[Environmental Health and Prevennve Medicine 5, 134-137, January. 2001]
Determination of the Platelet Activating Factor in Silicotic
Patients and its Effect on Fibroblasts
Qunwei ZHANG *~' 21 Yiqun MO .3, Jinpin LOU *~, Xinqiang ZHU .4, Zhimin CHEN .5,
Linyun HE*' and Huixian ZHONG*'
* l Institute of Hygiene, Zhejiang Academy of Medical Sciences, China
,k 2 Department of Environmental Medicme, School of Medicine, New York Universtty, U.S.A.
*3 Pharmacy College, The Ohio State University, U.S.A.
*,4 Department of Toxicology
~r Children's Hospital, School of Medicine, Zhejiang University, China
Platelet-activation factor (PAF), one of the potent proinflammatory mediators, is produced
from a large range of cells, induding polymorphonudear neutrophils, monocytes, and natural
killer cells. To study the role of PAF in the pathogenesis of silicosis, we determined the PAF in
silicotic patients and in healthy persons. The results showed that the concentration of PAF in
the plasma of silicotic patients was significantly higher than that of healthy persons. Our in
vitro experimental results showed that the total numbers of fibroblasts were markedly raised
with added PAF from 0 to 1/z g/ml. Adding 1/1 g/ml PAF significandy increased the total
numbers of fibroblasts after culture for 48, 72, 96 hrs. Therefore, we suggest that PAF be
possibly involved in the pathogenesis of silicosis. However, the mechanism remains to be
Key words: Platetet activating factor (PAF), Fibroblasts, Silicosis.
Platelet-activation factor (PAF, 1-O-alkyl-2-acetyl-sn-
glyvcero-3-phosphocholine) is a potent phospholipid
proinflammatory mediator that exerts its effects by binding to
specific receptors on responsive cell types *-~. Previous studies
showed that PAF mediates both the pulmonary microvascular
injury and acute inflammatory changes that follow administering
endotoxic or bleomycin to rats 4-5~
Inhalation of various dusts, such as silica and asbestos results
in pulmonary injury and fibrosis. Both in vivo and in vitro studies
have suggested that one mechanism underlying tissue injury
induced by these dusts involve the production of mediators by
activated lung phagocytes. Furthermore, reactive oxygen species
(ROS) have been recognized as injurious to many cell types via
modification of macromolecules such as DNA, and the effects of
oxidants on cells are complex and dose-related. Oxidants
generated by fibrogenic dusts may induce lipid peroxidation,
stimulation of cell-signaling cascades and transcription factor,
and release of cytokines such as tumor necrosis factor-alpha 6-7)~ A
number of studies have shown that in vitro exposure to 03 results
in increased production and relation of PAF by macrophages and
Received Dec. 17 1999/Accepted Apr. 3 2000
Reprint requests to: Qunwei ZHANG, M.D., Ph.D,
Department of Environmental Medicine, School of Medicine, New York
University, 57 Old Forge Road, Tuxedo Park, NY 10987, U.S.A.
TEL. +1(845)731-3526 FAX.+l(845)315-2118
epithelial cells, suggesting that this inflammatory mediator may
be important in the actions of 03 irritant ~. In macrophages and
neutrophils, PAF receptor binding induces a rapid and transient
mobilization of intracellular calcium'~-"'. This appears to be an
important component of the PAF signal transduction pathway
leading to increased arachidonic acid release and production of
eicosanoids ,2-,5~ as well as chemotaxis, degranulation, oxidative
metabolism, and adherence to vascular endothelium, all responses
associated with inflammation. However, there have been no
reports on the role of PAF ,which is an important inflammatory
mediator, in the pathogenesis of silicosis.
The aim of this study was to determine the PAF in the
plasma of silicotic patients, and try to find the effect of PAF on
the growth of fibroblasts. The data obtained from this study will
support some clues for seeking the pathogenesis of silicosis.
Subjects, materials and methods
There are thirty-one male silicotic patients, aged from 33 to
58 years old, with an average age 45 years. They were divided
into 3 categories with 10 category 1 patients, 11 category 2
patients, and 10 category 3 patients. None of these patients had
other diseases except silicotic disease. The diagnosis of silicosis
was based on a definite history of exposure to silica dust and
The Role of PAF in Sdmosis
silicotic findings on chest radiography according to the 1980
International Labor Office (ILO) Classification of Radiographs of
Pneumoconiosis"% And all of them were employed in a
fluorsparmine company. All the blood samples were collected
during a health examination.
