Journal of Clinical Laboratory Analysis 25 : 47–51 (2011)
Phagocytosis and Nitric Oxide Levels in Rheumatic Inflammatory
States in Elderly Women
Iêda Maria Martinez Paino, Julise Cunha Miranda, Cleni Mara Marzocchi-Machado,
Evandro José Cesarino, Fabı́ola Attié de Castro, and Ana Maria de Souza
Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto,
São Paulo, Brazil
Background: Very few studies have investigated, in the elderly, the effect of rheumatic inflammatory states on phagocyte
function and free radical production. The
objective of this article is to evaluate
phagocytosis by neutrophils and the production of nitric oxide ( NO) by monocytes
in elderly women recruited among patients
of the Brazilian Public Health System.
Methods: Forty patients aged more than
60 years with rheumatic inflammatory diseases were studied. Phagocytosis was
measured by flow cytometry. NO production was measured by the total nitrite assay
and conventional inflammation markers
were determined. Data were analyzed
with the Mann–Whitney nonparametric test
and Po0.05 was considered significant.
Results: C-reactive protein levels and white
blood cell counts were significantly higher
in inflammation than in the control group
(Po0.05). The phagocytosis fluorescence
intensity per neutrophil and the percentual
of neutrophils expressing phagocytosis were
significantly higher (Po0.05) in the test than
in the control group. Furthermore, there was
significant NO overproduction by monocytes, (Po0.05). Conclusion: Phagocytosis
and NO production are affected by rheumatic states. This suggests that the increased
NO levels may play a part in the increased
oxidative stress in rheumatic diseases in
elderly women. J. Clin. Lab. Anal. 25:47–51,
r 2011 Wiley-Liss, Inc.
2011.
Key words: nitric oxide; phagocytosis; elderly women; rheumatic diseases; oxidative stress
INTRODUCTION
A considerable amount of clinical research in geriatric
medicine has been conducted in recent years; yet, there
are few studies regarding the effect of rheumatic
inflammatory diseases on the functions of phagocytes
in elderly women.
Phagocytes, especially monocytes and neutrophils,
play an essential part in the innate immune system via
the oxidative burst, being involved in antimicrobial
activity, the eradication of pathogens, and the production of free radicals and reactive oxygen and nitrogen
species (1,2). However, some studies have revealed that
these free radicals, reactive oxygen species (ROS) and
reactive nitrogen species (RNS) are also involved in
deleterious effects in inflammation (1,3). Furthermore,
there are numerous discrepancies in the literature
between in vivo and in vitro results, as well as between
results for murine and human neutrophils (4).
The free radical nitric oxide ( NO) has an important
role in the innate immune system, but it has also been
found to play an important part in the pathophysiology
c
2011 Wiley-Liss, Inc.
of many human diseases. RNS derived from NO
interact with biomolecules, such as proteins, carbohydrates, and lipids, modifying both their structure and
function (5,6). The generation of NO by human
monocytes seems to be controversial, and it has not
yet been explored in the elderly, especially those with
rheumatic diseases.
Rheumatoid arthritis (RA) is a chronic joint disease
leading to severe erosion of an adjacent bone, which is
not observed in patients with osteoarthritis (OA) (7),
and neutrophils and monocytes play an important role
in overt inflammation. OA is the most common disease
of the joints. It is a chronic condition that has periods of
Correspondence to: Iêda Maria Martinez Paino, Faculty of
Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo,
Av. do Café, s/n. Monte Alegre, CEP 14040-903, Ribeirão Preto-São
Paulo, Brazil. E-mail: iedammp@hotmail.com
Received 19 May 2010; Accepted 21 October 2010
DOI 10.1002/jcla.20429
Published online in Wiley Online Library (wileyonlinelibrary.com).
48
Paino et al.
acute pain and affects the quality of life, owing to the
impairment of normal functional activity (8).
Thus, the aim of this study was to investigate the
phagocytosis in neutrophils and NO production by
monocytes isolated from peripheral blood, in elderly
women with OA and RA.
