Investigation of the relation of allergy and oxidative damage
by metallic elements using SR micro beam
K. Nakashimaa*, T. Shirakawaa, A. Ide-Ektessabib
a
Health Promotion and Human behavior, Kyoto University Graduate School of Public Health, Kyoto, JAPAN.
b
Kyoto University International Innovation Center, Kyoto, JAPAN.
ROS (reactive oxygen species) and free radicals are thought to modulate pathways in leukocytes mediated
inflammation, especially in T-lymphocytes. In this study, synchrotron radiation x-ray fluorescence (SRXRF)
spectroscopy was applied to non-destructive elemental mapping in the HL60 cells with O2- production.
There are several mechanisms of ROS production, and many researchers consider that excessive
accumulation of metallic elements has a role in its generative processes. Mapping of the elements with a
beam size of 4.5×6 µm and an energy of 14.2 keV was carried out in HL60 cells. The distribution of trace
elements in HL60 was obtained in an area of about 80×80 µm. There was clearly correlation between O2production and Fe distribution. Simultaneously, change of other trace elements could be detected. Our
results suggest that change of distribution or amounts of intracellular trace elements is important in
inflammation processes.
Key words: synchrotron radiation (SR) micro beam, HL60 cells, Reactive oxygen species (ROS), transition metals,
allergy, oxidative damage, trace elements
exogenous signals by chemotaxis, activation of the
respiratory-burst oxidase, exocytosis of their
cytoplasmic granules, and phagocytosis of foreign
particles. Studies of responses of neutrophils to
extracellular stimuli contributed to a better
understanding of diverse signal-transduction pathways
and their elements [4].
INTRODUCTION
Allergy is one of the common diseases. While the
cause of allergy remains unclear, there are some
indications that excessive accumulation of essential
element, and sometimes, incorporation of toxic foreign
elements in cells aggravate allergic reactions. During
the past decade, many researchers have investigated
the causative factors correlate between genetic and
environmental factors [1,2]. Since, transition metals
induce metal allergy, and it is known that transition
metals seem to play an important role in production of
reactive oxygen species (ROS) and free radicals [3], it
can be hypothesized that reactive oxygen may play a
causative factor in allergic reaction.
In order to detect the metallic elements in cultured
cell lines, we used establish human cell lines, ROS
induced by dimethyl sulfoxide; a differentiated
promyelocytic leukemia cell line HL60 (DMSOHL60) whose characteristics are similar to those of
human neutrophils [5]. Leukocytes release several
mediators, which play important role in mechanisms
of allergy. While synchrotron radiation (SR) analysis
is easy to prepare samples, and its detection level is
Neutrophils constitute the first line of defense
against invading microorganisms. They respond to
*
To whom correspondence should be addressed at Health Promotion and Human Behavior, Kyoto University Graduate School of Public Health,
Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, JAPAN. Tel: +81-75-753-4451. Fax: +81-75-753-4452. E-mail: [email protected].
CP680, Application of Accelerators in Research and Industry: 17th Int'l. Conference, edited by J. L. Duggan and I. L. Morgan
© 2003 American Institute of Physics 0-7354-0149-7/03/$20.00
517
containing 1.3% DMSO for 7 days as described [8]
and then washed twice with and resuspended in HBSS
containing 30 mM HEPES (pH 7.4; DMSO-HL60).
quite low, this technique has not yet been applied in
the allergy field.
ROS induce various types of oxidative damages
considered to be important in inflammation processes.
We had experimental system to measure biologically
generated ROS induced intracellular oxidative damage,
however we did not have method to measure
quantitatively the concentrations of intracellular
transition metals which seems to be more important
for the production of ROS as electron donor. Thus the
mechanisms of intracellular distribution of transition
metals in relation to biological events have not been
yet clarified so far.
