ELSEVIER
Biochimica et Biophysica Acta 1316 (1996) 210-216
Biochi~ic~a
et BlbphysicaA~ta
Neutrophils obtained from obliterative atherosclerotic patients exhibit
enhanced resting respiratory burst and increased degranulation in
response to various stimuli
Attila Mohhcsi a,*, Bertalan Kozlovszky
b,
Ildik?9 Kiss c, Ildik?~ Seres a, Tamhs FiiliSp, Jr. d
a First Department of Medicine, Unicersity Medical School of Debrecen, P.O. Box. 19, 4012 Debrecen, Hungary
b First Department of Surgery, Uniuersity Medical School ofDebrecen, Debrecen, Hungary
c Department of Biochemistry, Unit~ersity Medical School ofDebrecen, Debrecen, Hungary
d Unicerit~ de Sherbrooke, Centre de Recherche en G~rontologie et GJriatrie, H6pital d'Youcille, Sherbrooke, Qc, Canada
Received 4 April 1996; accepted 17 April 1996
Abstract
Tissue destruction in atherosclerosis is partly due to uncontrolled protease and oxygen radical release. In this study we investigated the
release of elastase and myeloperoxidase, as well as the production of reactive oxygen species by polymorphonuclear leukocytes (PMNLs)
obtained from patients with obliterative atherosclerosis of the lower legs. In addition we measured the plasma concentration of xanthine
oxidase. PMNLs of atherosclerotic patients have a greater ability to increase elastase and myeloperoxidase release after their stimulation
with formyl-methionin-leucyl-phenylalanin(fMLP) and calcium ionophore, A23187, independently of their age, than PMNLs of healthy
middle-aged subjects. Similarly to healthy elderly subjects there was an increased superoxide anion (O 2) production under basal
condition in both atherosclerotic patient age-groups. The activation of PMNLs with fMLP and A23187 enhanced 0 2 formation both in
healthy subjects and in patients with atherosclerotic disease of the lower legs, however the increase was significantly less in the latter
group. No biochemical parameters showed significant correlation with patient's risk factors, however myeloperoxidase production was
significantly higher in less severe stage of the disease (P < 0.05). We found that patients with atherosclerotic disease of the lower legs
have higher plasma xanthine oxidase level than control subjects. This study indicates an other piece of evidence suggesting the activation
and involvement of neutrophils in the pathogenesis of atherosclerosis of the lower legs. The similar tendencies in the reactivity of
neutrophils during aging and in atherosclerosis suggest that atherosclerosis may be an early aging process.
Keywords: Atherosclerosis; Neutrophil;Respiratory burst; Elastase; Xanthine oxidase
1. Introduction
Human phagocytic cells (monocytes and polymorphonuclear leukocytes) beside their role to eliminate microorganisms by phagocytosis, followed by intracellular
killing are also responsible for severe tissue destruction
under pathological conditions such as inflammation,
atherosclerosis and tumor spread. These deleterious effects
of phagocytic cells result from the release of free radicals
such as superoxide anion ( 0 2) and hydrogene peroxide
(H202), as well as of neutrophilic granular contents, including potent proteolytic enzymes e.g. elastase. The uncontrolled release of elastase is able to attack the elastic
Corresponding author. Fax: + 36 52 414227; e-mail: [email protected].
fibres of the arterial wall [1-4] which play an important
role not only in providing structural support, but also by
modulating cell functions [5]. This destruction results in
the liberation of soluble elastin peptides. It was demonstrated that elastin peptides have marked biological effects
on PMNLs as well as on monocytes, fibroblasts and
smooth muscle cells such as migration, aggregation, degranulation and generation of oxygen radicals, thus, suggesting that they may influence cells which are involved in
the pathogenesis of atherosclerosis [6-11]. Moreover,
PMNLs are able to cause arterial damage by an additional
mechanism, namely the released elastase enhances the
conversion of endothelial xanthin dehydrogenase (XD) to
xanthin oxydase (XO) [12,13]. XO in the presence of
xanthin reduces O z to O 2 which in turn may reduce
intracellular Fe 3+ to Fe z+, permitting the Fenton reaction
0925-4439/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved.
