Oxidative and nitrative modifications of enkephalins by human neutrophils: Effect of nitroenkephalin on leukocyte functional responses

Article abstract of DOI:10.1007/s00726-011-1147-0

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid

Full text of DOI:10.1007/s00726-011-1147-0

Amino Acids (2012) 43:875–884
DOI 10.1007/s00726-011-1147-0
ORIGINAL ARTICLE
Oxidative and nitrative modifications of enkephalins
by human neutrophils: effect of nitroenkephalin
on leukocyte functional responses
Elisabetta Capuozzo
Federica Giovannitti
Mario Fontana
•
Laura Pecci
Alessia Baseggio Conrado
•
•
•
Received: 2 July 2011 / Accepted: 3 November 2011 / Published online: 24 November 2011
Ó Springer-Verlag 2011
Abstract Neutrophils play a major role in acute inflam-
mation by generating reactive oxygen/nitrogen species.
Opioid peptides, including enkephalins, are present at
inflammation sites. Neutrophils contribute to protect against
inflammatory pain by releasing opioid peptides. In this
investigation, the ability of human polymorphonuclear cells
to induce oxidative and nitrative modifications of Leu-
enkephalin has been investigated in vitro. Activated human
neutrophils mediate the oxidation of Leu-enkephalin
resulting in the production of dienkephalin. In the presence
of nitrite at concentrations observed during inflammatory
and infectious process (10–50 lM), nitroenkephalin, a
nitrated derivative of Leu-enkephalin, is additionally
formed. The yield of nitroenkephalin increases with nitrite
concentration and is significantly inhibited by the addition of
catalase or 4-aminobenzoic acid hydrazide (ABAH), a spe-
cific inhibitor of peroxidases. These results suggest that
neutrophils induce nitration of Leu-enkephalin by a mech-
anism that is dependent on myeloperoxidase activity and
hydrogen peroxide. Oxidative/nitrative modifications of
Leu-enkephalin have been also evidenced when cells were
treated with the NO-donor molecule, DEANO. The nitrated
enkephalin has been examined for its effect on leukocyte
functional responses. The data reveal that nitroenkephalin at
micromolar concentrations inhibits superoxide anion gen-
eration and degranulation of azurophilic granules of human
polymorphonuclear cells. Moreover, nitroenkephalin inhib-
its spontaneous apoptosis of neutrophils, as evaluated by
measuring caspase-3 activity. Collectively, our data indicate
that the nitrated enkephalin attenuates neutrophil activation
and promotes the short-term survival of these cells, sug-
gesting a possible role of the nitrocompound in the efficiency
and resolution of inflammatory processes.
Keywords Enkephalin ꢀ Nitroenkephalin ꢀ Neutrophils ꢀ
Protein nitration ꢀ Inflammation ꢀ Apoptosis
Abbreviations
ABAH
4-Aminobenzoic acid hydrazide
Acetyl-Asp-Glu-Val-Asp-7-amido-
4-methylcoumarin (caspase-3
substrate)
Ac-DEVD-AMC
DEANO
fMLP
Diethylamine NONOate
N-Formyl-methionyl-leucyl-
phenylalanine
HRP
Horseradish peroxidase
Leucine enkephalin
Leu-enkephalin
MeO-Suc-Ala-
N-Methoxysuccinyl-Ala-Ala-Pro-
Ala-Pro-Val-AMC Val-7-amido-4-methylcoumarin
(elastase substrate)
MPO
ODQ
Myeloperoxidase
1H-[1,2,4]Oxadiazole[4,3-
a]quinoxalin-1-one
PMA
PMNs
SOD
Phorbol 12-myristate 13-acetate
Polymorphonuclear cells
Superoxide dismutase
E. Capuozzo ꢀ L. Pecci ꢀ F. Giovannitti ꢀ
A. Baseggio Conrado ꢀ M. Fontana (&)
Introduction
Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli’’,
Istituto di Biologia e Patologia Molecolari del CNR, Sapienza
Universit a` di Roma, P.le Aldo Moro 5, 00185 Rome, Italy
e-mail: mario.fontana@uniroma1.it
Enkephalins belong to a class of bioactive peptides known
for their endogenous opiate agonist activity (Frederickson
123

8
76
E. Capuozzo et al.
1
977; Hughes et al. 1975). Mainly enkephalins, endorphins
the structure and the function of numerous biomolecules
(Eiserich et al. 1998a; Lim et al. 2002; Vadseth et al. 2004;
Quijano et al. 2005; Souza et al. 2008). It has been
observed that various reactive nitrogen species react in
vitro with Leu-enkephalin. As a consequence of these
reactions, the tyrosine amino-terminal residue of enkeph-
alin is converted either to 3-nitrotyrosine producing nitro-
enkephalin or dityrosine with the production of an
enkephalin dimer, the dienkephalin (Fontana et al. 2006).
