Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Toxicology Program, University of Texas Medical School, Houston, Texas 77030
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ABSTRACT |
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Exposure to silica dust can result in lung
inflammation that may progress to fibrosis for which there is no
effective clinical treatment. The mechanisms involved in the
development of pulmonary silicosis have not been well defined; however,
most current evidence implicates a central role for alveolar
macrophages in this process. We have previously demonstrated that
fibrotic agents, such as asbestos and silica, induce apoptosis in human
alveolar macrophages. The goal of this study was to identify molecular
events in the silica-induced apoptotic process to better understand the
mechanism by which fibrotic agents may be inducing apoptosis in human
alveolar macrophages. To elucidate the possible mechanism by which
silica causes apoptosis, we investigated the involvement of the
interleukin-converting enzyme (ICE) family of proteases. Human alveolar
macrophages were treated with silica in vitro and were examined for the
involvement of ICE, Ich-1L, and
cpp32 in silica-induced apoptosis. Pretreatment of cells with 10 µM of the ICE inhibitor z-Val-Ala-Asp-fluoromethyl ketone and the
cpp32
inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone completely
blocked silica-induced apoptosis. Additionally, an increased formation
of the active p20 fragments of ICE and
Ich-1L as well as degradation of
the inactive zymogen form of cpp32
protein were observed in
silica-treated human alveolar macrophages, indicating activation of
these proteases. Furthermore, degradation of the nuclear protein
poly(ADP-ribose) polymerase was observed within 2 h of silica
treatment. These results suggest that silica-induced apoptosis involves
activation of the ICE family of proteases and is the first step in
elucidating the intracellular mechanism of particulate-induced
apoptosis in human alveolar macrophages.
interleukin-converting enzyme; Ich-1L; cpp32
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INTRODUCTION |
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SILICA IS A UBIQUITOUS occupational fibrogenic agent capable of inducing fibroblast proliferation and excess collagen production, causing lung fibrosis (silicosis; see Ref. 27). Silicotic lungs in experimental animals are characterized by macrophage aggregates, a significant increase in lymphocytes, and alveolar type II cell hyperplasia (31). It can therefore be concluded that a repertoire of cells, including lymphocytes, macrophages, and fibroblasts, are ultimately involved in the development of silicosis. Most current observations indicate that alveolar macrophages (AM) play a central role in the development of fibrosis (9, 24, 30). AM isolated from silicotic patients and animals are highly activated and release excessive amounts of fibrogenic factors and cytokines (6, 29). In vitro studies in our laboratory have established that treatment of human AM with particulates, such as silica and asbestos, results in apoptosis (14, 18).
Apoptosis or programmed cell death is a mechanism of cellular death believed to play an important role in a wide variety of physiological conditions (36). Deregulation of apoptosis has been proposed to contribute to the pathogenesis of many diseases ranging from cancer to acquired immunodeficiency syndrome (26). Shrinkage of cells accompanied by unique DNA fragmentation, about 180-200 bp, caused by the activation of endonucleases are characteristic features of cells undergoing apoptosis (38). Although the detailed mechanism of apoptosis has not been established, proteases are thought to play an important role in the regulation of programmed cell death (23).
It appears that a family of cysteine proteases of the
interleukin-converting enzyme (ICE) family is involved in programmed cell death or apoptosis (7, 23, 35, 37, 38). ICE is a cytoplasmic
cysteine protease synthesized as an inactive 45-kDa precursor that is
proteolytically cleaved to the active 20-kDa (p20) and 10-kDa (p10)
heterodimer form. The primary known function of ICE is to cleave the
inactive 31-kDa pro-interleukin (IL)-1 to generate the active
17.5-kDa form of IL-1
(25). In addition, several lines of evidence
indicate that proteases such as ICE may be important modulators of
apoptosis. A number of inhibitors of the caspase family have been used
to study the involvement of these proteases in apoptosis. Cytokine
response modifier A (CrmA) is a cytokine response modifier gene encoded
by cowpox virus that preferentially inhibits ICE over cpp32 and p35, a
baculovirus that inhibits both ICE and cpp32. Additionally, synthetic
tetrapeptides, such as Tyr-Val-Ala-Asp (YVAD) and Asp-Glu-Val-Asp
(DEVD), specifically inhibit ICE and cpp32, respectively. Studies with
inhibitors of ICE, such as cowpox virus protein CrmA, and the
tetrapeptides YVAD (7) and DEVD show that they dramatically block
apoptotic cell death (35, 37), implicating the involvement of ICE in apoptosis.
