ABBS 2005,38(06): Effect of Ozone Produced from Antibody-Catalyzed Water Oxidation on Pathogenesis of Atherosclerosis

Abstract        Recent studies have
suggested that antibodies can catalyze the generation of unknown oxidants
including hydrogen peroxide (H2O2) and ozone (O3) from singlet oxygen (1O2) and water.
This study aimed to detect the effect of antibody-catalyzed water oxidation on
atherosclerosis. Our results showed that both H2O2 and O3 were produced in human leukemia THP-1
monocytes incubated with human immunoglobulin G and phorbol
myristate acetate. In the THP-1 monocytes incubated
with human immunoglobulin G, phorbol myristate acetate and low density lipoprotein, the
intracellular accumulated total cholesterol, free cholesterol, cholesteryl ester and lipid peroxides clearly increased,
and a larger number of foam cells were observed by oil red O staining. The
accumulation of all intracellular lipids was significantly inhibited by
vinylbenzoic acid, and only slightly affected by catalase. These findings
suggested that the production of O3, rather than
H2O2, might be involved in the pathogenesis of
atherosclerosis through the antibody-catalyzed water oxidation pathway.

Key words        antibody; ozone; THP-1 monocyte; foam cell; atherosclerosis

Antibodies are classical
adaptor molecules of the immune system. In terms of the antibody effector mechanism, the central idea is that antibodies do
not have destructive abilities, but mark foreign antigens and pathogens for
removal by complement cascade and/or phagocytosis
[1]. However, the recent discovery of a new property of antibody molecules
suggests a previously unexplored effector function of
the immune system. All immunoglobulins (Ig), regardless of source or antigenic specificity, can
catalyze the reaction between singlet oxygen (1O2) and water
to facilitate the production of a number of water oxidants, such as hydrogen
peroxide (H2O2) and ozone (O3), which are highly bioreactive
and cytotoxic compounds, resulting in direct killing
of foreign antigens or pathogens [25]. The discovery of this pathway greatly
enriches our knowledge of the ability of antibodies to destroy foreign
pathogens. however, it also
suggests that there might be some potential pathophysiologic
effects in this antibody-mediated activity. O3 is a kind of gas
with high chemical reactivity, and H2O2 is a strong
oxidant. As well as the efficient killing of pathogens, they might also lead to
oxidative damage on tissues.

Atherosclerosis has been
considered a chronic inflammatory disease, in which the oxidative damage of
lipids and the consequent formation of foam cells are key steps in onset and
development [6]. It is now widely accepted that the inflammation associated
with the presence of leukocytes and antibodies is a crucial component in human
atherosclerosis [7]. It has been suggested that H2O2, as a strong
oxidant, is an important risk factor for atherosclerosis, and recent research
also reported that the O3 oxidation products of cholesterol triggered
the foam cell formation in tissue macrophages [8].

Can the
antibody-catalyzed water oxidation pathway, however, produce H2O2 and O3 at the same time, both of which play major
roles in the generation and development of atherosclerosis? And what impact
does H2O2 or O3 production in this pathway impose on the
pathogenesis of atherosclerosis? In this study, we investigated the production
of H2O2 and O3 by the antibody-catalyzed water oxidation
pathway, and their effects on intracellular accumulation of cholesterol and
lipid peroxides in human leukemia THP-1 monocytes and on foam cell formation.
The clarification of these questions will provide valuable information for
better understanding of atherosclerosis, as well as for searching for new ways
to prevent and treat this disease.

Materials and Methods

Materials

Human leukemia cell line
THP-1 has been widely used as a model for monocyte-macrophage
lineage. THP-1 monocytes were provided by the Department of Immunology,

Preparation of low
density lipoprotein (LDL)

Isolation of human LDL
was carried out using NaBr gradient
ultracentrifugation [9], followed by 48 h dialysis against phosphate-buffered
saline (PBS) containing 200 mM EDTA at 4
ºC. LDL was then passed through a 0.22 mm sterilized millipore filter,
followed by an agarose gel (0.5%) electrophoresis to
identify the purity. The protein content of LDL was determined using Coomassie brilliant blue G-250 staining.