Pll S0925-4439(96)00027-0
A. Mohhcwi et al. / Biochimica et Biophysica Acre 1316 (1996) 210-216
to occur, resulting in the generation of highly toxic hydroxyl radical (OH).
We found previously [14], that the susceptibility of
purified elastin to enzymatic hydrolysis increased with the
severity of atherosclerosis and in the mean time the amount
of soluble elastin peptides showed a marked enhancement
in the sera of atherosclerotic patients measured by ELISA
and DOT immunobinding [15-17], further supporting the
hypothesis that elastic fibres degradation may be accelerated during the atherosclerotic process. Beside the decrease
of the rate of elastin biosynthesis a concomitant progressive increase in elastase activity may explain the disappearance of crosslinked elastin from the vessel wall. Indeed, in our earlier work we showed a higher elastase
content in PMNLs of the atherosclerotic group compared
to the control group [18]. Furthermore, it is supposed that
during the proteolytic damage it is not the serum elastase
level, but the amount of the released elastase by stimulated
cells, such as PMNLs, that may be the crucial factor in
tissue damage. To confirm this altered process of degranulation in atherosclerosis we decided to investigate the
release of elastase from activated PMNLs, As the degranulation is accompanied by the generation in PMNLs of
other biochemical processes such as the oxidative burst,
leading to inflammatory reactions, we measured also the
superoxide anion and H 2 0 2 productions, as well as the
myeloperoxidase release after chemotactic peptides (fMLP
and K-elastin) and calcium ionophore, A23187 stimulations in patients who were suffering from atherosclerotic
vascular disease. There exists an immunochemical evidence for the presence of XO in the cytoplasm of vascular
endothelial cells which is released into the blood stream
only after disrupture of the cell, a well-known phenomenon during atherogenesis [19,20]. Thus, we also determined the activity of xanthin oxidase (XO) in the
plasma which could then reflect the degree of capillary
endothelial lesion.
2. Materials and methods
21 I
ing and pulsed Doppler system using ATL ultramark UV
Duplex scanner. Patients who had only peripheral obstructive arterial disease without any generalized manifestation
of the atherosclerotic process were included in the present
study. No patients took drugs known to influence PMNLs
functions. (2) No clinical and spirometric evidence for the
existence of chronic obstructive pulmonary disease; (3)
negative smoking history or a cease of smoking since at
least one year before taking part in the study and (4)
obtention of informed consent from all participants.
Claudicators were stratified into three categories corresponding to the disease severity. Claudication judged as
'mild', 'moderate' and 'severe' were classified using the
ankle/brachial pressure index (API = Doppler derived tibial arterial pressure indexed to the brachial arterial blood
pressure, normal value more than 1.0). API was less than
0.75 but more than 0.50 in cases of mild severity, while
API was between 0.49-0.20 in moderate cases. In the case
of severe claudication, API was less than 0.19. Distribution
of gender, risk factors and lipid values did not differ
significantly in the groups although age increased slightly
with disease severity without statistical significance. A
group of 40 healthy volunteers, matched for age, sex, lipid
values and smoking habits, was taken as a control group.
2.2. PMNLs
Separation of PMNLs was routinely carried out as
previously described [18,21]. Briefly: PMNLs were isolated from whole heparinized venous blood. Cells were
separated by Ficoll-Hypaque density centrifugation, following dextran sedimentation, of the PMNLs rich pellet.
Contaminating erythrocytes were removed by hypotonic
lysis. Neutrophils were then washed three times with
Hank's balanced salt solution and resuspended in HBSS,
and immediately tested for superoxide anion release.