Superoxide production assay
Cells were washed at two times with HEPES-HBSS
and resuspended at a concentration of 2.5 × 106
cells/ml in HEPES-HBSS containing 1.4 g/l CaCl2, 1.0
g/l MgCl2 and 1.0 g/l MgSO4. Control cells without
inhibitors were incubated in the same conditions. The
cell suspension, kept at 37 ℃ , were added to
prewarmed water containing 2mM of cytochrome c.
Superoxide formation was initiated by the addition of
PMA at the indicated concentrations. Cytochrome c
reduction was continuously monitored at 550 nm. For
the detection of SOD inhibitable cytochrome c
reduction, it added for the reference cuvette (final
concentration was 100 units/ml).
In this study, HL60 was used for measuring
intracellular superoxide anion (O2-). HL60 can be
induced to differentiate into neutrophils or
macrophages by DMSO or PMA, respectively [6-8].
The differentiated HL60s have similar functions to
their respective counterparts. Neutrophils play several
roles in inflammation. We, therefore, determined the
transition metals amounts in the cell line, and then
investigated the relationship between intracellular
concentrations of transition metals and ROS induction
to clarify the chemical state of the transition metals. In
addition, Electron spin resonance (ESR) spin trapping
is rather direct and useful assay for free radicals. Thus
we also used the cell as target and detected O2- release
by PMA, by the use of ESR with 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap. Using these
techniques, we studied the mechanisms of O2- release
in relation to with Habor-Weiss reaction, especially in
Fenton-reaction.
Electron spin resonance (ESR) analysis
Cells were washed two times with HEPES-HBSS
and resuspended at a concentration of 5×106 cells/ml
in HEPES-HBSS. Cells were mixed with DMPO (0.1
M), and the reaction was started with the addition of
PMA (1 nM). ESR spectra were recorded at 25℃ with
ESR spectrometer. ESR settings were microwave
power, 10 mW; modulation frequency, 100 kHz;
modulation amplitude, 0.8 G; response time, 1 s, 10×
103; sweep time, 12.5 G/min (11).
X-ray analyses
After stimulation, the cells were washed twice with
phosphate-buffered saline without Mg2+ and Ca2+; PBS
(-), and immediately immersed into an ice 99% ethanol.
Fixed samples were mounted on PET film for x-ray
analyses. Synchrotron Radiation X-ray Fluorescence
spectroscopy (SRXRF) analyses were performed the
beamline 4A at the Photon Factory, High Energy
Accelerator Research Organization (KEK), Tsukuba,
Japan. The x-ray beam was monochromatized with a
synthetic multilayer film. The x-ray energy was 14.2
keV. Monochromatized x-ray was focused with
Kirkpatrick-Baez optics. The cross-section of the beam
was 4.5×6 µm on the sample. The sample stage was
moved by x-y step pulse motors and the distributions
(x-ray intensity maps) of P, Cl, Ca, Cu, Fe, and Zn
were obtained. The scanning area was 80×80 µm and
was divided into 90×90 pixels. Each measurement
point of the sample was irradiated for 4 sec. The beam
current of the storage ring was about 350 mA.
Measurements were made in both air and vacuum. The
beamline was equipped with a CCD camera in front of
the sample holder. The image from this camera gave
visual information on the measuring points.
MATERIALS AND METHOD
Reagents.
Phorbol myristate acetate (PMA), 4-acetamido-4’isochiocyanatostillene 2,2’-disulfonic acid (STIS),
cytochrome c and deferoxamine were purchased from
Sigma Chemical Co. (St Louis, MO). PMA was
dissolved in dimethyl sulphoxide (DMSO). Catalase
was obtained from Roche (Mannheim, Germany).
Superoxide dismtase (Cu/Zn-SOD) was purchased
from Wako Pure Chemicals, Inc. (Osaka, Japan).
Cell Treatments.
HL60 was kindly supplied by Japanese Collection
of Research Bioresources (JCRB cell bank) and grown
in RPMI 1640 (Nikken, Japan) containing 10% heatinactivate fetal calf serum (Hyclone, USA) and
penicillin/streptomycin (GIBCO-BRL, Grand Island,
NY). HL60 was differentiated with culture medium
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(A)
RESULTS
Experimental results were presented on the relative
content of iron and other trace elements in DMSOHL60 cells. Emphasis was placed on a comparison
between the stimulated cells and control cells. Using
SR microbeam, the constituent elements and their
distributions in the cells were obtained.