Cell culture

The THP-1 monocytes were
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf
serum (56 ºC, 45 min), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 ºC in 5% CO2. The well-cultured cells were washed three times
with PBS, and shaken for 10 min each time to remove the IgG
bound nonspecifically. Except for the analysis of H2O2 and O3, cell
concentration was adjusted to 1´106 cells/ml in
RPMI 1640 medium supplemented with 0.2% bovine serum albumin, 100 U/ml
penicillin and 100 mg/ml
streptomycin. The cells were then divided into seven groups: research group,
0.5 mg/ml PMA, 100 mg/ml LDL, 1 mg/ml human IgG
in cultured cells; catalase group, supplementing 100 U/ml (final concentration)
bovine catalase to the research group; vinylbenzoic acid group, supplementing

Analysis of accumulation
of cholesterol and content of lipid peroxides in cells

The cultured THP-1
monocytes were collected by scraping using a cell scraper, and washed three
times with PBS. After re-suspension in 0.5 ml of sodium phosphate buffer
solution (For total cholesterol
(TC) determination, as described previously [10], 0.1 ml of the sample was
added to 0.9 ml of assay solution (0.1 U/ml cholesterol oxidase,
1 U/ml HRP, 0.01 U/ml CE hydrolase, 0.05% Triton
X-100, The content of lipid
peroxides was determined by estimating malonic dialdehyde using the thiobarbituric
acid method with an assay kit.

Oil red O staining for
foam cells

The cultured THP-1
monocytes were washed with PBS three times and fixed in 2.5% glutardialdehyde for 3 h, dipped in 2.5% potassium dichromate
for 16 h, and stained in 1% oil red O for 20 min to identify lipid droplets in
cytoplasm. Cell nuclei were then stained in hematoxylin
for a few seconds. For each procedure, the fixed cells were washed with
distilled H2O. THP-1 monocyte-derived foam cells were observed and photographed.
Semi-quantitative analysis of foam cells was evaluated by the percentage of
positive oil red O-staining cells.

Analysis of H2O2

After removing the IgG bound nonspecifically, the cultured THP-1 monocytes were
collected and diluted to 1.5´107 cells/ml in
PBS containing 1 mg/ml human IgG. After the cells
were incubated at 4 ºC for 1 h, they were activated at 37 ºC for 10 min by the
addition of PMA (1 mg/ml). Every
aliquot (1 ml) was removed from the cell suspension at 3 min intervals and
filtered through a 0.22 mm syringe.
Cell filtrates were collected at each time point during the reaction, and the H2O2 content of each filtrate was monitored by the
following method [11]: 2.6 ml of Tris-HCl buffer
solution (pH 7.4,

Analysis of indigo
carmine oxidation during THP-1 monocyte activation

After removing the IgG bound nonspecifically, the cultured THP-1 monocytes
were collected and diluted to 1.5´107 cells/ml in
PBS containing 1 mg/ml human IgG. After the cells
were incubated at 4 ºC for 1 h, they were activated at 37 ºC for 10 min by the
addition of PMA (1 mg/ml). As
described previously, a solution of the O3 probe, indigo
carmine, in PBS (pH 7.4) was added to the activated cells to give a final
concentration of 30 mM. Every
aliquot (1 ml) was removed from the cell suspension at 3 min intervals and
filtered through a 0.22 mm syringe.
Four comparing-control groups were set: group 1, supplementing a final
concentration of 100 u/ml bovine
catalase in the cell suspension; group 2, supplementing a final concentration
of

Statistical analysis

data were analyzed using spss
11.0 for Windows (SPSS,

Results

effect of antibody on H2O2 production by activated THP-1 monocytes

When human IgG-coated THP-1 monocytes were
activated by PMA, the production of H2O2 was significantly increased as time progressed. In
the absence of IgG, the production of H2O2 by PMA-activated THP-1 monocytes was much
lower than by IgG-coated THP-1 monocytes
activated with PMA (Fig. 1).

effect of antibody on O3 production
by activated THP-1 monocytes

The antibody-catalyzed O3 generation was
assayed by indigo carmine bleaching reaction, a sensitive chemical probe for O3. When human IgG-coated
THP-1 monocytes were activated by PMA, the indigo
carmine was markedly bleached, which was slightly enhanced by catalase and
significantly inhibited by vinylbenzoic acid. Omitting PMA or IgG produced no bleaching reaction of indigo carmine (Fig.
2).

effect of antibody on accumulation of cholesterol and
lipid peroxides in activated THP-1 monocytes

When THP-1 monocytes
were co-cultured with LDL, PMA and IgG, all the
intracellular lipids, including TC, FC, CE and lipid peroxides, increased
significantly compared with control groups (P<0.01 or P<0.05). The above parameters were clearly inhibited
by vinylbenzoic acid (P<0.01 or P<0.05), and only slightly affected by catalase (Table
1).

effect of antibody on foam cell formation from activated
THP-1 monocytes

When THP-1 monocytes
were co-cultured with LDL, PMA, and IgG, a great
number of foam cells were found by oil red O staining (Fig. 3), and the
percentage of foam cells was much higher than that of control groups (P<0.01). This process was also significantly
inhibited by vinylbenzoic acid (P<0.05), and only slightly affected by catalase (Table
2).