PMNLs purity over 95% and cell viability greater than
98% were microscopically determined by Giemsa staining
and trypan blue exclusion, respectively. All incubations
were performed in CO 2 (5%), air (95%), humidity (95%)
at 37°C.
2.1. Patients
2.3. Experimental conditions
52 patients with clinical signs of peripheral obstructive
atherosclerotic disease were selected from our outpatient
clinic to participate in this study. All patients met the
following criteria. (1) Peripheral obstructive atherosclerotic
disease had to be detected with Doppler ultrasonography
a n d / o r angiography before the patient entered into the
study. Nevertheless all patients underwent an extensive
vascular staging to determine whether the atherosclerosis
was generalized. All patients exercised on a bicycle ergometer according to the Bruce protocol. In case of incapacity a Dipyridamol-thallium imaging was performed to
detect coronary arter disease. The disease of the carotid
bifurcation was studied by a combination of B-mode imag-
To stimulate PMNLs we used fMLP (10 ~ M), a
synthetic chemotactic peptide. In nonadherent PMNL, microfilaments ordinarily restrain enzyme secretion. Since
cytochalasin interfere with the contractile forces generated
by cytoplasmatic actin this drug may allow to remove the
restraints, and thus permit the release of enzymes into the
cell's environment. Another agent that we used to stimulate the elastase release was calcium ionophore, A23187,
agent that is able to raise intracellular calcium level which
promotes secretion from azurophil granules. A23187 agent
in 1 ~M concentration mostly stimulates secretion from
specific granules that contain, e.g. lactoferrin, B~2 binding
212
A. Mohhcsi et aL / Biochimica et Biophysica Acta 1316 (1996) 210-216
protein, but not proteases and myeloperoxidase [22].
Therefore, 10 txM calcium ionophore A23187 was used
because it is enabled the release of substances from both
azurophil and specific granules.
Human PMNLs (2 × 10 6 cells/ml) were stimulated in
Hank's buffer with A23187 or cytochalasin B (Cyt B) plus
tMLP. We incubated PMNLs using different methods: (1)
in the presence or absence of 10 IxM A23187 for 30 min at
37°C; (2) in the presence or absence of 10 txM Cyt B for
80 min at 37°C; (3) in the presence of 10 IxM Cyt B for 20
min, and after adding 0.01 IxM fMLP, further 60 min at
37°C.
Incubated PMNLs were pelleted by centrifugation (500
g for 5 min at 4°C) and the supernatant were assayed for
neutrophil elastase and myeloperoxidase activity.
2.4. Assay of human leukocyte elastase (HLE) activity
Enzyme assay was carried out according to the method
of Hornebeck et al. [23,30] slightly modified by us [16].
Briefly: Elastase activity was measured with 1 mM MeoSuc-Ala-Ala-Pro-Val-pNa (Sigma) dissolved in dimethyl
sulfoxide in Hepes buffer at pH 7.5 containing 0.5 M NaCI
and 10% DMSO at 37°C. Elastase activity was expressed
as nmoles of substrata cleaved per minute assuming an
extinction coefficient of 8.8 x l03 M -~ cm -~ at 405 nm.
2.6. Superoxide anion O f production and hydrogen peroxide (H20 2 ) release
This was determined according to the method of Babior
et al. [25] as previously described. Briefly, neutrophils
( 2 . 5 X 10 6 cells) were incubated (37°C, 15 min) with 0.01
IxM fMLP (Sigma) or 10 IxM calcium ionophore, A23187
(Sigma) or kappa elastin in 1.0 ixg/ml concentration.
Kappa elastin was prepared from bovine ligamentum
nuchae elastic by alkaline hydrolysis in ethanol as previously described by L. Robert in 1968 [26]. Incubation was
carried out in the presence of 50 IxM ferricytochrome c
(Sigma, type III). Parallel tubes, with the same components, included also 300 U / m l superoxide dismutase (SOD,
type I, Sigma). After incubation, the tubes were subsequently centrifuged (400 X g, 10 min, 4°C). The superox±de production was determined from the O.D. 550 of the
supernatants without SOD minus the O.D. 550 of matched
samples with SOD, using an extinction coefficient of
2.1 × 10 4 M 1 c m - ' . The results were expressed as nmol
O~/106 cells per min [27].