All the 22 healthy subjects were also male, aged from 25 to
50, with an average of 43 years. Blood samples were collected
during a health examination.
Informed consent was obtained from all subjects, and all the
subjects were volunteers. The Ethics Committee of Zhejiang
Academic of Medical Sciences and the Public Health Department
of Zhejiang Province approved the study.
Sigma Company provided PAF standard.
Shanghai Cellular Biological Institute of Chinese Academy of
Sciences provided fibroblasts (WI-38 cells).
1. Determination of the concentration of PAF in plasma
The concentration of PAF was measured according to the
methods previously described ,5~. Briefly, 0.5-ml plasma was taken
from whole blood after centrifugation; 2 ml of distilled water and
4 ml of methanol were added and mixed , and then 2 ml of
distilled water and 2 ml of chloroform were added. Chloroform
phase was separated after centrifugation. Then PAF was separated
by thin-layer chromatography using activated silica gel G plates
developed in a Slovene system, chloroform/methanol/water
(65:35:6,vol/vol). The zone that comigrated with PAF standard
(Sigma Chemical Co.) was identified by iodine vapor, scraped,
and extracted by chloroform/methanol/water (l:2:0.8,vol/vol) as
described by Pinckard et a1,7). An aliquot was taken to calculate
the recovery; another aliquot of the sample was dried and used
for PAF assay.
2. Determination of the growth of fibroblasts
Fibroblasts were adjusted to a concentration of 2x105/ml
with RPMI-1640, and then 5 ml of cells with a concentration of
2x105 were placed in flask. Fibroblasts were cultured in an
incubator in a humidified atmosphere of 5%CO2/95% air at
3. Treatment of cells with PAF
Dose-response: PAF was added to the fibroblasts which were
attached in the flask at a final concentrations of 0, 0.01, 0.05, 1,
10, 100/1 g/ml. After culture for 48 hours, 0.25% trypsin was
added and the fibroblasts were collected. The cells were counted
in a hemacytometer by the conventional method.
Time-effect: 1/1 g/ml PAF was added to the fibroblasts
attached in the flask. After culture for 48, 72, 96 hrs, 0.25%
tryspin was added to collect the fibroblasts. The cells were
counted in a hemacytometer by the conventional method.
Value was expressed in terms of mean and standard errors.
The differences among groups were tested by one-way analysis of
variance (ANOVA). If a p value was less than 0.05, the difference
was considered significant. All the statistics were performed using
1. The concentration of PAF in plasma of healthy and silicotic
The concentration of PAF in plasma of categories 1,2, and 3
of the silicotic patients were 3.309 - 0.568 ng/ml, 3.197 --
0.740 ng/ml, and 3.635 - 0.568 ng/ml, respectively. Although
there were no significant differences among the categories, the
PAF in silicotic patients was significantly higher than that in
healthy persons (1.698 - 0.534ng/ml) (Figure 1)
2. Dose-response effect of PAF on the growth of fibroblasts
Figure 2 shows the growth of fibroblasts after adding various
doses of PAF. Each dose stimulated the population of fibroblast
increase, and there was a dose-related increase after adding PAF
from 0 to 1/1 g/ml. However, adding 10 or 100/~ g/ml of PAF
showed less growth enhancement than 1/L g/ml of PAF.
3.Time-response effect of PAF on the growth of fibroblasts
Figure 3 shows the growth of fibroblasts 48, 72 and 96 hrs
after adding PAF. The total numbers of fibroblasts in the control
and in that with added PAF increased within the experimental
period. However, adding 1 /1 g/ml PAF more significantly
increased the numbers of fibroblast cells than those of the control
within the experimental period.
Silicosis is a disease induced by inhalation of one of the
forms of crystalline silica. A number of studies have demonstrated
the relationship of pulmonary silica burden and silicosis. This
issue is complicated by a variety of factors, including type of silica
particle involved and co-exposures to other types of minerals. A
variety of cell types conventionally have been regarded as key
participants in the inflammatory process 77.