Up to now, no study has been published on nitrative bio-
chemistry of enkephalins with neutrophil-generated reac-
tive oxidants.
and dynorphins represent the endogenous opioid peptides.
Endorphins and enkephalins share a common amino-ter-
minal sequence and exhibit a tyrosine residue at the amino
terminus, which is essential for their biological activity
(
Terenius et al. 1976). The aromatic amino acid residues
are especially susceptible to oxidation by various forms of
reactive oxygen species (ROS). In particular, enkephalins
and opioid peptides react easily with ROS giving rise to
oxidized products (Fontana et al. 2001; Nagy et al. 2009;
Rosei 2001).
Opioid peptides, including enkephalins, are present at
inflammation sites (Padr o´ s et al. 1989; Sibinga and Gold-
stein 1988; Vindrola et al. 1990). During inflammatory
process, the migration of opioid-containing leukocytes to
inflamed tissues is induced. The secretion of opioid pep-
tides by polymorphonuclear cells (PMNs), monocytes and
lymphocytes can relieve inflammatory pain by interacting
with peripheral opioid receptors (Cabot 2001; Przewlocki
et al. 1992; Rittner et al. 2001; Stein et al.1990). Therefore,
inflammatory response can be associated with the release
of high levels of opioid peptides. It has been reported that
opioid peptides, such as methionine-enkephalin and beta-
endorphin, are involved in neutrophil priming and that
beta-endorphin also exerts immunosuppressive potency
In the present investigation, we explored the ability of
human PMNs to induce oxidative/nitrative modifications of
Leu-enkephalin. Moreover, the nitrated enkephalin has
been examined for its effect on leukocyte functional
response. The data reveal that nitroenkephalin inhibits the
respiratory burst and degranulation of azurophilic granules
of human PMNs and their spontaneous apoptosis in vitro.
These findings are of potential interest, inasmuch nitroen-
kephalin may act as modulator of tissue inflammatory
response.
Materials and methods
(
McCain et al. 1982; Pasnik et al. 1999). Opioid peptides
have been reported to bind to specific receptors on human
phagocytic leukocytes and to modulate the activation of
neutrophils (Diamant et al. 1989; Lopker et al. 1980;
Menzebach et al. 2003).
Chemicals
Leucine-enkephalin (Leu-enkephalin), horseradish peroxidase
(HRP), diethylamine NONOate sodium salt (DEANO),
Activated neutrophils produce a variety of powerful
inflammatory mediators such as reactive oxygen species,
enzymes and cytokines that play a key role in the host
defenses against invading microorganisms, but can also be
responsible for tissue damage. The reactive oxygen species
are produced by neutrophils as a result of NADPH oxidase
activation (reactions named ‘‘respiratory burst’’) (Hampton
et al. 1998; Witko-Sarsat et al. 2000). NADPH oxidase can
be activated by various stimuli such as chemotactic pep-
tide, N-formyl-methionyl-leucyl-phenylalanine (fMLP) or
phorbol 12-myristate 13-acetate (PMA). Neutrophil func-
tion in vivo can be also regulated through NO-dependent
mechanisms (Belenky et al. 1993; Fialkow et al. 2007;
Moilanen et al. 1993). Nitric oxide (NO) modulates neu-
trophil pro-inflammatory responses and plays a protective
role against priming and activation of these cells (Arm-
strong 2001; Clancy et al. 1992; Dal Secco et al. 2003;
Grisham et al. 1999). However, when generated at elevated
levels during inflammation by inducible NO synthase, NO
is readily transformed into potent nitrating and oxidizing
phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-
Phe (fMLP), N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-
methylcoumarin (MeO-Suc-Ala-Ala-Pro-Val-AMC, elastase
substrate), acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcou-
marin (Ac-DEVD-AMC, caspase-3 substrate), porcine
pancreas elastase, human recombinant caspase-3, naloxone
and 4-aminobenzoic acid hydrazide (ABAH) were obtained
from Sigma-Aldrich, Inc (St. Louis, MO, USA). 3-Nitroty-
rosine, potassium nitrite, hydrogen peroxide, cytochrome
c from horse heart and cytochalasin B were from Fluka
Chemie GmbH (Buchs, CH). 1H-[1,2,4]oxadiazole
[4,3-a]quinoxalin-1-one (ODQ, guanylate cyclase inhibitor)
was from Alexis Co (Lausen, CH). All other chemicals were
of analytical grade.
Synthesis and purification of nitroenkephalin
Nitroenkephalin was enzymatically synthesized via nitra-
tion of Leu-enkephalin by peroxidase-catalyzed oxidation
of nitrite. Briefly, Leu-enkephalin (2 mM) was incubated
with 1 lM horseradish peroxidase (HRP) and 10 mM
potassium nitrite in 20 mM K-phosphate buffer at pH 7.4.