2.5. Assay of neutrophil myeloperoxidase (MPO) act±riO,
Myeloperoxidase activity was measured using the
method of Bretz et al. [24]. with 0.32 mM o-dianisidine
(3,3-dimethoxybenzidine, Fluka) in citrate buffer, pH 5.5,
containing 0.08 mM hydrogen peroxide. The reaction was
stopped with 35% perchloric acid. The enzyme activity
was expressed as nanomoles of product per minute assuming an extinction coefficient of 2 × t 0 4 M - ~ cm- ~ at 560
rim.
2.7. Measurement of xanthine oxidase (XO) activi~
XO activity was measured according to the method of
Hainig and Legan [28] and Beckman et al. [29]. Briefly:
plasma samples were assayed for their XO activity using
2-amino-4-hydroxypteridine (AHP) as substrate in a spectrofluorimeter (Perk±n-Elmer MPF 44B) with excitation at
345 nm and emission at 390 nm. The volume of the assay
mixture was 2.0 ml in PBS which consisted of 200 ixl
substrate and putative enzyme from samples (25 txl). Reaction proceeded for 60 rain at 37°C. The concentration of
isoxanthopterin formed was calculated from the linear
relationship between fluorescence intensity versus its concentration in 0-10 nmol of the product. The buffer-substrate was prepared on the day of use by diluting a stock
Table 1
Secreted human leukocyte elastase and myeloperoxidase from granulocytes separated from healthy and atherosclerotic middle-aged subjects, stimulated
with various stimuli
Stimuli
Age (yrs)
Resting
CytB
A23187
CytB(+ fLMP)
Released protease by granulocytes:
human leukocyte elastase ( n m o l / m i n per 106 cells)
myeloperoxidase ( n m o l / m i n per 106 cells)
healthy (middle aged)
atherosclerotic (middle aged)
healthy (middle aged)
atherosclerotic (middle aged)
46.3 ± 3.4
1.8 ± 0.15
1.46 ± 0.09
7.56 + 1.06
11.10 _ 1.32
47.6 + 5.1
0.54 + 0.05
1.32 -+ 0.81
13.23 _+ 1.44 *
24.75 + 1.83 *
46.3 _ 3.4
1.34 ± 0.10
0.99 ± 0.07
4.865 +_ 0.43
5.86 ± 0.61
47.6 __. 5.1
0.95 ± 0.11
1.16 + 0.12
7.695 -/-_0.65
10.795 _+ 1.17 "
Human granulocytes (2 x 106 cells/ml) were stimulated in Hank's puffer with A23187 (10 p,M) for 30 min at 37°C or with cythochalasin B (0.1 ixM) in
the presence and absence of formyl-methionilleucyl-phenylalanine (0.01 IxM) for 80 min at 37°C. The incubated granulocytes were pelleted and the
supernatants were assayed for human leukocyte elastase with MeoSuc-Ala-Ala-Pro-Val-pNa (Sigma) and myeloperoxidase using 3,3-dimethoxybenzidine
(Fluka). Enzymes activity were expressed as nmol of substrate cleaved per minute or nmol of product per minute assuming extinction coefficients of
8.8 )< 103 M -I cm -] at 405 nm or 2 X 104 M -I cm -1 at 560 nm, respectively. Data represent mean + SD.
* P < 0.05 statistically significant as compared to healthy middle aged subject.
A. Moh{lcsi et al. / Biochimico et Biophy,*ica Acta 1316 (1996) 210-216
solution as desired with phosphate buffer (0.2 M, pH 7.2).
The stock solution was prepared by dissolving 14.68 mg
pulverized AHP with a few drops of 0.2 N NaOH, then
diluting to 100 ml with distilled water. The stock solution
9 × 10 4 M was kept frozen between usage.