MATERIAL AND METHODS
biochemical analyses were performed on the serum:
creatinine, fasting glucose, gamma-glutamyltransferase
activity, and C-reactive protein (CRP). Hematological
tests were performed on the EDTA whole blood:
white blood cell (WBC) counts and electrophoresis of
hemoglobin (data not shown). Phagocytosis assay and
isolation of mononuclear cells were analyzed on the
heparinized whole blood.
Subjects
We performed a prospective controlled study of 40
elderly women (60–84 years old). They were recruited
from patients attended by the Brazilian Public Health
System in a geriatric department in Ribeirão Preto city.
All subjects gave informed consent to participate in the
study, and the procedures were approved by the Ethics
Committee of Faculty of the Pharmaceutical Sciences of
Ribeirão Preto, University of São Paulo, in keeping with
the principles enunciated in the Declaration of Helsinki.
Tobacco users and patients with malignancies, impaired
kidney function, hemoglobinopathy, decompensated diabetes mellitus, bone marrow proliferative disorders, or
infectious diseases were excluded from the study.
Although the patients were under treatment with an
antihypertensive (enalapril) and a hypocholesterolemiants
(simvastatin) at enrollment, none had been treated with
immunosuppressive drugs. Thus, these drugs had the same
(homogeneous) distribution in both groups. All patients
presenting hypertension and dyslipidemia had them
controlled with medication. Under instructions from the
leading cardiologist, blood pressure assessments were
taken three times before venopunction at 15 min intervals.
The control group of elderly women (n 5 20) had normal
complete hemograms and no signs of inflammation.
The inflammation group (n 5 20) was composed of
elderly women with rheumatic disorders, specifically RA
(77%) and OA (23%). They took Nonsteroidal Antiinflammatory Drugs, ibuprofen, diclofenac, and meloxican.
Materials
Lipopolysaccharides (LPS), fluorescein isothiocyanate
(FITC), Ficoll-Hypaque (Histopaques, Sigma Aldrich,
St. Louis, Missouri), RPMI 1640 medium, propidium
iodide, trypan blue, fetal calf serum (FCS), neutral red
solution, streptomycin, penicillin, and 2-mercaptoethanol
were purchased from Sigma-Aldrich (St. Louis, MO).
Blood Samples
Human peripheral blood samples were drawn in the
morning on a routine basis from volunteers who had
fasted for 12 hr. Whole blood and serum samples
were collected into tubes in the presence or in the
absence of the anticoagulant, respectively. The following
J. Clin. Lab. Anal.
Analytical Methods
The laboratory biochemical and hematological tests
(Table 1) were performed in duplicate for each patient
and reviewed by a physician to exclude subjects with
hemoglobinopathy, kidney disorders, infection, liver
disorders, or bone marrow proliferative disorders.
WBC counts were obtained with an automated Micros
45-ABXs counter (France), and differential counts were
done by examining blood smears stained with Leishman.
The high sensitivity CRP was measured by immunoturbidimetric assay (BioTécnicas, Brazil). The biochemical analyses were performed with colorimetric
kits (Labtests, Brazil) in a Beckman DUs-70-USA
(Maryland) spectrophotometer.
Phagocytosis Function
Heparinized whole blood (100 ml) was collected from
the peripheral blood of volunteers and incubated with
100 ml of FITC-labeled bacteria (E. coli) (9) for 10 min at
371C. After addition of 50 ml quenching solution, the
leukocytes were washed twice with phosphate-buffered
saline (PBS) by centrifuging at 250 g, 4–81C for 5 min.