-
species, including peroxynitrite (ONOO ), nitrogen diox-
ꢀ
ide radical ( NO ), and nitrous acid (HONO) (Pryor and
2
Squadrito 1995; Radi et al. 2001), which can modify both
1
23

Oxidative and nitrative modifications of enkephalins
877
The reaction was started by the addition of 1 mM H O and
2
Respiratory burst of neutrophils
2
allowed to proceed at 37°C. The reaction was stopped after
h by the addition of 10 nM catalase. Aliquots of the
1
Superoxide productionby NADPH oxidase was estimated by
measuring the rate of superoxide dismutase-inhibitable
reduction of cytochrome c at 550 nm (e = 21,100
reaction mixture were chromatographed on HPLC to
accomplish the purification of nitroenkephalin. Elution was
-
1
-1
performed on
7.8 9 300 mm; 6 lm particle size). The mobile phases
were—A: 0.2% aqueous trifluoroacetic acid containing
0% acetonitrile; B: acetonitrile:water (50:50, v/v); linear
gradient: from A to 100% B over 30 min; and flow rate:
.5 mL/min. The eluent was monitored at 274 and 360 nm
a
Prep Nova-Pak HR C18 column
M
cm for ferrocytochrome c) by a modification of the
method described by Lehmeyer et al. (1979). The incubation
(
6
mixture contained 2 9 10 cells/mL, 80 lM cytochrome
1
c in phosphate-buffered saline containing 0.5 mM MgCl
,
2
0.5 mM CaCl and 5 mM glucose. After 3 min of preincu-
2
1
bation at 37°C, the reaction was started by adding 1 lg/mL
PMA or 0.1 lM fMLP. Cytochalasin B (1 lg/mL) was
added to fMLP-stimulated PMNs. The controls contained, in
addition, 20 lg/mL superoxide dismutase. Steady-state
velocity of superoxide production was estimated from the
linear part of the reaction curve. Where used, the soluble
guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-
a]quinoxalin-1-one (ODQ, 4 lM) was preincubated 20 min
with cells before the assay.
and the fractions eluting between 28 and 30 min, corre-
sponding to nitroenkephalin, were pooled and lyophilized
(
freeze/dried). The product was analyzed by HPLC as
already described (Fontana et al. 2006) and nitroenkephalin
was quantified from peak area using 3-nitrotyrosine as
reference standard. Nitroenkephalin was stored in
K-phosphate buffer, pH 7.4, at -20°C until use.
Isolation of neutrophils
Determination of neutrophils degranulation by elastase
release
Leukocytes were purified from heparinized human blood
freshly drawn from healthy donors. Leukocyte preparations
containing 90–98% neutrophils and apparently free of
contaminating erythrocytes were obtained by a one-step
procedure involving centrifugation of blood samples lay-
ered on Ficoll-Hypaque medium (Polymorphprep, Axis-
Shield, Oslo, Norway) (Ferrante and Thong 1980). The
cells were suspended in isotonic phosphate-buffered saline,
pH 7.4, with 5 mM glucose and stored on ice. Each prep-
aration produced cells with a viability higher than 90% up
to 6 h after purification. The viability of the cells was
measured by trypan blue exclusion test.
Degranulation of azurophilic granules was determined by
elastase release (Sklar et al. 1982). Elastase release was
measured by hydrolysis of the elastase substrate (MeO-
Suc-Ala-Ala-Pro-Val-AMC). Briefly, isolated neutrophils
6
(2 9 10 cells/mL) were resuspended in phosphate-buf-
fered saline containing 0.5 mM MgCl , 1 mM CaCl ,
2 2
5 mM glucose and 1 lg/mL cytochalasin B at 37°C. The
elastase substrate (MeO-Suc-Ala-Ala-Pro-Val-AMC) was
added at a final concentration of 40 lM. After 3 min of
preincubation at 37°C, the reaction was started by adding
1
lM fMLP, and elastase activity was monitored fluoro-
Oxidative/nitrative modifications of Leu-enkephalin
by neutrophils
metrically (excitation wavelength 380 nm, emission
wavelength 460 nm).