3. Statistical analysis
All biochemical values were expressed as means _+ SD.
Statistical difference was determined by Student's t-test,
with significance defined as P < 0.05.
4. Results
4. l. Elastase and myeloperoxidase release
Our results documenting the elastase and myeloperoxidase release from PMNLs under various stimuli are shown
in Table 1. In our present experiment, Cyt B did not cause
significant enzyme release from PMNLs obtained from
both healthy and atherosclerotic patients. A23187 alone,
and fMLP in the presence of Cyt B, induced a significant
release of elastase ( P < 0.05) in atherosclerotic patients in
contrast to the age-matched controls. In studying the agerelated association of this parameter, a marked aged dependent increase of the elastase release was found in healthy
subjects ( r = 0.91, P < 0.05) (Fig. 1). Furthermore, this
response was more pronounced in the subgroup of healthy
elderly of 75 yr and over, and did not differ from the
response of PMNLs derived from elderly atherosclerotic
patients (data not shown). The stimulated release of
myeloperoxidase was significantly higher in atherosclerotic than in healthy middle aged subjects ( P < 0.05).
Similar to the elastase, the release of MPO was also
enhanced during the aging process (data not shown).
nmol/min/10~'cells
35
•
r=0.91
>
25
5(
v
20
15
30
Doppler index
0.75-0.50
0.49 >> 0.20
< 0.20
No. ( m a l e / f e m a l e )
Age (yr)
Human leukocyte elastase
( n m o l / m i n per 10 ~ cells)
IMLP
A23187
Myeloperoxidase
( n m o l / m i n per I() ~ cells)
tMLP
A23187
19 ( 1 4 / 5 )
52.5 + 7.9
22 ( 1 8 / 4 )
53.6 ± 11.3
11 ( 7 / 4 )
56.5 ± I I
26.61 _+2.76
16.39_+1.46
20.19_+2.47
10,87_+0.96
19.33±2.5
7.45+0.66
12.68_+ 1.17
11.16+1.71
9.14_+0.97
6.215_+2.56
8.82_+ 1.45
4.76_+0.44 ....
Patients suffered from obliterativ atherosclerotic disease of the lower legs
were stratified into the mild (0.75-0.50), moderate (0.49 >> 0.20) and
severe ( << 0.20) stages of the disease according to the a n k l e / b r a c h i a l
pressure index (Doppler derived tibial pressure indexed to the brachial
arterial pressure). All values represent the mean + SD.
P < 0.01. Difference was significant as compared to the group of
patients with a n k l e / b r a c h i a l pressure index above 0.50.
To find out, whether risk factors and severity of the
disease have an influence on the quantity of HLE or MPO
released, we analyzed the correlation between protease
release and the former parameters. An inverse correlation
was found between protease secretion and disease severity.
In the group of patients with mild claudication, as determined by ADI, the quantity of released HLE as well as
MPO was the most pronounced as compared to that in the
moderate and severe stages of the disease. Statistically
significant differences were only found in the case of MPO
release in mild claudicators as compared to the values of
severe claudicators ( P < 0.05). The quantity of released
HLE was also higher in PMNLs obtained from mild
claudicators than that obtained from severe claudicators.
but the difference was not statistically significant (Table
2).
4.2. O~ production and H,O~ release
i
20
Table 2
Clinical characteristics of the studied patient populations and the release
of elastase and myeloperoxidase from stimulated PMNLs in relation to
severity of the disease stages
p<0.05
X r=0.12 n.s.
30
213
40
50
Fig. I. Correlation of released
phenylalanine (fMLP) and age of
tase from granulocytes of healthy
stars: atherosclerotic subjects ( r =
60
70
80
90<
(yearsl
elastase by formyl-methionin-leucylsubjects. Black triangle: released elassubjects ( r = 0.91, 17= 40, solid line),
0.12, n = 52, dotted line).
