In vitro cytotoxicity of Manville Code 100 glass fibers: Effect of fiber length on human alveolar macrophages
Particle and Fibre Toxicology
In vitro cytotoxicity of Manville Code 100 glass fibers: Effect of fiber length on human alveolar macrophages
Patti C Zeidler-Erdely 2
William J Calhoun 1
Bill T Ameredes 1
Melissa P Clark 1
Gregory J Deye 0
Paul Baron 0
William Jones 3
Terri Blake 2
Vincent Castranova 2
0 Division of Applied Research and Technology, National Institute for Occupational Safety and Health , Cincinnati, OH , USA
1 AAARC, Division of Pulmonary , Allergy , and Critical Care Medicine, University of Pittsburgh , Pittsburgh, PA , USA
2 Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Morgantown, WV , USA
3 Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health , Morgantown, WV , USA
Background: Synthetic vitreous fibers (SVFs) are inorganic noncrystalline materials widely used in residential and industrial settings for insulation, filtration, and reinforcement purposes. SVFs conventionally include three major categories: fibrous glass, rock/slag/stone (mineral) wool, and ceramic fibers. Previous in vitro studies from our laboratory demonstrated length-dependent cytotoxic effects of glass fibers on rat alveolar macrophages which were possibly associated with incomplete phagocytosis of fibers ≥ 17 µm in length. The purpose of this study was to examine the influence of fiber length on primary human alveolar macrophages, which are larger in diameter than rat macrophages, using length-classified Manville Code 100 glass fibers (8, 10, 16, and 20 µm). It was hypothesized that complete engulfment of fibers by human alveolar macrophages could decrease fiber cytotoxicity; i.e. shorter fibers that can be completely engulfed might not be as cytotoxic as longer fibers. Human alveolar macrophages, obtained by segmental bronchoalveolar lavage of healthy, non-smoking volunteers, were treated with three different concentrations (determined by fiber number) of the sized fibers in vitro. Cytotoxicity was assessed by monitoring cytosolic lactate dehydrogenase release and loss of function as indicated by a decrease in zymosan-stimulated chemiluminescence. Results: Microscopic analysis indicated that human alveolar macrophages completely engulfed glass fibers of the 20 µm length. All fiber length fractions tested exhibited equal cytotoxicity on a per fiber basis, i.e. increasing lactate dehydrogenase and decreasing chemiluminescence in the same concentration-dependent fashion. Conclusion: The data suggest that due to the larger diameter of human alveolar macrophages, compared to rat alveolar macrophages, complete phagocytosis of longer fibers can occur with the human cells. Neither incomplete phagocytosis nor length-dependent toxicity was observed in fiber-exposed human macrophage cultures. In contrast, rat macrophages exhibited both incomplete phagocytosis of long fibers and length-dependent toxicity. The results of the human and rat cell studies suggest that incomplete engulfment may enhance cytotoxicity of fiber glass. However, the possibility should not be ruled out that differences between human versus rat macrophages other than cell diameter could account for differences in fiber effects.
Synthetic vitreous fibers (SVFs) are inorganic
noncrystalline materials widely used in residential and industrial
settings for insulation, filtration, and reinforcement
purposes. SVFs conventionally include three major
categories: fibrous glass, rock/slag/stone (mineral) wool, and
ceramic fibers . The chemical composition of fibrous
materials is known to play a role in fiber-induced toxicity
as fiber biodurability directly correlates with pathogenic
potential in rodents , but it has also been suggested that
fiber length is an important factor. In the past, the study
of fiber length as a cause of toxicity has been complicated
by the inability to obtain pure size-selected fiber samples.
However, the development of the dielectrophoretic
classifier by Baron and colleagues has aided in the study of
monodisperse size-selected fiber samples on lung cell
activation and toxicity . This classifier separates fibers by
length using dielectrophoresis that involves the
movement of neutral particles in a gradient electric field [3,4].
Rodent macrophage toxicity and activation have
previously been demonstrated in vitro in our laboratory using
these length-classified fibers and indeed, fiber length was
an important determinant [5-7].