Several ICE homologs have been identified, such as cpp32, Mch2,
Mch3, Mch4, Tx (ICE rel III), Ich-2 (ICE rel II),
Ich-1S, and
Ich-1L. ICE and cpp32
have been
demonstrated to play an important role in Fas-induced apoptosis, the
latter also being inhibitable by CrmA. An overproduction of
Ich-1L has been shown to induce apoptosis, whereas Ich-1S serves
to protect against cell death (37). Each member of this family of
proteases is synthesized in a pro (zymogen) form, which is required to
be cleaved for activation (37). Therefore, multiple members of the ICE
family appear to be cleaved and activated in the apoptotic process (4,
8). It has been proposed that members of the ICE family are involved in
a chain of events leading to apoptosis (35, 37). For example, ICE has
been shown to process cpp32
to its active form in vitro (35).
Additionally, Ich-1L appears to be
also processed by ICE (22). Therefore, all of these proteases are
capable of autocleavage and/or can serve as substrates.
As a consequence of the activation of the ICE family of proteases, a
number of nuclear proteins such as nuclear lamins, U1 ribonucleoprotein, and poly(ADP) ribose polymerase (PARP) have been
reported to be degraded in apoptosis. PARP, a 116-kDa nuclear protein,
is cleaved to an 85-kDa fragment in apoptosis induced by a number of
agents (19). Studies have demonstrated that members of the ICE family,
such as cpp32 (35) and Ich-1L
(22), may be responsible for the formation of the 85-kDa PARP fragment.
Previous studies have demonstrated that silica treatment of human AM
results in a slightly increased release of IL-1 (15). This increased
IL-1
release may have been due to the activation of ICE by silica.
The purpose of this study was to test the hypothesis that
silica-induced apoptosis of human AM may involve the activation of ICE
and/or other members of its family.
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MATERIALS AND METHODS |
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Cell cultures. Human AM were obtained by bronchoalveolar lavage of normal, nonsmoking adult volunteers of either gender as previously described (5). This procedure has been approved by the University of Texas Committee for the Protection of Human Subjects. Installations of sterile saline resulted in recoveries of 240-260 ml of lavage fluid that was kept at 4°C until cells were isolated from the lavage fluid by centrifugation. The saline supernatant was aspirated and discarded, and the cell pellet was resuspended in a small volume (1-5 ml) of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium 199 (GIBCO-BRL, Gaithersburg, MD) with 10% fetal bovine serum (Sigma) and antibiotics (50 U/ml penicillin, 50 µg/ml gentamicin, and 50 µg/ml streptomycin). The cell count was determined with a ZBI Coulter Counter (Coulter Electronics, Hialeah, FL). Lavages yielded an average of 20 × 106 cells that were >92% AM, as verified by leukostat staining (Fisher Scientific, Houston, TX). Viability was >90% as determined by trypan blue exclusion.
Particulate. Crystalline silica (average size 5 µm, acid-washed Min-U-Sil-5 from Pennsylvania Glass Sand, Pittsburgh, PA), at a concentration of 133 µg/1 × 106 cells, was used in all experiments. We have established that 133 µg/ml is a bioactive but nonnecrotic dose of crystalline silica (18).
Cytocentrifugation and morphological differentials. Immediately after cell culture, 30 × 103 cells were incubated with phosphate-buffered saline (PBS) for 5 min in sterile disposable cytofunnels (Shandon, Pittsburgh, PA) and were centrifuged at 1,500 revolutions/min (rpm) for 5 min on positively charged glass slides (Probe On Plus; Fisher Scientific, Pittsburgh, PA) using a Shandon cytospin 2 (Shandon). Slides were stored at 25°C until leukostat fixation, and staining was performed.
Leukostat staining. After cytocentrifugation, cells were fixed in cold methyl alcohol for 5 min, stained in leukostat (Fisher Scientific) eosin stain for 2 min, and then stained in leukostat methylene blue stain for 4 s. The slides were air-dried and were examined by light microscope at ×630 (dry objective).