Red blood cells were lysed (NH4Cl lysis solution, 0.83%,
pH 7.2). The leukocytes were washed once with PBS
(centrifuged at 4–81C, 250 g for 5 min) and resuspended
in 500 ml of PBS-D. The cell viability evaluations were
analyzed and supplemented with propidium iodide
(16.6 mg/ml). Phagocytosis was reported as percentual
(%) of cells containing fluorescence and the median
fluorescence intensity per cell. Tubes were kept at 4–81C
TABLE 1. Age, Biochemical and Hematological Parameters
of Patients
Parameters
Age
Fasting glucose (mg/dl)
Serum creatinin (mg/dl)
GGT (U/l)
WBC/ml
CRP (mg/l)
Control group
Inflammation group
68 (65.5–75.0)
85 (77.4–95.5)
0.9 (0.8–1.2)
(21.6) (17.7–29.2)
5,800 (4,750–6,450)
0.2 (0.2–0.9)
69 (68.5–75.0)
88 (81.7–110.8)
0.92 (0.8–1.2)
(17.5) (8.5–24.4)
6,100 (5,600–7,550)
4.3 (3.5–5.1)
Data are reported as medians from 25th to 75th percentiles in brackets.
Significantly different from control, Po0.0001 (Mann–Whitney test).
Phagocytosis and Nitric Oxide Levels
in the dark, until being read in a flow cytometer
(FacScans, Becton-Dickinson, Bioscience, San José, CA).
Data were taken from 10,000 cells per sample in the region
for neutrophils, as well as monocytes.
49
results were considered significant when Po0.05. All
tests were analyzed by the program GraphPad Prism
version 4.02.
RESULTS
Isolation of Cells
Heparinized peripheral blood was collected from
volunteers. The mononuclear cells isolation was made
by the difference of gradient density Ficoll-Hypaque
(Histopaques) 1077. After centrifugation (400 g;
30 min at room temperature), the monocytes were found
at the plasma/1077 interphase. Monocytes were collected carefully with a Pasteur pipette and washed in
PBS twice (240 g for 10 min), and suspended in RPMI
1640 medium with 10% FCS.
Monocyte Cell Culture
Cells (1 105 monocytes/well) were counted in neutral
red solution, plated in 24 well-culture plates, and
allowed to adhere at 371C in a 5% CO2 atmosphere
for 2 hr. The supernatant medium and nonadherent cells
were washed off and the wells recharged with medium at
371C in a 5% CO2 atmosphere. Cell preparations
contained more than 98% viable monocytes, as assessed
by the trypan blue exclusion method. The cells were
cultured for 24 hr at 371C in a 5% CO2 atmosphere, with
(test) or without (control) 1 mg/ml LPS as stimulus, to
analyze the NO production.
. NO Production
Concentration of nitrite (NO2 ) was taken as an
indirect measurement of NO produced by the monocytes. It was determined by the Total Nitric Oxide/
Nitrite/Nitrate Assay kit (R&D Systemss, MN), a
colorimetric method that determines nitric oxide concentrations by the enzymatic conversion of nitrate to
nitrite by nitrate reductase. Absorbance at 540 nm was
measured in duplicate with a Spectra Max Plus spectrophotometer (Molecular Devicess, Sunnyvale, CA),
according to the manufacturer’s instructions. Briefly,
sodium nitrate (2,000 mmol/l) and sodium nitrite
(2,000 mmol/l) solutions were provided as standards.
The average duplicate readings for each standard and
sample were subtracted from the average blank optical
density. The measurement of the total endogenous
nitrite concentration is the measurement of the total
nitrite concentration after the conversion of nitrate
to nitrite by the nitrate reductase.
Age, hematological, and biochemical parameters of
patients are shown in Table 1. The CRP was significantly higher in the inflammation group than in the
control group (Po0.05).
The percentage of neutrophils expressing phagocytosis
and the phagocytosis fluorescence intensity per neutrophil were significantly higher in the inflammation group
than in the control group (Figs. 1 and 2, respectively).
The viability of cells in the phagocytosis assay was
9673% (mean7SD).
The production of NO2 was significantly higher in the
inflammation group than in the control group (Fig. 3).
DISCUSSION
There are very few studies assessing the effects of
inflammatory states on phagocytosis in elderly women.