Frustrated or incomplete phagocytosis has been
implicated as a mechanism of fiber-induced cytotoxicity. This
process involves repeated attempts by a phagocyte to
engulf a fiber longer than its diameter, thereby possibly
enhancing its respiratory burst activity . In comparison
to short fibers that are fully engulfed, longer fibers may
cause sustained cellular activation and increase phagocyte
recruitment into the airspace, subsequently increasing
lung oxidant burden [9-11]. Indeed, several in vivo and in
vitro rodent studies suggest longer fibers are more
pathogenic than short fibers [12-14]. However, macrophage
size is relevant when investigating fiber toxicity because
human alveolar macrophages are larger in size than rat
alveolar macrophages, approximately 18 and 13 µm in
average diameter, respectively . Therefore, the
purpose of this study was to examine the influence of fiber
length on isolated primary human alveolar macrophages,
which are larger in diameter than rat macrophages, using
length-classified Manville Code 100 (JM-100) glass fibers
(8, 10, 16, and 20 µm). Respiratory burst activity and
leakage of cytosolic lactate dehydrogenase (LDH) were used as
parameters of activation and toxicity, respectively.
Microscopic analysis was also conducted to determine if
frustrated phagocytosis had occurred. A comparison to results
obtained using the rat alveolar macrophage is made. Since
this investigation employed a static rather than a flow
system, issues of fiber solubility were not addressed.
L1Fma0iagc0cturagrotlaepshds1aefgihbeyesdr(rs1ofgo×ern11a08s5echeroelullser/swaseellf)rofomllopwrimngareyxphousmuarne atolv eJMol-ar
Lactate dehydrogenase release from primary human alveolar
macrophages (1 × 105 cells/well) following exposure to
JM100 glass fibers for 18 hours. Data are presented as percent
control from 100%. Bars represent mean values ± S.E. of
three independent experiments. No significant difference was
found among fiber length groups of a given concentration.
Glass fiber induced LDH
Figure 1 shows glass fiber-induced cytotoxicity on human
alveolar macrophages as measured by the LDH assay 18
hours post-treatment in vitro. The fiber lengths tested (8,
10, 16, and 20 µm) all exhibited equal cytotoxic effects.
These effects were not significantly different among fiber
length groups of the same concentration, i.e., fiber length
did not affect cytotoxicity over the range of lengths
evaluated. A trend toward a concentration-response was
observed although this did not achieve statistical
significance. Table 1 depicts the effects of glass fiber length on
rat alveolar macrophages as reported previously .
Significant toxicity, measured as % of total LDH, occurred
with the long (17 µm) glass fiber, but a shorter fiber
(7µm) showed little or no toxic effect.
Glass fiber induced inhibition of zymosan-stimulated
Zymosan-stimulated chemiluminescence, reported as %
control, is depicted in Figure 2. Zymosan, a particulate
βglucan from yeast cell walls, was used to stimulate reactive
species production in the human alveolar macrophages
exposed to glass fibers for 18 hours in vitro. Data show a
concentration-dependent decrease in macrophage
activation (i.e. reactive species production) with increasing fiber
concentration. Fiber length did not significantly affect
human alveolar macrophage function. It is of interest to
note the lowest concentration of fibers caused a priming
(71% increase in activation) of the human cells to release
reactive species in response to zymosan while the highest
Note: Adapted from Blake et al., 1998; ND is not detected ; LDH is
lactate dehydrogenase; * represents a significant difference from
concentration caused an 84% loss of activation. Table 2,
adapted from Blake et al., represents the percent decreased
activation versus control observed in rat macrophages
following glass fiber exposure for 18 hours . In contrast to
the human cells, the rat macrophages show distinctly
decreased oxidative function with increasing fiber length,
with the 17 µm fiber exhibiting a stronger effect (100%
loss of activation) than the 7µm fiber (15% reduction of
activation). In addition, no cellular priming was observed
in the rat macrophages as compared to the human.
Microscopic examination of primary human alveolar
Figure 3, panel A, demonstrates the ability of human
macrophages to engulf 8 µm glass fibers in vitro. Figure 3,
panel B, demonstrates that human macrophages can
successfully engulf 20 µm glass fibers. Data from Blake et al.
revealed incomplete phagocytosis by rat alveolar
macrophages occurred with glass fibers ≥ 17 µm in length .
These results suggest human alveolar macrophages can
completely phagocytize fiber lengths that rat alveolar
macrophages can not. Therefore, frustrated phagocytosis
and its possible effects do not appear to be a factor with
human alveolar macrophages exposed to fibers ≤ 20 µm
The present study employed an in vitro system to expose
primary human alveolar macrophages to monodisperse
JM-100 glass fibers of different target lengths. Using this
system, human alveolar macrophages were assessed for
length-dependent cellular effects and results were
compared to previous data obtained with rat alveolar
macrophages. The data presented here reveal differences in the
responses of rat versus human macrophages to glass fibers
of various lengths.