Necrosis assay (trypan blue exclusion). Cells were exposed to trypan blue dye (0.04% in PBS), placed on a hemocytometer, and examined under light microscopy. Only necrotic cells internalize the dye. Two-hundred random cells were counted after each treatment, and the percentage of "blue" cells was expressed as the percentage of necrotic cells for any given condition. There was no evidence of significant necrosis in silica-treated human AM at 6 h as seen by trypan blue staining.
DNA fragmentation assay.
Particulate-treated cells were washed one time with PBS before DNA
isolation. Genomic DNA was isolated by using the DNA isolator (Genosys,
Woodlands, TX). The isolated genomic DNA was dissolved in 10 mM
tris(hydroxymethyl)aminomethane (Tris) and 1 mM EDTA buffer and was
3'-end labeled with
[-32P]dCTP (ICN
Pharmaceuticals, Costa Mesa, CA) by incubation of 1 µg of DNA in 50 µl of reaction buffer (50 mM Tris · HCl, pH 7.6, 10 mM MgCl2, 200 µM dATP, 200 µM
dGTP, 200 µM dTTP, 2 µl [
-32P]dCTP, and 2 units klenow) at 37°C for 30 min. The
[
-32P]dCTP-labeled
DNA was mixed with 10 µl loading buffer (0.25% bromphenol blue, 100 mM EDTA, and 30% glycerol). The same amount of
[
-32P]dCTP-labeled
DNA (50 ng) for each sample was loaded onto a 2% agarose gel and was
run at 5 V/cm for 5 h in 40 mM Tris-acetate buffer, pH 8.0, with 1 mM
EDTA. The gel was dried at 60°C for 4 h under vacuum in a gel drier
and was exposed to X-ray film for detection of labeled DNA fragments.
Cell death enzyme-linked immunosorbent assay. Cytosolic histone-bound DNA fragments were detected by cell death enzyme-linked immunosorbent assay (ELISA). Cells from control and particulate treatments were processed according to the manufacture's protocol and were analyzed for cytosolic histone-bound DNA fragments using the cell death ELISA kit (Boehringer Mannheim, Indianapolis, IN). Briefly, 1 × 105 cells from each condition were washed one time with PBS; cells were then lysed in 1 ml lysis buffer, incubated on ice for 30 min, and then centrifuged at 14,000 rpm for 15 min at 4°C. Lysate were retained, and 100 µl of lysate were used for each reaction. A 96-well ELISA plate was coated with blocking buffer for 2 h, wells were washed, and 100 µl of anti-histone antibody were added per well and incubated for 90 min at room temperature; wells were washed, and 100 µl of cell lysate (triplicate assays were performed for each condition) were added and incubated for 90 min at room temperature. Wells were washed, and 100 µl of developing reagent were added. The reaction was allowed to occur for 15 min, and optical density was read at 405 nm using an Emax precision microplate reader (Molecular Devices, Sunnyvale, CA).
ICE and cpp32 in silica-induced
apoptosis. Cells were pretreated with and without ICE
and cpp32
inhibitors z-Val-Ala-Asp-fluoromethyl ketone (zVAD-FMK; 10 µM; Enzyme Systems, Livermore, CA) and Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-FMK; 10 µM; Enzyme Systems) at 25°C for 30 min.
Cells were cultured at 1 × 106 cells/ml in the presence or
absence of silica for 6 h at 37°C. Cells were maintained in
suspension by slow end-over-end tumbling in sterile polypropylene
tubes. Cultures were assayed for apoptosis by cell death ELISA and DNA
fragmentation assay.
Immunoprecipitation. Approximately 3 × 106 cells were cultured at 1 × 106 cells/ml with or without zVAD-FMK in the presence or absence of silica for 4 h at 37°C. Cells were lysed with 250 µl sodium dodecyl sulfate (SDS) lysis buffer, incubated for 30 min on ice, and centrifuged at 14,000 g for 15 min, and supernatant was retained. Rabbit immunoglobulin G was added (for nonspecific binding) and was incubated for 1 h after which 20 µl of 50% Protein A/G beads were added and allowed to incubate at 4°C for 1 h with slow end-over-end tumbling. Samples were centrifuged, and beads were discarded. Antibody to the protein of interest (anti-ICE p20 or anti-Ich-1L p20, Santa Cruz Biotechnologies, Santa Cruz, CA; anti-cpp32 p20, Transduction Laboratories, Lexington, KY) at a concentration of 20 µg/ml was added to the supernatant and was incubated overnight at 4°C. The p20 protein-antibody complex was isolated by incubation with 50% Protein A/G beads for 1 h at 4°C. Samples were washed and centrifuged three times, and the Protein A/G beads with the bound p20 protein were retained. Protein was denatured with denaturing buffer and was boiled for 5 min to dissociate the p20 from the beads.