In this investigation, this question was examined by
studying the effects of rheumatic inflammatory conditions on phagocytosis by neutrophils and NO generation by monocytes, isolated from elderly women.
ROS and RNS are produced at high levels by phagocytes
in the course of the inflammatory response. Free radicals are
thought to contribute to the pathology of many inflammatory conditions. Leukocytes generate large amounts of ROS
via activation of NADPH oxidase, neutrophils accumulate
in large numbers, and are stimulated to produce superoxide
and other reactive oxidants (10).
Statistical Analysis
The Mann–Whitney nonparametric test was used to
analyze the data and values are given as medians. The
Fig. 1.
Percentage of neutrophils expressing phagocytosis in control
and inflammation groups. Data are reported as medians. Statistical
difference vs. control group: Po0.05 (Mann–Whitney test).
J. Clin. Lab. Anal.
50
Paino et al.
Fig. 2.
Phagocytosis fluorescence intensity per neutrophil in control
and inflammation groups. Data are reported as medians. Statistical
difference vs. control group: Po0.05 (Mann–Whitney test).
Fig. 3.
NO production (mmol/l) by monocytes in control and
inflammation groups. Monocytes were cultured at 1 105 cells/ml for
24 hr at 371C in a 5% CO2 atmosphere. Total nitrite concentration in
the samples was determined in the cell culture supernatant. Data are
reported as medians. Statistical difference vs. control group: Po0.05
(Mann–Whitney test).
In this study, both the phagocytosis fluorescence
intensity per neutrophil and the percentage of neutrophils
expressing phagocytosis in the inflammation group were
significantly higher than in the control group. The reason
for this may involve the participation of phagocytes in host
defense mechanisms, owing mainly to proinflammatory
cytokines, such as TNF-a and IL-6, produced in large
amounts during inflammation (11) and these proinflammatory cytokines are likely to be encountered in the
circulation in peripheral blood neutrophils of RA patients
as priming agents (12). Thus, in our study, it is probable
that proinflammatory cytokines may be modulating and
increasing phagocytosis (13).
The direct reaction of NO with most biological
molecules does not occur at low concentrations
J. Clin. Lab. Anal.
(100–500 nmol/l) in vivo, but NO can be harmful at
concentrations up to 1–3 mmol/l (6). There are reports
that activated human neutrophils can catalyze the
formation of nitrosated products (5,14), an extensive
event during oxidative stress. This shows that the NO2
production in our results (Fig. 3) represents overproduction of NO (above 50 mmol/l). It is believed to
play a critical role in mediating inflammatory tissue
injury. Several studies have reported increased endogenous NO synthesis. Overproduction of NO may be
important in the pathogenesis of RA contributing to the
inflamed joint and periarticular bone loss observed in
RA (15). Recent evidence suggests that NO contributes
to T-cell dysfunction in autoimmune diseases, such as
RA and systemic lupus erythematosus (16). Significantly,
higher NO levels occur in acute appendicitis, which is
strongly correlated with oxidative stress and conventional
inflammation markers (17).
All the patients were under treatment with enalapril,
an ACE inhibitor, and simvastatin at enrollment. These
drugs have antiinflammatory effects and may interfere
with the study. In fact, the use of the antiinflammatory
drugs could characterize a limitation of the study. This
finding should be investigated further, as this study was
not designed to evaluate oral drugs action. However, the
drugs have the same distribution in both groups, in
order to minimize this effect.
Phagocytosis is an ancestral/innate component of the
immune system that seems to be relatively preserved
during aging. Taken together, these results suggest that
NO is essential for the upregulation of the rheumatic
inflammatory response in the target population. It
suggests that the increased NO may play a role in the
increased oxidative stress in rheumatic inflammation
diseases in elderly women. This may reflect a characteristic of the inflammatory rheumatic condition and/or a
current status in behavior and pathophysiology of the
inflammatory rheumatic states.
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