Our studies showed that the PAF in the plasma of silicotic
patient was higher than that in healthy subjects. This study does
not elucidate the mechanism where PAF is synthesized nor the
role of PAF in the development and progression of silicotic
nodules and fibrosis. However, PAF is now recognized as a
Healthy sub~.s CateL, ory I Category 2 Category 3
Fig. 1 The concentration of PAF in the plasma of healthy subjects
and the different categories of silicotic patients.
* Significantly different from the healthy subjects,P<O.05.
The Role of PAF in Silicosts
Different of doses r
48 72 96
Different times (hrs)
Fig. 2 Numbers of fibroblasts after treatment with different doses of PAF.
Note. Values are mean • SE of three experiments
* Significantly different from the control group P<0.01.
Fig. 3 Numbers of flbroblasts after treatment with PAF at different times.
Note. Values are mean • SE of three experiments
* Significantly different from the control group P<0.01.
phospholipid synthesized in response to specific stimuli by many
target cells in the lung, including the neutrophil, endothelial cells,
mast cells, and macrophages, and others. There are substantial
data to support the hypothesis that PAF is an important lipid
mediator in the inflammatory lung" 2~. Intratracheal instillation of
PAF induced a dose-dependent acute pulmonary inflammation
characterized by an accumulation of macrophages in the alveolar
space, as well as degenerative and necrotic changes of alveolar
epithelium, and an accumulation of macrophages of
polymorphonuclear leukocytes *~). PAF is also involved in
bleomycin-induced lung fibrosis and PAF-receptor antagonist
ameliorated the fibrosis ,91. Furthermore, PAF has been shown to
mediate both pulmonary microvascularinjury and acute
inflammatory changes where an endotoxin was administered to
rats ~"-~'. PAF has also shown to be a potent mediator of increased
vascular permeability in the bronchial circulation of the guinea
pig, which can be abolished using a PAF-receptor antagonist 22~.
The biological effect of PAF may be amplified by the presence of
other mediators released from activated neutrophils and platelets.
Such mediators include lysosomal enzymes, toxic oxygen radicals,
thromboxane, and leukotrienes ~' 2.~. Cytokine-mediated
interactions are known to regulate the proliferation of fibroblasts
under in vitro conditions and the monocyte/macrophage figures
prominently as an initiator ~324).
Our in vitro studies also showed that PAF could increase the
fibroblast populations. However, adding 10 or 100/~ g/ml of
PAF showed less growth enhancement than 1/1 g/ml of PAF.
The reason is still unknown. Probably when the fibroblast was
stimulated with a high concentration of PAF, PAF itself has a
cytotoxic effect on the fibroblast. As the principal cell type
responsible for collagen formation, the fibroblast becomes the
prime focus of attention, and its participation involves functional
augmentation in existing cells, with which an increase in
population may be associated ~4). Increase in the fibroblast
population may then contribute to connective tissue formation ~3).
Enhanced growth of fibroblasts and collagen production were
promoted by blood monocytes via mediators or by a factor
released from human alveolar macrophages after stimulation by a
nonmineral ~. However, this macrophage-derived growth factor
was unable by itself to stimulate fibroblast replication, for which
initiating factors provided by fibroblasts or platelets were required
to establish competence zs~. Regulation of fibroblast growth by this
means has exposed complexities. And implication of mineral
particles in the macrophage regulation of fibroblast proliferation
stems from observation on both compact and fibrous dusts.
Silica-treated macrophage extract enhanced collagen synthesis
under in vivo conditions and, reversing the arrangement, an
extract from silicotic rat lung stimulated proline incorporation by
granulation tissue fibroblasts ~). Furthermore, impaired
chemotaxis of macrophages by inhaled particles was confined to
those that were fibrogenic and proved to be independent of dust
burden, but the agent responsible was not identified 2~-~5k
In conclusion, our studies confirmed that PAF is involved
with the development of silicosis and can stimulate an increased
growth of fibroblasts.
This work was supported by a grant from The Public Health
Department of Zhejiang Province, People's Republic of China,
(Zhang QW). Authors gratefully acknowledge Dr. Yan DL, Dr.
Zhang ZH for they kind encouragement and help.
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