SDS-polyacrylamide gel electrophoresis and
immunoblotting. Aliquots of the above denatured protein
were run on 12% SDS Ready Gels (Bio-Rad, Hercules, CA) in a mini-gel
apparatus (Bio-Rad). Resolved proteins were transferred using a wet
minitransfer unit (Bio-Rad) to a nitrocellulose membrane (Amersham,
Arlington Heights, IL). Membranes were blocked by overnight incubation
at 4°C in blocking buffer (5% dry nonfat milk in 10 mM Tris and
150 mM NaCl, pH 7.2). Membranes were then incubated with the relevant
antibodies (anti-ICE p20,
anti-Ich-1L p20 from Santa Cruz
Biotechnologies; and anti-cpp32 from Transduction Laboratories) at 1 µg/ml dilution in blocking buffer for 16 h at 4°C and were washed
extensively with Tris-buffered saline [0.05% Tween 20, pH 8.0 (TBST)]. Blots were incubated with peroxidase-linked
anti-rabbit/mouse immunoglobin (Amersham) in TBST for 1 h at 25°C
and then was washed extensively with TBST. Blots were placed in
enhanced chemiluminescence (ECL) reagents (Amersham) for 1 min,
followed by exposure to autoradiographic film (ECL film; Amersham) for
3 min, and were developed by an automated film processor (Kodak).
Cleavage of PARP and cpp32
degradation. Approximately 2 × 106 cells were cultured at 1 × 106 cells/ml in the presence or absence of silica
(133 µg/ml) for 2, 4, and 6 h at 37°C. Samples were denatured,
and aliquots of the denatured protein were run on 12% SDS Ready Gels
(Bio-Rad) in a mini-gel apparatus (Bio-Rad). Immunoblotting was
performed as described above using the anti-cpp32
monoclonal
antibody (Transduction Laboratories) at 1 µg/ml and anti-PARP
monoclonal antibody (Enzyme Systems, Livermore, CA) at 1:1,000
dilution.
Statistical analysis. Values are presented as means ± SE. The number of individuals whose cells were used for a given experiment is denoted by n in the legends for Figs. 1-9. For each experiment, statistical treatment included a one-way analysis of variance followed by a Student-Newman-Keuls test for post hoc pairwise comparisons.
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RESULTS |
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ICE in silica-induced apoptosis. In a previous study, preliminary data were presented demonstrating the partial inhibition of silica-induced apoptosis by the ICE inhibitor zVAD-FMK (2 µM) in human AM (18). This result suggested the involvement of ICE. To establish more firmly the role of ICE in silica-induced apoptosis, a higher concentration of zVAD-FMK was used. Human AM were treated with 133 µg/ml silica with and without 10 µM zVAD-FMK for 6 h at 37°C and were evaluated for apoptosis by cell death ELISA (Fig. 1). The cell death ELISA assay detects cytosolic histone-bound DNA fragments formed in cells undergoing apoptosis. The results revealed that treatment of human AM with silica resulted in a significant increase in cytosolic histone-bound DNA fragments compared with control. However, significant inhibition of silica-induced apoptosis was observed in cells incubated with 10 µM zVAD-FMK.
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To confirm the cell death ELISA results, silica-treated cells were also examined morphologically. Figure 2 is a representative photomicrograph of cells incubated for 4 h and represent control (A), 133 µg/ml silica treated (B), 10 µM zVAD-FMK treated (C), and 10 µM zVAD-FMK treated and 133 µg/ml silica treated (D). Control AM are rounded with uniformly large, light purple nuclei and normal cytoplasms. In contrast, Fig. 2B shows that human AM treated with silica have dark, shrunken nuclei indicative of nuclear condensation commonly seen in apoptosis. Nuclear disintegration is also apparent in some cells, which is characterized by a very dark, condensed, segmented nucleus. In contrast, cells treated with silica in the presence of zVAD-FMK have a morphology similar to control cells, indicating the absence of apoptosis (Fig. 2D). Therefore, zVAD-FMK was effective in blocking silica-induced apoptosis, further implicating ICE involvement in the apoptotic process.
