specific substrate peptide by crude neutrophil lysates was
Ca2+-dependent, as well as insensitive to
okadaic acid, a potent and specific inhibitor of phosphatases 1A and 2A
(reviewed in Ref. 47). When 500 nM okadaic acid was
included in the assays, nearly all of the remaining phosphatase
activity was Ca2+-dependent and could be
eliminated by substituting 5 mM EGTA for Ca2+
(Fig. 2). In contrast, the okadaic acid-sensitive component was resistant to EGTA, which is consistent with the reported
Ca2+ independence of phosphatases 1A and 2A (17). CN
activity was abrogated in nominally calcium-free medium and in the
presence of a known inhibitor of calmodulin, trifluoroperazine (48). However, trifluoroperazine did not inhibit calcium-independent, okadaic
acid-sensitive phosphatases from neutrophil lysates (Fig. 2). Taken
together, these data indicate that a specific
Ca2+/calmodulin-dependent phosphatase activity
is present in the neutrophil lysates.

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Fig. 1.
Specific dephosphorylation of a synthetic
peptide substrate by crude neutrophil lysates. Calcineurin
phosphatase activity was measured as indicated under "Experimental
Procedures." Reaction mixtures contained 2 µM
32P-labeled phosphopeptide and 500 nM okadaic
acid in a total volume of 60 µl of assay buffer C. A,
calcineurin activity was assayed using increasing amounts of neutrophil
lysates during 15 min of incubation at 30 °C. B,
calcineurin activity was assayed during the indicated times with 50 µg of protein from neutrophil lysates. Inset,
immunoblotting analysis of calcineurin expression. Proteins were
resolved by SDS-PAGE (12% polyacrylamide gel), transferred to
nitrocellulose, and probed with an antiserum that recognizes both the A
and B chains of calcineurin. Lane 1, blood human
lymphocytes (50 µg of protein); lanes 2-4,
crude neutrophil lysates (25, 50, and 100 µg of protein,
respectively). Size markers are indicated on the left.
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Fig. 2.
Effect of calcium, okadaic acid, and
trifluoroperazine on calcineurin activity from crude neutrophil
lysates. Reaction mixtures contained 20 µl of crude neutrophil
lysate (80 µg of protein), 2 µM 32P-labeled
phosphopeptide, and 40 µl of assay buffer. Other additions were as
follows: 0.1 mM CaCl2, 5 mM EGTA,
500 nM okadaic acid (OA), or 200 nM
trifluoroperazine (TFP), as indicated. CN activity was
assayed during 15 min at 30 °C. Phosphatase activity is expressed as
picomoles of phosphate released per min per mg of protein.
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Inhibition of Calcineurin Activity in Isolated Neutrophils Treated
with Immunosuppressive Drugs--
As previously indicated (1-4), CsA
and FK 506 can now be used as tools to elucidate the participation of
CN on signal transduction processes. To assess whether treatment with
these drugs inhibits CN activity, neutrophils were incubated with
different concentrations of FK 506 and CsA for 2 h, and
phosphatase activity was measured in cell lysates. Both agents
effectively inhibited Ca2+-dependent
phosphatase activity, as shown in Fig. 3.
These results indirectly suggested that the drug-sensitive phosphatase
present in neutrophils is CN. Furthermore, in drug titration
experiments both FK 506 and CsA inhibited CN activity in a
concentration-dependent fashion. IC50 values
determined for CN inhibition were approximately 0.5 ng/ml for FK 506 and 5 ng/ml for CsA. This greater sensitivity to FK 506 than to CsA
exhibited by neutrophil CN is similar to that previously described for
lymphocyte CN (1-4).

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Fig. 3.