A significant difference ( P < 0.05) was found between
the basic levels of O,- of healthy middle-aged subjects and
of atherosclerotic patients. In the case of middle aged
atherosclerotic subjects the resting 0 2 production was
enhanced, while stimulation was decreased using various
stimuli such as fMLP and K-elastin. Stimulation remained
unaffected when we used calcium ionophore (Fig. 2). The
0 2 production was also significantly ( P < 0.05) different
between healthy middle aged and healthy aged subjects.
No differences were found between healthy and atherosclerotic elderly subjects. The profile of O ; generation of
healthy elderly and middle aged atherosclerotic subjects
was quite similar.
H202 production which is not linked to receptor stimulation was diminished in the case of atherosclerotic pa-
A. M ohg~csi et al. / Biochimica et Biophysica Acta 1316 (1996) 210-216
214
5°I
30
4O
T
20
c,~ 20
I
O
10
10
middle aged H
elderly H
middle aged ats
elderly ats
l r e s t i n g l K a p p a elastin []FMLP [--']A23187
Fig. 2. Superoxide anion generation in resting and stimulated granulocytes obtained from healthy middle aged (n = 24, average age: 46.3 + 3.4
yr) and elderly healthy ( n = 16, 67.5_+4.5) subjects and middle aged
(n = 25, 47.6+_5.1) and elderly (n = 27, 70.3 + 5.5) atherosclerotic patients. The stimulating agents were as follows: KE = K-elastin 1.0 ixg/ml,
fMLP = formyl-leucyl-phenylalanin 0.0l ~M, A23187 = calcium
ionophore, 10 IzM. * P < 0.05 It was compared to healthy middle aged
subjects. Each column represents the mean+ S.D. H = healthy, ats =
atherosclerotic.
tients, and only a slight increase of H202 generation was
detected in the case of healthy elderly subjects without
statistical significance (Fig. 3.). K-Elastin significantly ( P
< 0.05) stimulated the H202 production in case of middle-aged healthy subjects.
4.3. Xanthin oxidase activity
XO activity was raised in the plasma of atherosclerotic
patients of both ages. The elevation was the most marked
Middle aged H
12[
I. . . . .
I
-. 10
~
6
0
2
Fig. 3. H202 release from granulocytes obtained from healthy and
atherosclerotic subjects of various ages induced by K-elastin (1.0 Ixg/ml).
Middle aged H = middle aged healthy (n = 24) or Elderly H = elderly
healthy subjects (n = 16), Middle aged ats = middle aged atherosclerotic
patients (n ~ 25) and elderly ats = elderly atherosclerotic patients (n =
27). Each column represents the mean _. S.D. * P < 0.05 when compared
to healthy middle-aged subjects.
0
Middle aged H Elderly H Middle aged ats Elderly ats
Fig. 4. Plasma xanthin oxidase activity in patients with obliterative
atherosclerotic disease of the lower legs and healthy subjects. Each
column represents the mean ± S.D. * P < 0.05 It was compared to healthy
middle aged subjects.
in middle aged atherosclerotic patients and differed statistically significantly from the healthy middle aged subjects
( P < 0.05), however, plasma XO activity does not increase
with age in healthy subjects (Fig. 4).
5. Discussion
One of the most important pathological feature of
atherosclerosis is the degradation of elastin fibers.
Hornebeck et al. [23] have correlated the increased elastase
type activity in human aortic tissue with the severity of
atherosclerosis, however the cellular origin of elastase is
still an unresolved question. Although, vascular cells, such
as smooth muscle cells are able to modulate the structure
and composition of the extracellular matrix by the release
of proteolytic enzymes (e.g. elastase), PMNLs remain the
main source of elastase. As the released elastase is rapidly
dissolved in the circulation, the serum elastase may not
represent adequately the total elastase activity in pathological conditions [31,32]. Therefore, we choose an alternative
approach to confirm the role of elastase in atherosclerosis.