Previous studies have shown that rat alveolar and mouse
peritoneal macrophages attempt to engulf glass fibers and
that successful phagocytosis is influenced by fiber length
[5,7]. Short (7 µm) glass fibers were completely engulfed,
fporlilmowarinyghaunm1a8nhaolvueroelxarpoInhibition of zymosan-stimulated primary human alveolar
macrophage chemiluminescence following an18 hour
exposure to JM-100 glass fibers. Data are presented as percent
control from 100%. Bars represent mean values ± S.E of
three independent experiments. * - indicates a significant
difference between the lowest (4 × 105 fibers/ml or 1:1
fiber:cell) and intermediate (4 × 106 fibers/ml or 8:1 fiber:cell)
fiber concentrations, †- between the lowest and highest (4 ×
107 fibers/ml or 80:1 fiber:cell) fiber concentrations, and $
between the intermediate and highest fiber concentrations of
the same fiber length (p ≤ 0.05). Fiber length did not affect
human alveolar macrophage function.
whereas long (17 µm) glass fibers were only partially
engulfed. Human alveolar macrophages have a larger
diameter than the rat counterpart; therefore, it was
hypothesized that these phagocytes would completely
engulf longer fibers and the absence of frustrated
phagocytosis would attenuate cytotoxicity. Overall, the data
supported this hypothesis.
Human alveolar macrophage function, measured as
zymosan-stimulated chemiluminescence, was
significantly affected by fiber concentration but not fiber length
over the range of 8–20 µm (Figure 2). The lowest of three
fiber concentrations (4 × 105 fibers/ml or 1:1 fiber:cell
ratio) primed the cellular response to zymosan while the
highest concentration (4 × 107fibers/ml or 80:1 fiber:cell
ratio) decreased this response from control. The activation
level of cells exposed to the intermediate fiber
concentration (4 × 106 fibers/ml or 8:1 fiber:cell ratio) was similar
to that of controls). In contrast, our previous findings
showed no increased priming effect of JM-100 glass fibers
in rat alveolar macrophages at any concentration. This
differential response between human and rat cells may be
the result of several possible factors. First, fiber to cell ratio
has been shown in our laboratory to be important in the
activation of rodent macrophages. Prior studies revealed a
fiber to cell ratio of at least 5:1 is required for significant
Note: Adapted from Blake et al., 1998; ND is not detected;*
represents a significant decrease from control
tumor necrosis factor-α (TNF-α) production in mouse
macrophages . The effective fiber to cell ratio found to
elicit human macrophage cellular priming in this study
was 1:1. Approximately a 1:2.5 fiber to cell ratio (106
fibers/ml) was used as a low concentration in the rat
macrophage studies reported by Blake et al., which may not have
been high enough to prime the cellular response to
zymosan above control following short fiber phagocytosis
. Secondly, luminol was used as the light enhancer in
the present study, but lucigenin was used in the rat cell
study. Luminol detects multiple reactive species
compared to lucigenin, which is a superoxide specific light
enhancer [16-18]. Most likely, superoxide is not the sole
oxidant produced in glass fiber-exposed cells. Therefore,
this experimental system may have an enhanced detection
level compared to the past system. Finally, the result may
reflect inherent differences in the oxidant production of
rat and human alveolar macrophages. Human alveolar
macrophages are reportedly more active than the rat in the
production of reactive species following particulate
exposure . Whether this is the case with glass fibers would
need further investigation.