Calcineurin phosphatase activity in CsA and
FK 506-treated neutrophils. Neutrophils (7 × 106
cells/ml) were incubated in 1 ml of incubation medium with the
indicated doses of CsA and FK 506 for 2 h at 37 °C. Then the
cells were harvested and washed, and the pellet was lysed in buffer B. Calcineurin activity was assayed in the cell lysates (80 µg of
protein) as indicated under "Experimental Procedures." Phosphatase
activity is expressed as picomoles of phosphate released per min per mg
of protein. Three experiments were performed with similar
results.
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Effect of Oxidants on Calcineurin Activity--
As previously
indicated, there is a clear relationship between the oxidative stress
and activation or suppression of transcription factor activity
(27-29). Since it has been shown that H2O2
suppresses the transcriptional activation of NF-AT (35) and that NF-AT is able to directly interact with CN (18, 19), we have explored whether
H2O2 could alter CN activity. Fig.
4 illustrates that preincubation of
neutrophils with H2O2 alone had no effect on this phosphatase. However, the preincubation of cells with AMT, an
inhibitor of catalase, for 30 min and the further addition of
H2O2 produced a clear decrease in CN activity.
These data suggest that intracellular catalase in neutrophils (49)
diminishes the effective concentration of exogenously added
H2O2 and also indicate a potential protective
role of catalase against CN inactivation. The preincubation of cells
with AMT/H2O2 and PDTC, an antioxidant, partially ameliorated the inhibitory effect of
H2O2 on CN activity. Neither AMT nor PDTC added
by themselves had any effect on CN activity. The antioxidant action of
PDTC against AMT/H2O2 inhibition was found to
occur only when equimolar concentrations of
H2O2 and PDTC were used; at 500-1000
µM H2O2 the preincubation with 100 µM PDTC was without effect (data not shown). A
similar inhibitory effect of AMT/H2O2 on CN
activity was observed to occur in isolated human lymphocytes. The
treatment of these cells with 500 µM
H2O2 or 50 µM pV(phen) for 1 h resulted in a marked decrease in CN activity (54 and 43% inhibition
after H2O2 and pV(phen)-treatment, respectively). To explain this inhibitory effect of
H2O2 on CN activity two possibilities may be
raised: (i) H2O2 alters the CN polypeptide
structure, and (ii) H2O2 damages CN prosthetic group structure. To test the first hypothesis, lysates from neutrophils previously incubated with AMT and H2O2 were
analyzed by immunoblotting using a specific CN antibody. Fig.
5 shows that these agents did not alter
the ability of the A and B subunits to be recognized by the
anti-calcineurin antibody, suggesting that the CN protein structure
remained intact. However, the modification of prosthetic groups
associated with the catalytic center of CN remains a possibility. Previous data (50) describe a protective role of superoxide dismutase
on partially purified CN from brain, suggesting that the environment
redox can alter the structure of Fe-Zn center of CN in a reversible
manner. In agreement with this possibility we have observed (Fig.
6) that the inhibition of CN by
H2O2 in a neutrophil cell-free system can be
partially reversed by the further addition of Fe2+, DTT, or
ascorbate, all of which act as reductants. A previous report on the
protective effect of these agents was documented in a partially
purified CN assay activity from brain (50). Thus, our results are more
consistent with a reversible modification of the catalytic center of CN
elicited by H2O2.

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Fig. 4.
Inhibition of calcineurin activity in
neutrophils treated with hydrogen peroxide. Neutrophils (7 × 106 cells/ml) were incubated at 37 °C under the
following conditions: with 100 µM
H2O2 alone ( ); preincubated with 25 mM AMT for 30 min and then with 100 µM
H2O2 ( ); preincubated with 25 mM
AMT for 30 min, then 100 µM PDTC for 10 min, and finally
treated with 100 µM H2O2 ( ).
The times of incubation with H2O2 are
indicated. Incubation of cells was stopped at different times, and
calcineurin activity was assayed in cell lysates (80 µg of protein)
as indicated under "Experimental Procedures." Phosphatase activity
is expressed as picomoles of phosphate released per min per mg of
protein. Means ± S.E. values from three separate experiments
performed in triplicate are presented.
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Fig. 5.