O2 - generation from DMSO-differentiated HL60.
When DMSO-HL60 was stimulated with PMA, we
detected an ESR spectrum consisted of DMPO-OOH
and spin trapped adducts of hydroxyl radical (H2O2),
DMPO-OH. In this time, we did ESR analysis as
confirmation of O2 - production. We compared these
waves with standard added to H2O2 only. From result
of ESR analysis (Data not shown), O2 - were released
in this system. Addition to PMA, O2 - generation from
DMSO-differentiated HL60 was 9.11±0.96 (mean±
S.E.) µM/ml. After PMA and SOD stimulate, it was
0.21 ± 0.09 µM/ml. After catalase addition, 8.24 ±
1.21 µM/ml After addition of STIS as an anion
channel blocker, 4.96 µM/ml, addition of
Deferoxamine, Fe chelater, O2 - generation was 3.89
µM/ml. It is cheater of free ion even inside the cells,
thus speed of O2 - generation became slow. O2 generation was depressed by addition to SOD.
(B)
Elemental content in stimulated and control cells.
XRF spectra were obtained at (A) control, (B)
PMA stimulated. Typical spectrum of each group is
shown in FIGURE 1. Comparing these groups, Fe and
Zn are clearly increased in amounts at intracellular
region. Iron was detected inside the stimulated cells at
high concentrations.
FIGURE 1. Typical spectra of intracellular and extracellular
region (A) Control, (B) PMA stimulation.
XRF Imaging of HL60 cells
Distribution of Sodium and Calcium and Chlorine
X-ray intensity maps of major elements of HL60 in
are shown in FIGURE 3. In each condition, several
cells were obtained in the scanning area. These XRF
imaging show that metal elements in the (A) control
and (B) PMA stimulated. (a) Optical microscopic
photograph. (b-f) Elemental maps of (b) Zn, (c) Fe, (d)
Ca, (e) Cu and (f) P. Amount of Fe and Zn are
increased after stimulation. Intracellular and
extracellular amount of Fe were same, however after
PMA stimulation, intracellular amount of Fe increased.
Other trace elements also increased in intracellular
amounts.
Sodium and calcium balance is important elements
in the cell signaling. Their balances seem to be
important for transfer information. When the amount
of chlorine was X axis, distributions of Ca/K ratio with
control and PMA stimulation are clearly different.
After PMA stimulation, Ca/K ratio did not change but
the amount of intracellular chlorine increased.
Intracellular change of several elements could be seen
simultaneously by this technique. (FIGURE 2).
Cl channel is important for the system and
construction of various cells. Change of Cl distribution
effected on regulation of cell capacity, which is
important in apoptosis or necrosis. In this experience,
intracellular chlorine distribution became higher after
PMA stimulation, in other words, after O2 - release.
Production of O2 - and the PMA stimulus were
correlated.
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DISCUSSION
In this study, states and amounts of trace elements in
DMSO-HL60 to generate O2- is detectable. The cell,
similar characteristics with those of neutrophils,
generated O2- upon stimulation with PMA. This system
is though to be the model of neutrophil oxidation.
Changes of intracellular trace elements we could
detect in immune cells. We also demonstrated that
PMA induced O2- release from the cells by ESR with
DMPO as a spin trap, and pointed out the importance
of serum factors and transition metals for the O2release. Extracellular stimulation increased O2-,
intracellular existence states or amounts of trace
elements are changeable. Our results suggest that
chemical amounts of intracellular trace elements play
an important role in mechanisms of immune cells. On
the contrary, undifferentiated HL60 did not release any
oxygen radicals by the stimulation. To stimulate by
PMA, the size of the cell becomes smaller. Change of
cell size may also effect on the change of trace
elements. In this study, we have not detected the
intracellular chemical state of trace elements yet. Thus,
several insights can be made from these findings.