It was found that no fiber length or concentration induced
significant cellular membrane damage as assessed by
cytosolic LDH release into the cellular supernatant
although a concentration-dependent trend was apparent
(Figure 1). Castranova et al. reported effects of metallic
ions on particle-stimulated oxygen consumption and
chemiluminescence in alveolar macrophages, i.e.,
functional assays, occurs at concentrations that do not affect
the integrity of the cellular membrane . Therefore, a
reason for the lack of a significant LDH
concentrationresponse may be that the cellular function is typically
affected before membrane damage making the
chemiluminescence response a more sensitive measure of
In summary, this study reported no effects of fiber length
over the range of 8–20 µm on the human alveolar
macrophage response. This conflicts with previous data where
increased glass fiber concentration decreased
chemiluminescence and increased LDH in rat alveolar macrophages
but major length-dependent effects were evident. The
reason for this discrepancy most likely is due to the larger
diameter of human alveolar macrophages, resulting in the
lack of frustrated phagocytosis in the present experimental
system. Microscopic analysis verified these cells were able
to completely engulf even the longest fiber sample tested
(20 µm) (Figure 3B). Blake et al. demonstrated cellular
effects on rat alveolar macrophages (diameter ~13 µm)
using 17 and 33 µm glass fibers, approximately 4–20 µm
longer than the cells (multiple rat macrophages were
often observed adhered to a long fiber with the fiber
appearing to protrude through the cellular membrane)
. In the present study, the longest fiber sample used was
20 µm, only 2 µm longer than the human alveolar
macrophage cellular diameter; therefore not a sufficient length
to cause frustrated phagocytosis. Consequently, further
studies are needed using fiber samples over a range of 20–
40 µm in length. Assuming similar mechanisms are
involved in human and rat fiber phagocytosis, results
most likely would agree with those reported by Blake and
This study showed: 1.) an absence of length-associated
cytotoxicity in primary human alveolar macrophages that
was previously observed in rat alveolar macrophages
treated in vitro with length-classified glass fibers 2.) a
possible mechanism for this absence of length-dependent
cytotoxicity may be the lack of frustrated phagocytosis in
the human macrophages versus the rat 3.) human alveolar
macrophages appear to be activated by glass fibers of
monodisperse lengths at a fiber to cell ratio of
approximately 1:1, while significant cytotoxicity was observed
only at an excessively high fiber:cell ratio of 80:1. In
conclusion, the use of monodisperse length-classified fiber
samples will aid in the determination of specific fiber
lengths that macrophages can engulf. In addition, these
preliminary data may aid in the design of future in vivo
experiments using fibrous particles.
Bulk samples of JM-100 glass (Manville code 100
supplied by John Mansville Corporation) were first milled,
aerosolized, and separated into length categories using
dielectrophoresis as previously described [3,4]. The
dielectrophoretic classifier was operated in a differential
mode so that fibers with narrow length distributions were
extracted in an air suspension at the end of the classifier.
These length-classified fiber samples were collected on
polycarbonate (Nuclepore) filters at rates up to 1 mg/day.
Fibers were scraped off the filters for microscopic analysis
and for biological experiments.
eFMniiggcurolrfisencgo3pgliacsasnfaiblyesris of primary human alveolar macrophages
Microscopic analysis of primary human alveolar macrophages
engulfing glass fibers. Panel A demonstrates the ability of the
alveolar macrophages to engulf 8 µm glass fibers. Panel B
verifies effective phagocytosis of 20 µm glass fibers by human
Samples of the length-classified fibers were prepared for
size and count analysis by adding weighed portions of the
dusts to freshly filtered water. These samples were then
diluted and filtered through polycarbonate filters.
Measurements of length, width, and fiber count/mass were
made using a JEOL JSM-6400 scanning electron
microscope (Table 3). Measurements at each magnification
were referenced to a National Institute of Standards and
Technology electron microscopy standard rule.
All fiber samples were heat-sterilized and stored at 4–6°C.
Prior to each experiment, the fibers were suspended in
sterile Ca+2 + Mg+2 free phosphate-buffered saline (PBS).
Human bronchoalveolar lavage
Human alveolar macrophages were collected by a
standard procedure at University of Pittsburgh. Briefly, a total of
six male healthy subjects between the ages of 20 and 40
years were recruited for study. All signed a statement of
informed consent approved by the University of
Pittsburgh Institutional Review Board for Biomedical
Research. Subjects were screened by history, physical
examination, and spirometry. All subjects had to exhibit
normal spirometry, and have no history of asthma or
allergic diseases. Skin test responses to a panel of 15
common allergens, plus histamine phosphate as a positive
control (Greer Laboratories, Lenoir, NC), were
determined using the prick-puncture method. Any subject
demonstrating a positive skin test to an allergen was
deemed to have atopic disease and was excluded.
Bronchoscopy and bronchoalveolar lavage (BAL) were
performed according to published methods [21-23]. Briefly,
subjects were lightly sedated with midazolam 1.0 mg and
atropine 0.5 mg IM, and meticulous local anesthesia of
the nasopharynx was obtained using 4% cocaine solution,
1% lidocaine by gargle, and 1% benzocaine aerosol.