Immunoblot analysis of the stability of
calcineurin structure. Neutrophils (1 × 107
cells/ml) were incubated under the following conditions:
lane 1, no additions (control); lane
2, 0.5 mM H2O2 for 30 min;
lane 3, 25 mM AMT for 30 min and then
0.5 mM H2O2 for 30 min;
lane 4, 100 µM PDTC for 10 min and
then 0.5 mM H2O2 for 30 min;
lane 5, 25 mM AMT for 30 min and then
100 µM PDTC for 10 min; lane 6, 25 mM AMT for 30 min followed by 100 µM PDTC for
10 min and then 0.5 mM H2O2 for 30 min; lane 7, T cells without treatment. Proteins
were resolved by SDS-PAGE (12% polyacrylamide gel), transferred to
nitrocellulose, and probed with a polyclonal antiserum against
calcineurin. The band at 28 kDa is probably a proteolytic fragment of
calcineurin A generated during the preparation of cell lysates.
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Fig. 6.
Reversibility by Fe2+, ascorbate,
and dithiothreitol of H2O2-inhibited
calcineurin in neutrophil lysates. Cell lysates (80 µg of
protein) were incubated with 5 mM AMT for 5 min, followed
with 100 µM H2O2 for 10 min. When
present, 0.5 mM ferrous sulfate (Fe2+), 5 mM DTT, or 5 mM sodium ascorbate
(Asc) were added, either alone or altogether, for 20 min
before the peptide substrate for the assay of calcineurin activity was
added. Phosphatase activity is expressed in relation to the value
obtained without cell treatment (Control).
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H2O2 rapidly diffuses away from and into cells.
In order to analyze the ability of neutrophils to internalize exogenous
H2O2, intracellular oxidant levels were
monitored by measuring the fluorescence of H2DCFDA, a
highly fluorescent probe sensitive to peroxides (51). Cells labeled
with H2DCFDA displayed a significant increase in DCF
fluorescence upon incubation with H2O2
(154.5 ± 3.2 fluorescence units in 100 µM
H2O2-treated cells versus 12.7 ± 1.1 units in control cells). In other experiments, the treatment of
neutrophils with a variety of physiological and pharmacological stimuli
revealed that agents that promoted an increase of intracellular
H2O2 also induced a dose-dependent
inhibition of CN activity in human neutrophils. Notably, both tumor
necrosis factor-
(50 ng/ml) and interferon-
(50 units/ml)
elicited an inhibition of CN activity of about 25% after 2 h of
treatment. Increased times of tumor necrosis factor-
treatment
(e.g. 8 h) resulted in an enhanced inhibitory effect (of about 35%) on CN activity. However, LPS (100 ng/ml),
platelet-activating factor, or glucocorticoids were without effect on
CN activity (data not shown).
To investigate whether the H2O2 effect was
specific, we also analyzed the CN activity with a new peroxovanadium
compound, pV(phen), which causes intracellular oxidative stress and
induces strong protein tyrosine phosphorylation (37). The treatment of
neutrophils with pV(phen) caused a dose- and time-dependent inhibition of CN activity (Fig. 7). After
60 min of incubation of the cells with 50 µM pV(phen), an
inhibition of about of 60% was observed, with an apparent
IC50 of about 30 µM. Therefore, we provide
evidence that the observed inhibition of CN by
H2O2 could be the result of oxidative stress.
Under these conditions (e.g. in the presence of
H2O2 and pV(phen)), the cell viability was
determined to be about 90%, and hence significant cytotoxic effects
can be ruled out. Also, a strong increase in the intracellular phosphotyrosine levels was detected in neutrophil lysates after incubation of the cells with pV(phen),2 in agreement with
previous reports on B lymphocytes (37).

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|
Fig. 7.