FIGURE 2. Distribution of chlorine and Ca/K ratio in the
HL60 cells. Ca/K ratio is amount of Ca (ppm) divided by
that of K (ppm).
(A)
Free radicals or ROS are also biochemically
dangerous; for example, Iron can damage tissues by
catalyzing the conversion of hydrogen peroxide to free
radical ions that attack cellular membranes, protein
and DNA. This threat is reduced in the normal state
where, because of the fine iron metabolism regulation,
there is never appreciable concentration of ‘free iron’.
Under pathological conditions, iron metabolism and
superoxide metabolisms are clearly interactive. Each
can exacerbate the toxicity of the other. Iron overload
may amplify the damaging effects of superoxide
overproduction in a very broad spectrum of
inflammatory, both acute and chronic, conditions [9].
Furthermore, chronic oxidative stress may modulate
iron uptake and storage, leading to a self-sustained and
ever-increasing spiral of cytoxic and mutagenic events
[10].
(B)
Trace elements are seems to be important in immune
cell function. Calcium plays an important role in the
regulation of cellular differentiation and desquamation
of epidermal keratinocytes. The improvement of the
stratum corneum barrier properties in vitro is
concurrent with the normalization of the epidermal
calcium gradient, whereas deregulation of terminal
differentiation correlates with an accumulation of
calcium ions within incompletely differentiated
corneocytes [11]
FIGURE 3. SRXRF imaging of HL60. (A) Control (B)
PMA stimulated. (a) Optical microscopic photograph. (b-f)
Elemental maps of (b) Zn, (c) Fe, (d) Ca, (e) Cu and (f) P.
The scanning area was 80×80µm2. The image was obtained
from HL60 cells cultured in HEPES-HBSS for 15 minutes.
The ranges of measured fluorescent intensities are from 0 to
70 photons for Zn, from 0 to 70 photons for Fe, from 0 to 40
for Ca, from 0 to 18 for Cu and from 0 to 45 for P. Each
range is divided into sixteen levels. Each level has been
assigned a shade of red, blue and green respectively.
HL60 cells can also be induced to differentiate into
macrophages by PMA. Inflammatory mediator; IL-1,
520
IL-6 and tumor necrosis factor alpha (TNF-alpha),
induce the differentiation of the cells into macrophages
and arrested proliferation. However, IL-4 suppresses
these mediators induced differentiation of the cells and
restored the arrested proliferation with themselves [11].
Thus, further research is also required in the viewpoint
of immune secretion.
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Some techniques applied to x-ray microanalysis in
allergy field [13]. Electron microprobe and proton
microprobe x-ray analysis have been used in several
areas of dermatological research. EPMA is also useful
in single cell analysis, however it destroy the sample.
Whereas, this method could be a model to investigate
oxidative damage to cause inflammation as a part of
allergic reaction, and these studies are now in progress.
Since the SR analyses performance is improving, we
can expect to detect in the near future.
Zinc is required for the structure and activity about
300 metalloenzymes. Therefore, every system could
be affected functionally or structurally by that defect.
In these results, intracellular amount of zinc was also
correlated to O2- production. Not only Cu and Fe, the
amounts of other transition metals are also changed by
stimulation, it is thought by various intracellular
transition metals concerned with generating of ROS.
They may act as electronic supply in the organisms. If
changes metal is working also as an activity center of
various enzymes and it can act as the monitor of the
change of the intracellular metals simultaneously with
work of various enzyme, it will be leading to the
elucidation of a more detailed mechanism.
Our results suggest that distribution or amounts of
intracellular trace elements play an important role in
inflammation responsible for allergy reaction.
ACKNOWLEDGMENTS
The SRXRF experiments were performed at the
Photon Factory, Tsukuba, Japan. The authors thank
Professor A. Iida, KEK, and T. Kawakami, Kyoto
University Graduate School of Engineering, for their
support and valuable discussions during experiments.
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