We have demonstrated that marked difference exists in the
responsiveness of PMNLs, concerning the elastase release,
obtained from healthy and atherosclerotic middle-aged
subjects. PMNLs obtained from atherosclerotic patients
released significantly more elastase under fMLP and
A23187 stimulations than those from healthy middle-aged
subjects. Activation of PMNLs by various stimuli e.g.
growth factors, endothelial cells, was demonstrated to induce their adhesion to endothelial cells [33-39]. The activated and recruited PMNLs may be highly disruptive for
the integrity of the vessel wall rendering them more susceptible to further tissue destruction. Therefore our results
reinforce the supposition that PMNLs may be transiently
A. Mohhcsi et aL /Biochimica et Biophysica Acta 1316 (1996) 210-216
present in diseased artery wall at the very beginning of the
pathological process of atherosclerosis.
The difference found in elastase release between healthy
and atherosclerotic middle-aged subjects disappeared when
we compared healthy and atherosclerotic aged subjects.
Indeed, the elastase release showed an age related pattern
and the disease could not further modulate it. With age
decreased rate of elastin synthesis in the arterial wall as
well as altered effector functions of PMNLs were demonstrated [40-43]. Thus, the demonstrated gradual increase in
the elastase activity with age can further contribute to the
disappearance of the crosslinked elastin content of the
arterial wall. Our results suggest that these processes might
be accelerated during atherosclerosis already at an earlier
age. It is of interest, that we found an inverse correlation
between the elastase release from PMNLs and the clinical
severity of the disease. The most marked release of elastase was shown in individuals who were at the earliest
stage of the disease. Gminsky et al. [44] demonstrated that
an enhanced elastin metabolism followed by an autoimmunization process towards elastin could be already detected
in healthy children of atherosclerotic patients, underlining
the importance to determine the role of elastase before the
clinical manifestations of atherosclerosis. As the initial
stages of atherosclerosis are strongly related to clinical risk
factors and the reduction of these factors may result in
slowing of the pathological process, the use of synthetic,
orally active protease inhibitors could have therapeutic
value [45]. The properties of these inhibitors allow them to
effectively supplement the activity of natural in vivo inhibitors of PMNLs elastase particularly in primary or
secondary (e.g., smoking) deficiency of c~l-antitrypsin
[46,47]. Moreover, antioxidants can also provide significant protection suggesting an interaction between reactive
oxygen species (e.g., MPO-H202-CI system) and the elastase-antielastase (a~-antitrypsin) system.
Another deleterious consequence of the PMNLs degranulation is the production of reactive oxygen species (ROS)
such as O 2 that are essential for host defense, but may
also induce tissue damage and are frequently observed in
inflammation and atherosclerosis. We could demonstrate
that the resting level of O ] was increased, while its
stimulation was less effective in both age-groups of atherosclerotic patients, as well as in that of healthy elderly
subjects. In contrast, PMNLs derived from middle-aged
healthy subjects showed low resting 0 2 production with
marked increase in 0 2 production after stimulation by
various stimuli. After its release the O 2 rapidly dismutates
to H202 and 02. This increased basal production of 0 2
and the decreased stimulability of PMNLs with aging and
in atherosclerosis suggest that a disturbed transmembrane
signal transduction exists through receptors e.g. fMLP at
least for phospholipase C activation. These observations
are however not contradictory to the finding that protease
secretion could be still induced by fMLP. This may support the proposal that beside G~ protein which is very
215
important during the turning of the extracellular signal into
intracellular one for O 2 production, at least two other
different GTP binding proteins e.g. Gp or G e proteins
could be involved in NADPH oxidase activation and in
exocytosis thus, allowing alternative signaling pathways to
occur [48].