6
Human PMNs (2 9 10 cells) suspended in phosphate-
Detection of neutrophil apoptosis by caspase-3 activity
assay
buffered saline containing 0.5 mM MgCl , 0.5 mM CaCl
2
2
and 5 mM glucose were incubated with 1 mM Leu-
enkephalin at 37°C in the absence or in the presence of
different concentrations of potassium nitrite. After 5 min of
incubation, PMNs were stimulated by the addition of 1
lg/mL PMA or 0.1 lM fMLP. Cytochalasin B (1 lg/mL)
was added to f-MLP-stimulated PMNs. Reactions were
terminated by placing tubes on ice and immediately adding
SOD (90 U/mL) and catalase (500 U/mL) to scavenge
residual oxidants. PMNs were pelleted by centrifugation
Caspase-3 activity was tested in neutrophil lysates by mea-
suring the release of the fluorescent 7-amino-4-methyl-
coumarin (AMC) moiety from the synthetic substrate
acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-
6
DEVD-AMC). Neutrophils (5 9 10 cells), preincubated at
37°C for 3.5 h in phosphate-buffered saline containing
0.5 mM MgCl , 1 mM CaCl and 5 mM glucose were col-
2 2
lected by centrifugation and lysed in 0.5 mL volumes of
50 mM HEPES, 5 mM 3-[3-(cholamidopropyl)dimethyl-
ammonio]-1-proanesulfonate (CHAPS), 5 mM dithiothrei-
tol (DTT), pH 7.4, 10 lM 4-amidinophenylmethanesulfonyl
fluoride (APMSF), 10 lg/mL pepstatin and 10 lg/mL
aprotinin. The reaction was started by adding 100 ll aliquots
(
1,200g, 5 min, 4°C) and the products in the supernatant
determined by HPLC as already described (Fontana et al.
006). When used, ABAH (100 lM final concentration)
2
was added to the incubation mixture 5 min before neutro-
phil activation.
123

8
78
E. Capuozzo et al.
of the lysates in 2 mL solutions containing 16 lM Ac-
DEVD-AMC, 20 mM HEPES, 0.1% CHAPS, 5 mM DTT-
and 2 mM EDTA, pH 7.4. The assay mixture was incubated
at 20°C in the dark for 1 h. The fluorescence (excitation
wavelength 360 nm, emission wavelength 460 nm) increase
was compared with an appropriate blank control containing
1
0 lM acetyl-Asp-Glu-Val-Asp-al, a specific caspase-3
inhibitor (Nicholson et al. 1995) or a standard preparations of
recombinant caspase-3 (Sigma). A calibration curve
obtained with standard AMC solutions was employed for
quantitative analysis.
Fig. 1 Oxidative/nitrative modification of Leu-enkephalin by PMA-
stimulated neutrophils. Leu-enkephalin (1 mM) was incubated with
Statistical analysis
6
2
5
9 10 neutrophils at 37°C and potassium nitrite (0–200 lM). After
min, neutrophils were stimulated by the addition of 1 lg/mL PMA.
Results are expressed as mean ± SD for at least three
separate experiments. Graphics and data analysis were
performed using GraphPAD prism 4 software. Statistical
analyses were performed using the ANOVA and Bonfer-
roni post hoc test. p B 0.05 was deemed significant.
After 30 min, the reaction was stopped by the addition of SOD
90 U/mL) and catalase (500 U/mL). Neutrophils were pelleted by
centrifugation and the reaction products in the supernatant were
(
determined by HPLC. Inset, effect of SOD, catalase and ABAH on Leu-
enkephalin nitration by PMA-stimulated neutrophils. Leu-enkephalin
6
(
1 mM) was incubated with 2 9 10 neutrophils at 37°C and 100 lM
potassium nitrite in the presence of SOD (90 U/mL), catalase
500 U/mL) or ABAH (100 lM). The reaction was stopped after
0 min as above and the reaction products were determined by HPLC
(
3
Results
Oxidative/nitrative modifications of Leu-enkephalin
by neutrophils
MPO (Kettle et al. 1995), completely abolished nitroen-
kephalin formation. Collectively, these data support a
mechanism for PMN-mediated nitration of enkephalin that
is dependent on active MPO and hydrogen peroxide.
The oxidative/nitrative modifications of Leu-enkephalin
by human neutrophils have been also investigated in the
presence of an NO-producing system. PMNs activated with
PMA or fMLP were exposed to a continuous flux of NO,
using the NO-donor molecule DEANO. The yields of
dienkephalin and nitroenkephalin produced after 30 min of
incubation with 1 mM Leu-enkephalin are reported in
Fig. 2. It can be seen that PMNs activated with PMA are
capable of nitrating and oxidizing enkephalin with higher
efficiency compared with fMLP-activated PMNs.
Human neutrophils utilize myeloperoxidase (MPO)/H O /
2
2
-
NO2 system to generate reactive nitrogen species (Bren-
nan et al. 2002; Eiserich et al. 1998b). Recently, we
reported that oxidants formed by this enzymatic system are
able to convert the tyrosine amino-terminal residue of Leu-
enkephalin either to 3-nitrotyrosine producing nitroen-
kephalin or to dityrosine with the production of an
enkephalin dimer, the dienkephalin (Fontana et al. 2006).
Figure 1 shows that 30 min incubation of 1 mM Leu-
enkephalin with human PMNs activated with PMA yielded
a detectable level (0.82 ± 0.10 lM) of dienkephalin.