Inhibition of calcineurin activity in
neutrophils incubated with pV(phen). A, neutrophils
(7 × 106 cells/ml) were treated with 50 µM pV(phen) at 37 °C at different times. B,
neutrophils (7 × 106 cells/ml) were treated with
different pV(phen) concentrations at 37 °C for 1 h. Calcineurin
activity was assayed in cell lysates (80 µg of protein). Phosphatase
activity is expressed as picomoles of phosphate released per minute per
mg of protein. Means ± S.E. values from three separated
experiments performed in triplicate are presented.
|
|
EMSA analyses were carried out in order to test whether the changes in
CN activity promoted by H2O2 were accompanied
by an alteration of the DNA binding activity of two transcription
factors, namely NF-AT and NF-
B. No binding activity was detected on
neutrophil nuclear extracts when the NF-AT probe was used. However,
activated NF-
B was detectable as a uniquely positioned band in
assays of nuclear extracts from human neutrophils stimulated with LPS
(1 µg/ml) (Fig. 8). On the basis of the
results from McDonald et al. (46) and of supershift assays
using anti-p50 and anti-p65 antibodies (data not shown), we interpreted
the upper band as corresponding to the activated form of NF-
B
(i.e. the p50/p65 tetramer). Gel retardation analysis of
extracts from neutrophils stimulated with LPS and different doses of
H2O2 demonstrated that H2O2 selectively inhibits the activation of
NF-
B, resulting in a gradual decrease of the intensity of the
p50/p65 band, as H2O2 was increased. As a
negative control, we also analyzed the effect of CsA (1 µg/ml)
(lane 1). In agreement with previous data from other cell
lines (1-4), the p50/p65 band was only barely detectable in extracts
from CsA/LPS-treated neutrophils. These results are consistent with an
NF-
B activity regulated by CN in neutrophils, in agreement with
previous reports (20-22). Present data concerning H2O2 inhibition of CN in neutrophils and
lymphocytes suggest that previous evidence on
H2O2-stimulated NF-
B functional activity (27, 29) can be interpreted as the result that peroxides, above a
threshold level, are able to bypass the CN modulatory step and regulate
transcription factors activity independently of CN.

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|
Fig. 8.
Reduced binding activity of NF- B in
H2O2-treated human neutrophils. Binding
activity of nuclear proteins (5 µg) from human neutrophils to a
digoxigenin-labeled oligonucleotide containing the consensus NF- B
binding sequence was assessed by EMSA (see "Experimental
Procedures"). The cells were untreated (lanes 1 and 7) or incubated with H2O2 (0.1, 0.5, 1, and 2 mM; lanes 3,
4, 5, and 6, respectively) for 15 min
prior to LPS stimulation (1 µg/ml) for 10 min. Before treatment with
H2O2, cells were incubated with AMT (25 mM) for 30 min. CsA (1 µg/ml, lane
9) was added 30 min prior to LPS stimulation (1 µg/ml)
without AMT. A control including a 100-fold excess of unlabeled
oligonucleotide in the reaction performed with nuclear extracts from
LPS-treated human neutrophils is also shown (lane
10). The figure shows a representative experiment out of
three carried out with identical results.
|
|
 |
DISCUSSION |
Only a few reports concerning CN in neutrophils and presenting
indirect evidence of its presence in these cells have been published
(23-26). All of them deal with neutrophil motility on vitronectin and
its inhibition by CsA and FK 506 (23-26), a couple of well known
inhibitors of CN activity. Here we have analyzed accurately, using a
synthetic peptide corresponding to the phosphorylation site of the RII
subunit of cyclic AMP-dependent protein kinase, the
presence of CN in human neutrophils. Lysates from neutrophils dephosphorylate this substrate in a dose- and
time-dependent manner. CN activity in neutrophils is
insensitive to okadaic acid, whereas other phosphatases
(e.g. 1A and 2A) are sensitive to this inhibitor (47). In
the presence of okadaic acid the CN activity from neutrophil lysates is
both Ca2+-dependent and
Ca2+-sensitive to inhibition by trifluoroperazine, a
calmodulin inhibitor. In addition, we present evidence that two well
known immunosuppressants and inhibitors of CN activity in lymphocytes,
CsA and FK 506 (1-4), potently depressed CN activity in neutrophils.