Additional topical 1% lidocaine, via the bronchoscope
working channel, was applied to the vocal cords and airway
mucosa. Total lidocaine dose delivered below the vocal
cords was in all cases less than 200 mg. The bronchoscope
was advanced to the right upper lobe, and wedged in a
subsegment of the anterior segment. BAL was then
performed using two aliquots of sterile saline solution (0.9%
NaCl, 37°C, 60 ml each), recovered by hand suction. The
bronchoscope was then moved to a subsegment of the
right middle lobe, medial segment, and BAL was again
performed. Cells were recovered from BAL fluid by
centrifugation at 400 × g for 15 minutes and the resulting cell
pellet was washed once with Hanks Balanced Salt
Solution (Sigma, St Louis). Cells were counted using a
hemocytometer and adjusted to 2 × 106 cells/ml in Hanks
Balanced Salt Solution. Differential cell counts were
performed on Diff-Quick stained cytocentrifuge slides, and
≥300 cells were identified as alveolar macrophages,
lymphocytes, eosinophils, or neutrophils. Viability by trypan
blue exclusion always exceeded 95%, and cell populations
were in excess of 95% alveolar macrophages, with the
balance of the population comprised of lymphocytes.
Neutrophils were rarely observed as were eosinophils. In
healthy volunteers, purification over discontinuous
Percoll gradients is unnecessary .
Primary human alveolar macrophage zymosan-stimulated
Human alveolar macrophages were plated in a white
opaque 96-well plate at 1 × 105 cells/well in 200 µl of
sterile Eagle's modified essential medium (Biowhittaker,
Walkersville, MD) without phenol red supplemented
with 2% heat-inactivated fetal bovine serum, 1 mM
glutamine, 100 units/ml penicillin/streptomycin, and 10
mM HEPES at pH 7.2. After a 2 hour incubation at 37°C,
the adherent cells were washed three times with warm
media. Glass fibers (8,10,16, or 20 µm) were then added
to the cells at three different concentrations determined
by fiber number: 4 × 105, 4 × 106, and 4 × 107 fibers/ml.
Cultures were brought to a final volume of 200 µl and
each treatment was done in duplicate. After 18 hours at
37°C, the plate was centrifuged and the supernatant
harvested and kept on ice for later analysis. Warm
HEPESbuffered solution (pH = 7.4) was then added to the
alveolar macrophages on the plate and zymosan-stimulated
chemiluminescence was measured. The lumigenic
substance luminol (1 mM) was added to all wells of the assay
plate followed by zymosan (2 mg/ml) or HEPES-buffered
solution to a final volume of 220 µl/well.
Chemiluminescence was measured at 460 nm using a microplate
chemiluminometer (ML 3000 Microtiter Plate Luminometer,
Dynatech Laboratories, Chantilly, VA) for 30 minutes and
total cpm per well per 30 minutes was recorded.
Zymosan-stimulated chemiluminescence was calculated
as the stimulated (with zymosan) value minus the
corresponding unstimulated (without zymosan) value.
aLength measurements reported for the 16 and 20 µm fiber samples exclude short fiber populations that occasionally adhered to the long fibers
during classification. Prior to use, fiber suspensions were gently vortexed rather than sonicated to minimize release of these short fibers. Inclusion
of short fibers in the analysis slightly lowered the mean fiber length in the distribution for the 16 and 20 µm fiber samples. Note: Values for length
and width are means ± SD
Lactate dehydrogenase activity
LDH activity was determined in the culture supernatant
by monitoring the LDH catalyzed oxidation of pyruvate
coupled with the reduction of NAD at 340 nm using a
commercial kit and a Cobas Mira Plus Transfer Analyzer
(Roche Diagnostics Systems, Montclair, NJ).
Photographs of human alveolar macrophages were taken
Olympus IX70 inverted light
equipped with a Dage camera.
All data were analyzed using a one-way analysis of
a significant difference
defined as p ≤ 0.05. Pairwise differences were assessed
through appropriate contrasts.
BAL Bronchoalveolar Lavage
JM-100 Manville Code 100 Glass Fibers
LDH Lactate Dehydrogenase
PBS Phosphate Buffered Saline
SEM Scanning Electron Microscope
SVFs Synthetic Vitreous Fibers
The author(s) declare that they have no competing
PCZE carried out the in vitro cytotoxicity studies, analyzed
the data, and drafted the manuscript. WJC, BTA, and MPC
provided the human macrophages and provided
assistance in study design and coordination. GJD and PB
provided the length-classified fiber samples and aided in fiber
analysis. WJ performed the fiber analysis and provided
assistance in the study design. TB conducted studies with
the rat alveolar macrophages. VC conceived of the study,
participated in its design and coordination and helped in
manuscript preparation. All authors read and approved
the final manuscript.
the authors and do not necessarily represent the views of
the National Institute for Occupational Safety and Health.
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