-
Addition of NO2 to this reaction system produced nitro-
enkephalin as an additional product. The extent of the
conversion of Leu-enkephalin to the nitroderivative was
found to increase with the concentration of nitrite added,
Effect of nitroenkephalin on superoxide generation
by human PMNs
while dienkephalin production leveled off at approximately
-
2
5 lM NO2 . At higher nitrite concentrations ([200 lM),
To investigate if enkephalin, undergoing oxidative/nitrative
modification, could influence neutrophil functions, the
nitrated peptide was explored for its effect on respiratory
burst. Addition of 100 lM Leu-enkephalin or nitroen-
kephalin to human PMN in suspension did not stimulate
superoxide generation. However, nitroenkephalin added to
neutrophils 2 min before activation by 1 lg/mL PMA or
0.1 lM fMLP led to a concentration-dependent inhibition
of superoxide generation (Fig. 3b). Nitroenkephalin half
maximal inhibition of superoxide anion generation was
48.4 ± 1.3 and 66.9 ± 1.3 lM for PMA- and fMLP-
the yield of nitroenkephalin exceeded that of dienkephalin
data not shown). Unstimulated PMNs did not induce any
modification of Leu-enkephalin.
(
-
To elucidate the mechanism by which NO2 participates
in PMN-mediated nitration reaction, the system was
investigated in the presence of SOD and catalase. As seen
in Fig. 1 (inset), SOD exhibited only a marginal effect on
the production of nitroenkephalin, whereas catalase
strongly reduced the yield of the nitropeptide. In addition,
4
-aminobenzoic acid hydrazide (ABAH), an inhibitor of
1
23

Oxidative and nitrative modifications of enkephalins
879
Fig. 3 Inhibition of neutrophil superoxide anion generation in
response to either PMA or fMLP by nitroenkephalin. Superoxide
anion generation was estimated by measuring cytochrome c reduction
6
as described in ‘‘Materials and methods’’. Neutrophils (2 9 10 cells)
in 1 mL phosphate-buffered saline containing 80 lM cytochrome c at
Fig. 2 Oxidative/nitrative modification of Leu-enkephalin by PMA-
or fMLP-stimulated neutrophils in the presence of the nitric oxide
donor, DEANO. Leu-enkephalin (1 mM) was incubated with 2 9 10
3
7°C were activated by 1 lg/mL PMA or 0.1 lM fMLP and the
absorbance at 550 nm was monitored. Nitroenkephalin was added
min before activation. Steady-state velocity of superoxide produc-
6
2
neutrophils at 37°C and 1 mM DEANO. After 5 min, neutrophils
were stimulated by the addition of 1 lg/mL PMA or 0.1 lM fMLP.
Cytochalasin B (1 lg/mL) was added to f-MLP-stimulated PMNs.
After 30 min, the reaction was stopped by the addition of SOD
tion was estimated from the linear part of the reaction curve. a Time
course of superoxide anion generation by PMA-activated neutrophils
with/without 20 lM nitroenkephalin. b Dose-dependent inhibition of
PMA- and fMLP-stimulated neutrophil superoxide anion generation
by nitroenkephalin. b Inset, dose-dependent inhibition of PMA-
stimulated neutrophil superoxide anion generation by B5 lM nitro-
enkephalin. **p \ 0.05, compared with the control value
(
90 U/mL) and catalase (500 U/mL). Neutrophils were pelleted by
centrifugation and the reaction products in the supernatant were
determined by HPLC; n.d. not detected. Control values are without
DEANO. a Nitroenkephalin; b Dienkephalin
(
PMA-stimulated neutrophil superoxide anion generation in the
absence of nitroenkephalin)
stimulated human PMNs, respectively. Control experi-
ments, using the enzymatic system xanthine/xanthine oxi-
dase to generate superoxide anion, indicated that
nitroenkephalin did not act as a scavenger of superoxide
anion (not shown). In Fig. 4a, it is also shown that Leu-
enkephalin and the free nitrated amino acid, 3-nitrotyro-
sine, did not affect the oxidative burst of PMA-stimulated
neutrophils.
NO/cGMP system in the mechanism of inhibition exerted
by nitroenkephalin, fMLP-stimulated human PMNs were
incubated with nitroenkephalin in the presence of the sol-
uble guanylate cyclase inhibitor, ODQ. It can be observed
that the inhibitory effect of nitroenkephalin was not
reversed by the presence of ODQ indicating that the inhi-
bition of superoxide anion generation is not cGMP medi-
ated (Fig. 4b).
Previous investigations indicated that NO or NO-
releasing compounds increased cyclic GMP (cGMP) in
human neutrophils with concomitant inhibition of PMN
functions including superoxide anion generation (Moilanen
et al. 1993). To evaluate the possible involvement of the
Enkephalins are known to bind to specific opioid
receptors on human neutrophils (Menzebach et al. 2003).