It is well accepted that CN plays an important role as a prominent
component of the calcium signaling pathway in T cells, by acting as an
obligatory step between immunosuppressive drugs (e.g. CsA
and FK 506) and some transcription factors (e.g. NF-AT and
NF-
B) (1-4, 20, 21). The mechanism by which CN activates NF-AT
seems rather complex. It has been shown that the coexpression in
transfected cells of the activated CN and activated p21ras
could mimic T cell receptor signaling during NF-AT induction, both
acting as cooperative partners during T cell activation (52). Also, recent data show that CN forms a complex with cytosolic NF-AT4 (an
isoform of NF-AT), which is transported to the nucleus where CN
continues to dephosphorylate NF-AT4 (53). On the other hand the
important role of ROI in the regulation of some transcription factors,
mainly NF-
B, is stressed by its activation in response to the
addition of H2O2 (29), although doubts that it
represents a universal phenomenon has been raised (30-32, 34).
Moreover, an inverse relationship between ROI and NF-AT has also been
pointed out, since low levels of H2O2 can
actively suppress the transcription activity of NF-AT and the
expression of interleukin-2 mRNA (35). These studies indicate that
CN is a key component of the T cell signal transduction cascade and
that oxidative signals can positively or negatively regulate
transcription factor activity (54, 55). However, a deep knowledge of
the molecular mechanism connecting both components (i.e. CN
and oxidative signals) is lacking. We present here evidence for the
first time that human neutrophils treated with
H2O2 or pV(phen) exhibit a suppression of CN
activity and that H2O2 effect required the
previous inhibition of catalase activity. Only when catalase was
inhibited by AMT a clear decrease of CN activity in the presence of
exogenous H2O2 was observed. As expected, we
have observed that the preincubation of neutrophils with the
antioxidant PDTC cancelled the inhibition of CN activity by
H2O2. As a preliminary effort toward the
elucidation of the functional consequences of
H2O2-dependent CN inhibition, we
focused our attention on the transcription factor NF-AT as a potential target. However, we were unable to find any NF-AT DNA binding activity
in nuclear extracts from neutrophils (data not shown). This fact
closely agrees with previous results from immunoblotting analysis
pointing out the absence of NF-AT proteins in neutrophils (56).
Subsequent experiments were thus addressed to analyze whether
H2O2-dependent CN inactivation
could affect NF-
B activation. Conflicting results have been
published on the presence or absence of NF-
B in human neutrophils.
Browning et al. (57) did not observe any NF-
B activation
in these cells. However, Cassatella and co-workers (46) described the
presence of NF-
B subunits as well as the existence of NF-
B DNA
binding activity in human neutrophils. We have followed the methodology
described by the latter authors, with minor modifications, and have
detected NF-
B DNA binding activity in fresh human neutrophils,
together with its inhibition by H2O2. These
data indicate that NF-
B activation is modulated by CN activity in
human neutrophils.
Indirect evidence that CN is an enzyme sensitive to its redox
environment has been reported, based on the fact that superoxide dismutase protects CN from spontaneous inactivation in brain crude extracts (50). This inactivation was interpreted as resulting from
oxidative damage of the Fe-Zn active center of CN (50). Since there is
a good evidence that CN is an Fe-Zn-containing enzyme (16), the
hypothetical mechanism that can be proposed for this oxidative damage
is that H2O2 and pV(phen) could modify the
redox state of the Fe-Zn center in the catalytic site and thereby
inactivate the enzyme. This mechanism is in agreement with the
observation that the reactivation of
H2O2-inhibited neutrophil CN requires the
addition of reducing agents, such as Fe2+, ascorbate, and
DTT (Fig. 6), as it was also previously demonstrated for a preparation
of CN from brain (50). The observed inhibition of CN in intact
neutrophils by oxidants, such as H2O2 and
pV(phen), represents a novel mechanism of action for these agents. The
pV(phen) molecule presents a dual activity, acting both as an
intracellular oxidant and as an inhibitor of phosphotyrosine
phosphatase (37). Evidence also has been presented that the activities
of both protein tyrosine phosphatase and protein phosphatase 2A were
reduced after H2O2 treatment of intact Jurkat T
cells (58). Previously it has been described that CN has a regulatory
phosphorylation site that is phosphorylated by the
Ca2+-independent form of calmodulin-kinase II. This
phosphorylated CN exhibits a 50% decrease in its
Vmax and 2-fold increase in the
Km values (59). Thus, a hypothetical model in which oxidants modulate CN activity through phosphorylation of its regulatory site can therefore be postulated in the light of present data.