The H 2 0 2, which is generated secondary from the
dismutation of 0 2 may be further connected to the forming of hypochlorous acids (HOC1, HOBr) in the presence
of chloride and bromide during interaction with myeloperoxidase (MPO) [1,22]. The production of the H202 originating directly from stimulation with KE was found to be
lower in atherosclerotic patients compared to healthy subjects. Actually we do not have any plausible explanation
for this result. Theoretically, the presence of an intracellular H2Oz-scavenging system or an enhanced MPO
activity could lead to an underestimation of the amount of
H202. The MPO, which is also released from lysosomes
during stimulation was found significantly higher after
fMLP stimulation in both middle-aged atherosclerotic patients and healthy elderly subjects, compared to healthy
middle-aged subjects. We can not say actually that the
cause of the lower H202 value might be the result of the
elevated intracellular MPO content, but further investigations are needed to confirm this hypothesis. According to
our results, the xanthin oxidase (XO) activity could be
demonstrated in the plasma of both healthy and atherosclerotic subjects, however the XO activity was significantly higher in middle-aged atherosclerotic patients than
in healthy control subjects. These results could be further
confirmed by the following evidences. 1. We measured
whether the XO activity could have come from PMNLs or
other blood cells, but we failed to show any XO activity. 2.
We studied the inhibition of the reaction in the presence of
the specific XO inhibitor, the allopurinol, which was able
to effectively prevent the XO activity. 3. We tested our
method in case of hepatocellular damage, where our method
could effectively demonstrated the well known elevated
XO activity. Despite of the fact that, Jarash et al. [19]
failed to show any xanthin oxidase activity in human sera
even in patients with diabetic microangiopathy, XO appeared in the effluent blood of the involved tissue during
ischemia reperfusion [49]. Moreover, antibodies against
XO could arise during myocardial infarction, suggesting a
pathogenic role of XO in these disease states. We need
further studies to determine the exact pathologic significance of the plasma XO in atherosclerosis.
In summary, our results indicate that the amount of
elastase as well as of myeloperoxidase released by various
stimuli increased in middle-aged atherosclerotic patients.
We found that the elastase release was also enhanced in
healthy elderly subjects, demonstrating an age-related association between these parameters. According to our results, we suggest that if enhanced proteolytic activity is of
pathogenic importance in atherosclerosis it is likely to be
due to an increased quantity of released proteolytic en-
216
A. Moh{tcsi et al. / Biochimica et Biophysica Acre 1316 (1996) 210-216
z y m e f r o m p e r i p h e r a l P M N L s , a l r e a d y at the early stage o f
the disease. O u r results c o n c e r n i n g the s u p e r o x i d e a n i o n
p r o d u c t i o n i n d i c a t e that the e l e v a t e d r e s t i n g s u p e r o x i d e
a n i o n release m i g h t also c o n t r i b u t e to the p u t a t i v e h a r m f u l
effects o f P M N L s in a t h e r o s c l e r o s i s . T h i s h i g h X O level in
the p l a s m a o f a t h e r o s c l e r o t i c p a t i e n t s m i g h t r e p r e s e n t an
i n c r e a s e d e n d o t h e l i a l d a m a g e , b u t this h y p o t h e s i s s h o u l d
be f u r t h e r e l u c i d a t e d . Finally, o u r f i n d i n g s d e m o n s t r a t i n g
s i m i l a r p a t t e r n s for p r o t e a s e release a n d R O S p r o d u c t i o n
by P M N L s , after f M L P s t i m u l a t i o n , o b t a i n e d f r o m h e a l t h y
elderly a n d m i d d l e - a g e d a t h e r o s c l e r o t i c s u b j e c t s s u g g e s t
that a t h e r o s c l e r o s i s m i g h t b e an early a g i n g p r o c e s s .
Acknowledgements
This
tion o f
1459).
Mozga
r e s e a r c h w a s s u p p o r t e d by the R e s e a r c h F o u n d a the H u n g a r i a n M i n i s t r y o f W e l f a r e ( O T K A No.
T h e e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f Mrs. Gy.
a n d Mrs. M. N a g y are t h a n k f u l l y a c k n o w l e d g e d .
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