To evaluate whether the inhibitory activity of
123

8
80
E. Capuozzo et al.
addition of 100 lM naloxone did not modify the inhibitory
effect of nitroenkephalin on superoxide anion release,
suggesting that the mechanism of inhibition does not
involve opioid receptors.
Nitroenkephalin attenuation of fMLP-induced
degranulation of human PMNs
Pretreatment of neutrophils with 100 lM nitroenkephalin
strongly reduced fMLP-induced azurophilic degranulation
as evaluated by measuring the activity of released elastase
(Fig. 5a). Figure 5b shows that the inhibitory effect of
nitroenkephalin was concentration-dependent with half
Fig. 4 Comparison of the effect of nitroenkephalin with various
bioactive molecules on PMA- or fMLP-stimulated neutrophil super-
oxide anion generation. Superoxide anion generation was estimated
by measuring cytochrome c reduction as described in ‘‘Materials and
6
methods’’. Neutrophils (2 9 10 cells) in 1 mL phosphate-buffered
saline containing 80 lM cytochrome c at 37°C were activated by
1 lg/mL PMA or 0.1 lM fMLP, and the absorbance at 550 nm was
monitored. Nitroenkephalin and other test compounds were added
2
min before activation. ODQ was preincubated 20 min with cells
Fig. 5 Inhibition of fMLP-stimulated neutrophil degranulation by
nitroenkephalin. Degranulation acitivity was determined by elastase
release. Elastase activity was measured by hydrolysis of the elastase
substrate (MeO-Suc-Ala-Ala-Pro-Val-AMC) as described in ‘‘Materi-
before the assay. Steady-state velocity of superoxide production was
estimated from the linear part of the reaction curve. a Comparison of
the effect of 100 lM nitroenkephalin, 100 lM Leu-enkephalin and
1
00 lM 3-nitrotyrosine on PMA-stimulated neutrophil superoxide
6
als and methods’’. Neutrophils (2 9 10 cells) were incubated with
1
anion generation. b Comparison of the effect with/without nitroen-
kephalin of 100 lM naloxone and 4 lM ODQ on fMLP-stimulated
neutrophil superoxide anion generation. ***p \ 0.001, compared
with the corresponding control (the first column of each panel)
lg/mL cytochalasin B and 40 lM MeO-Suc-Ala-Ala-Pro-Val-AMC
at 37°C for 3 min before activation with 1 lM fMLP, as indicated by
arrows. Nitroenkephalin was added 2.5 min before activation. a Time
course of elastase release by fMLP-activated neutrophils with/without
1
00 lM nitroenkephalin. b Dose-dependent inhibition of fMLP-
stimulated neutrophil elastase release by nitroenkephalin. b Inset,
comparison of elastase release by fMLP-stimulated neutrophils in the
absence (control) and presence of 100 lM nitroenkephalin, 100 lM
Leu-enkephalin and 100 lM 3-nitrotyrosine. *p \ 0.05 and
***p \ 0.001, compared with the control (the first column of the panel)
nitroenkephalin is mediated by its binding to opioid
receptors, the effect of nitroenkephalin on respiratory burst
was examined in the presence of naloxone, a competitive
opioid receptor antagonist. Figure 4b shows that the
1
23

Oxidative and nitrative modifications of enkephalins
881
maximal inhibition of 13.8 ± 1.5 lM. Leu-enkephalin
Discussion
(
100 lM) was ineffective and 3-nitrotyrosine (100 lM)
showed a 37% inhibition of elastase release (Fig. 5b, inset).
Addition of nitroenkephalin to unstimulated PMNs had no
effect on their degranulation activity. Control experiments
using commercial elastase from porcine pancreas indicate
that nitroenkephalin (100 lM) did not affect the elastase
activity (not shown).
In a recent paper, we showed that reactive nitrogen species,
such as peroxynitrite or nitrite/singlet oxygen or peroxidase-
catalyzed oxidation of nitrite, are capable of nitrating and
oxidizing the bioactive peptide Leu-enkephalin to give
nitroenkephalin and dienkephalin (Fontana et al. 2006). The
results presented in this paper demonstrate that activated
human neutrophils, in the presence of nitrite, mediate
nitration and dimerization of Leu-enkephalin leading to
nitroenkephalin and dienkephalin formation. It should be
noted that addition of nitrite at concentrations observed
during inflammatory and infectious process (10–50 lM)
(Farrel et al. 1992; Torre et al. 1996) induced both nitration
and dimerization of Leu-enkephalin. The relative yields of
nitroenkephalin and dienkephalin produced by human PMNs
depend on nitrite concentration. At low concentrations of
nitrite, dimerization of enkephalin is the predominant path-
way, whereas at higher concentration of nitrite ([200 lM),
nitroenkephalin is the main product. These findings suggest
that enkephalin dimerization competes with nitroenkephalin
formation, which becomes more significant with increasing
levels of nitrite. As already reported, PMN-mediated nitra-
tion reactions involve reactive oxygen/nitrogen species and
myeloperoxidase activity (Brennan et al. 2002; Eiserich et al.