An apparent discrepancy between CN inhibition by
H2O2 and other oxidant species, described here,
and the protection exerted on CN by superoxide dismutase (50), which
converts anion superoxide on H2O2, may be
raised. In this context, however, the role of catalase and peroxidases,
as detoxicant enzymes that degradate H2O2,
should be introduced. These enzymes are also mutually protective, and
therefore synergistic, when both O
2 and
H2O2 are being made. In fact, experimental
evidence (Fig. 4) illustrates the absence of effect by exogenous
H2O2 added alone and the requirement of AMT to
inhibit intracellular catalase and to detect
H2O2-dependent CN inactivation.
These data indirectly provide the notion of catalase as an additional
protecting enzyme for CN against ROI inactivation. The implication of
this suggestion is that, under normal physiological conditions, the
cellular CN may be relatively less susceptible to molecular oxidative
damage by ROI. Conversely, in pathological process, such as
inflammation and reperfusion injury, or situations characterized by an
inhibition of ROI-detoxicant enzymes, the CN inactivation by oxidant
species may take place, and it could be a relevant process in the
context of the oxidative stress state.
In summary, available evidence indicates that CN activity is a
redox-sensitive step in cellular signaling cascades and suggests that
the inhibition of NF-AT from lymphocytes (35) and NF-
B from
neutrophils (present work) by H2O2 is elicited
through inactivation of CN.
 |
ACKNOWLEDGEMENT |
We thank Dr. C. B. Klee for
anti-calcineurin antiserum and for the critical reading of manuscript.
 |
FOOTNOTES |
*
This work was supported in part by Fondo Investigaciones
Sanitarias Grants 94/1484 and 97/1289 (to F. S.) and Grant 97/207 (to
J. C.) and the Fundation of SEAIC of Spain.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
The first two authors contributed equally to this work.
¶
Recipient of a grant from the Fondo Investigaciones Sanitarias
of Spain.

To whom correspondence should be addressed: Dept. de
Bioquímica Médica y Biología Molecular, Facultad
de Medicina, Ave. Sánchez Pizjuan 4, Sevilla-41009, Spain. Fax:
34-95-4907041; E-mail: fsobrino{at}cica.es.
2
M. Carballo and F. Sobrino, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
CN, calcineurin;
NF-AT, nuclear factor of activated T cells;
NF-
B, nuclear factor
B;
CsA, cyclosporin A;
CNA, calcineurin A subunit;
CNB, calcineurin
B subunit;
ROI, reactive oxygen intermediates;
H2O2, hydrogen peroxide;
pV(phen), sodium
oxodiperoxo(1,10-phenanthroline)vanadate(V);
HBSS, Hanks' balanced
salt solution;
BSA, bovine serum albumin;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
PDTC, pyrrolidine dithiocarbamate;
AMT, 3-amino-1,2,4-triazole;
PAGE, polyacrylamide gel electrophoresis;
DFP, diisopropyl fluorophosphate;
H2DCFDA, 2',7'-dichlorodihydrofluorescein diacetate;
EMSA, electrophoretic
mobility shift assay;
LPS, lipopolysaccharide..
 |
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