Discussion

It was considered that
the generation of O3 and H2O2 by
antibody-catalyzed water oxidation needs the participation of 1O2 [3,4], as follows (Equation 1):

Eq.  1

If the antibody-mediated
oxidation has any significant role in vivo, what is the origin of the
high-energy 1O2 molecule that
is required for initiation of the pathway? The primary source of 1OIn this study, we
detected O3 production from THP-1 monocytes using the
indigo carmine bleaching reaction. It is important to reiterate here that
indigo carmine, although a sensitive probe for O3 detection [3], is not
very specific, because 1O2 can also
bleach indigo carmine. So vinylbenzoic acid, a more specific ozone trap [12],
was used for distinguishing the possible involvement of 1OOur study further showed
that PMA-activated THP-1 monocytes, when cultured with IgG
and LDL, were clearly transformed into foam cells, and the contents of
intracellular TC, FC, CE and lipid peroxides in THP-1 monocytes increased
significantly. It has been proposed that foam cell formation is the result of
internalization of oxidation-modified LDL (Ox-LDL) by monocyte-derived
macrophages through scavenger receptors on the surfaces of these cells. The
internalization leads to the formation of lipid peroxides and facilitates the
accumulation of cholesterol esters, resulting in the formation of foam cells
[18]. Therefore, our findings implied that antibody-catalyzed water oxidation
could have participated in the oxidative modification of LDL and the formation
of macrophage-derived foam cells. This would be of profound significance to the
pathogenesis of atherosclerosis, because it is now widely believed that the
foam cell formation by Ox-LDL is a major pathologic characteristic of
atherosclerosis [19]. The accumulation of foam cells leads to the formation of
fatty streak lesions, which is a critical event in the early stages of
atherosclerosis. In the advanced stages of atherosclerosis, the death and
necrosis of foam cells facilitates the development of vulnerable
atherosclerotic plaques with large lipid cores and very thin fibrous caps, the
rupture of which leads to thrombus formation followed by clinical
manifestations of coronary heart disease, such as myocardial infarction [20].
Thus, the foam cell formation represents a key event in the development and
progression of atherosclerosis. The known biological activities of Ox-LDL, in
addition to its role in the formation of foam cells, also include: the chemotactic activity for circulating monocytes, a cytotoxic effect on endothelial cells; the induction in
endothelial cells of several factors such as granulocyte macrophage
colony-stimulating factor, cell adhesion molecules, platelet-derived growth
factor and heparin-binding epidermal growth factor-like protein; the induction
of platelet-derived growth factor in smooth muscle cells; the impairment of
endothelium-dependent arterial relaxation; and the stimulation of platelet
aggregation [21]. All of these events are involved in the development and
exacerbation of atherosclerosis. Taken together, our work provided novel
insights into the pathogenetic role of
antibody-catalyzed water oxidation in atherosclerosis.

The antibody-coated
THP-1 monocytes, after activation with PMA, can produce large amounts of O3 and H2O2, both of
which are strong oxidants, and might induce the oxidative modification of LDL.
Thus, they might be involved in the process of intracellular accumulation of
lipids and foam cell formation. However, which one is the main factor in the
oxidative damage of lipids and the formation of foam cells? This study showed
that the intracellular accumulation of TC, FC, CE and lipid peroxides, and the
formation of foam cells induced by antibody-catalyzed water oxidation, were
significantly inhibited by vinylbenzoic acid, a specific ozone trap. In
contrast, the above parameters were hardly affected by catalase, which can
remove H2O2. This finding suggests the involvement of OIn conclusion, these
results suggested that the production of O3, not H2O2, through the antibody-catalyzed water oxidation
pathway, could be an important mechanism for the pathogenesis and development
of atherosclerosis. The recent evidence of O3 formation in human
atherosclerotic arteries reported by Wentworth et al. [8] provides
strong support for this probability. Further study in vivo is necessary
to elucidate the positive evidence of the endogenous production of O3 through the
antibody-catalyzed water oxidation pathway. As well as a highly chemically reactive
character, O3 also elicits the production of cytokines,
including tumor necrosis factor-a and
interleukin-8 [22], thereby allowing the amplification of the inflammatory
cascade, which is a contributory factor in atherosclerosis.

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