1998b). Accordingly, our data show a critical role of
hydrogen peroxide as revealed by inhibition of the nitroen-
kephalin formation in the presence of catalase. Furthermore,
inhibition of myeloperoxidase by ABAH completely
abolishes the conversion of enkephalin to nitroenkephalin.
Collectively, these results support a mechanism for PMN-
mediated modification of Leu-enkephalin that is dependent
on myeloperoxidase activity and hydrogen peroxide pro-
duction. Nitrative/oxidative modifications of tyrosine by
MPO in the presence of hydrogen peroxide and nitrite has
been attributed to direct action of MPO Compound I and II on
both nitrite and tyrosine producing nitrogen dioxide radical
Influence of nitroenkephalin and Leu-enkephalin
on neutrophil spontaneous apoptosis
Neutrophil spontaneous apoptosis was evaluated by mea-
6
suring caspase-3 activity in lysates of neutrophils (5 9 10
cells/mL) that were preincubated at 37°C for 3.5 h. When
the preincubation step was performed in the presence of
Leu-enkephalin (5–100 lM), no considerable effect on the
caspase-3 activity was observed (Fig. 6). Conversely, the
addition of nitroenkephalin induced a decrease of caspase-
3
activity that was concentration dependent. With 50 lM
nitroenkephalin, the reduction of caspase-3 activity was
close to 40% (p \ 0.05). Control experiments indicated
that neither nitroenkephalin nor the parent peptide Leu-
enkephalin affected the activity of recombinant caspase-3
(
not shown).
To rule out toxic effects exerted by nitroenkephalin, cell
viability was measured by the trypan blue exclusion test.
Neutrophil viability was evaluated after incubation of the
cells at 37°C for 3.5 h. Nitroenkephalin showed no effect
in the range 10–100 lM (Fig. 6).
ꢀ ꢀ
NO ) and tyrosyl radical (Tyr ), respectively (Burner et al.
2
(
ꢀ ꢀ
000). Then, Tyr can either react with NO (k = 1.3
2
2
9
-1 -1
9
10 M
s ) to form nitrotyrosine or dimerize
-1 -1
s ) to give dityrosine (Hunter et al.
8
k = 2.25 9 10 M
(
1989; Prutz et al. 1985). With enkephalin as a target, the
tyrosine amino-terminal residue of the peptide can be con-
verted either to 3-nitrotyrosine producing nitroenkephalin or
to dityrosine, with the production of an enkephalin dimer, the
dienkephalin.
Fig. 6 Effect of Leu-enkephalin and nitroenkephalin on neutrophil
6
caspase-3 activity. Neutrophils (2 9 10 cells) were incubated in the
Recently, it has been reported that activated neutrophils
use MPO to oxidize Leu-enkephalin to their corresponding
tyrosyl free radical, which preferentially reacts with respi-
absence (control) and in the presence of various concentrations of
Leu-enkephalin or nitroenkephalin at 37°C for 3.5 h. Intracellular
caspase-3 activity was determined as described in ‘‘Materials and
methods’’. Cell viability (filled diamonds) was estimated by the trypan
blue exclusion test and is expressed as percentage of untreated cells.
Values are given as mean ± SD (n = 4). *p \ 0.05, and
9
ratory burst-derived superoxide anion (k = 1.5 9 10
-1 -1
M
s ) (Jin et al. 1993) to form a hydroperoxide derivative
(Nagy et al. 2010). Here, we demonstrate that stimulated
*
**p \ 0.001, compared with the control value
neutrophils, in the presence of nitrite, promote as a further
123

8
82
E. Capuozzo et al.
modification of enkephalins nitration of amino-terminal
tyrosine residue to give nitroenkephalin. As the rate constant
for the reaction of tyrosyl radical with superoxide anion is
Neutrophil apoptosis is an important process because it
provides a signal for PMN removal (Savill and Fadok
2000) and it results in the loss of functional neutrophil
responsiveness (Lee et al. 1993; Savill et al. 2002; Simon
2003). Thus, modulation of apoptosis may have a major
effect on the inflammatory process. As several studies
suggested a critical role of caspase-3 in both spontaneous
and Fas receptor-mediated apoptosis in neutrophils (Daigle
and Simon 2001; Khwaja and Tatton 1999; Ottonello et al.
2002; Pongracz et al. 1999; Weinmann et al. 1999), we
studied the effect of nitroenkephalin and the parent peptide
on spontaneous apoptosis by measuring the caspase-3
activity in cell lysates (Fig. 6). Our results indicate that the
nitroenkephalin influences the life span of human PMNs by
inhibition of spontaneous apoptosis.
ꢀ
9
-1 -1
similar to that with NO (k = 4.5 9 10 M
2
s ) (Gold-
stein et al. 1998), it is possible (but remain to be proved) that
Leu-enkephalin may be partly oxidized via superoxide
anion-mediated mechanism also under our experimental
conditions.
Nitrite is the major end product of NO metabolism, and
inducible NO synthase is contained in neutrophil primary
granules (Evans et al. 1996). To simulate a physiological
mechanism of nitrite formation, activated neutrophils were
exposed to a continuous flux of NO, using the NO-donor
molecule, DEANO. These conditions also promoted
nitration and dimerization of Leu-enkephalin. Although
NO readily reacts with superoxide anion to form the potent
oxidative/nitrative agent, peroxynitrite, it has been previ-
ously reported that NO fluxes induce tyrosine nitration
predominantly via MPO/H O -dependent oxidation of
It is recognized that the production of reactive oxygen
species by activated cells accelerate the apoptosis and that
superoxide release is required for spontaneous apoptosis
(Kettritz et al. 1997; Ottonello et al. 2002; Scheel-Toellner
et al. 2004). In agreement, blood neutrophils from patients
with a genetic defect of NADPH oxidase, thus not gener-
ating reactive oxygen species, demonstrate delayed spon-
taneous apoptosis (Kasahara et al. 1997; Hampton et al.
2002; Savill et al. 1989). Thus, it is possible that the
nitrated enkephalin decreases spontaneous apoptosis of
human neutrophils by inhibiting superoxide anion pro-
duction. Accordingly, the parent Leu-enkephalin, which
does not inhibit superoxide anion generation, does not
decrease spontaneous apoptosis.
2
2
ꢀ
nitrite to NO . In line with this, we found that with fMLP-
2
activated PMNs, the yield of nitroenkephalin was 1.6-fold
less, a consequence possibly related to decreased MPO
release (Eiserich et al. 1998b).
In our studies on the PMNs-mediated nitrative reactions,
we used enkephalin concentrations much higher than those
present in plasma. Circulating enkephalins are normally in
the nanomolar range; however, their amount can signifi-
cantly increase under some pathological conditions
(
Clement-Jones et al. 1980; Pierzchala and Van Loon 1990;
Semmoum et al. 2001). Moreover, locally synthesized
enkephalins in inflamed tissues could produce interstitial
enkephalin concentrations much higher than those
observed in plasma. The concept of local enkephalin is
supported by the finding that inflammatory cells contain
enkephalins and the mRNA for their precursor, proen-
kephalin (Padr o´ s et al. 1989; Przewlocki et al. 1992; Ritt-
ner et al. 2001). Furthermore, enkephalins undergo rapidly
hydrolysis, underestimating the actual concentration.
It has been shown that opioid peptides may prime or
modulate the activity of leukocytes (McCain et al. 1982;
Menzebach et al. 2003; Pasnik et al. 1999; Rabgaoui et al.
The cellular mechanism by which nitroenkephalin exerts
an inhibitory effect on neutrophil functional responses
requires further study. At the moment, the observation that
naloxone, a competitive opioid receptor antagonist, failed
to reverse the inhibition of superoxide anion generation by
nitroenkephalin, indicates that the opioid receptor is not
involved in the mechanism of inhibition. Furthermore,
cGMP has been observed to have an inhibitory effect on
fMLP-induced responses (Ervens et al. 1991). Under our
experimental conditions, the soluble guanylate cyclase
inhibitor, ODQ, did not interfere with the inhibitory effect
of nitroenkephalin providing evidence that the inhibition
on fMLP-induced respiratory burst occurs through a
cGMP-independent pathway.
1
993; Sulowska et al. 2002). To characterize a possible
bioactivity of the nitrated enkephalin, its influence on
human neutrophil functional responses has been examined.
Nitroenkephalin at micromolar concentrations inhibits
neutrophil activation in response to PMA or fMLP, as
indicated by the inhibition of superoxide anion generation
and of degranulation of azurophilic granules. Under the
same experimental conditions, the parent peptide, Leu-
enkephalin, showed no inhibitory activity on these human
neutrophil functions.
In conclusion, the results of our in vitro studies indicate
that Leu-enkephalin, present at inflammation sites, might
exert regulatory effects on the efficiency of inflammatory
processes, after its conversion to nitroenkephalin, induced
by activated human neutrophils. Although the bioactivity
of nitroenkephalin has been observed at concentrations
higher than those eventually formed in vivo, our findings
may lead to further understanding of biochemical
1
23

Oxidative and nitrative modifications of enkephalins
883
inflammatory mechanisms and open the route to the
development of new therapeutic strategies for
inflammation.
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