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Because internucleosomal DNA fragmentation is a characteristic feature of apoptotic cells, a DNA agarose gel was used to detect the presence of DNA fragments in 133 µg/ml silica-treated human AM with and without 10 µM zVAD-FMK. As can be seen in Fig. 3, treatment of human AM with silica resulted in significant DNA ladder formation at 6 h. However, pretreatment with 10 µM zVAD-FMK blocked silica-induced DNA fragmentation. Consequently, the results from cell death ELISA assay (measuring cytosolic histone-bound DNA), morphological characterization, and the DNA ladder formation demonstrated that zVAD-FMK was an effective inhibitor of silica-induced apoptosis. Taken together, these results demonstrate the involvement of ICE in regulating silica-induced apoptosis of human AM.
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Formation of the p20 fragment of ICE. As described earlier, the zymogen form of ICE is cleaved to a heterodimeric form (p20 and p10) to be activated (10). Therefore, to determine whether ICE is activated in silica-induced apoptosis, the formation of the active component of ICE, i.e., p20, was assessed. This was accomplished by immunoprecipitation with ICE p20 antibody followed by Western analysis as described in MATERIALS AND METHODS. The level of the p20 "active" form of ICE was determined in human AM treated with silica with or without 10 µM zVAD-FMK pretreatment. As shown in Fig. 4, there was an increase in the level of p20 after silica treatment within 1 h. In contrast, when cells were stimulated with silica in the presence of zVAD-FMK, the level of p20 was only marginally higher than control. These results provide evidence for silica-induced activation of ICE and for ICE activation being one of the crucial events in the induction of apoptosis by silica. Collectively, the above results (Figs. 1-4) strongly support a central role for ICE in silica-induced apoptosis.
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Involvement of Ich-1L. In a similar manner to other members of the ICE family, Ich-1L is present as an inactive p48 zymogen form that is cleaved to the active p20 and p10 heterodimer form. Therefore, the involvement of Ich-1L activation in silica-treated human AM cells was examined, using the same procedure as used for detecting ICE activation (immunoprecipitation and Western analysis). As shown in Fig. 5, an increase in Ich-1L p20 levels was detected as early as 1 h in the 133 µg/ml silica-treated cells. Furthermore, pretreatment (30 min) of human AM with 10 µM zVAD-FMK blocked the silica-induced increase of Ich-1L. These results suggest that silica increased Ich-1L activity in human AM, implicating the involvement of this protease in the sequence of events that result in silica-induced apoptosis.
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DISCUSSION |
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In previous studies, we demonstrated that in vitro treatment of human AM with fibrogenic particulates such as silica (18) and asbestos (14) resulted in cell death by apoptosis, whereas nonfibrogenic particulates did not induce apoptosis. The results suggested that the fibrogenecity of a particulate correlated with its ability to cause apoptosis of AM. This was further supported by studies demonstrating that other fibrotic agents also induce apoptosis in human AM (13). In addition, BéruBé et al. (1) reported that asbestos induced apoptosis in rat mesothelial cells. However, the possible mechanism by which fibrotic agents such as silica induce apoptosis is unclear. This is the first study that provides insight to the possible mechanism by which fibrotic particulates, such as asbestos and silica, induce apoptosis.
Results from a number of laboratories have demonstrated that cysteine
proteases play an important role in the regulation of programmed cell
death by some apoptotic agents (7, 23, 35, 37, 38). In this study, we
provide results supporting the direct involvement of the ICE family of
proteases in silica-induced apoptosis. Figures 1-3 show a
significant inhibition of silica-induced apoptosis in cells pretreated
with the ICE inhibitor zVAD-FMK. The ICE inhibitor blocked
silica-induced DNA cleavage, DNA ladder formation, and morphological
changes characteristic of apoptosis. The involvement of ICE was further
confirmed by detecting increased levels of ICE p20 (Fig. 4) in
silica-treated cells, implying the activation of ICE by silica. These
results directly implicate ICE in silica-induced apoptosis. In addition
to ICE, other members of the ICE family of proteases also appeared to
contribute to silica-induced apoptosis. Both cpp32 and
Ich-1L have been proposed to be
activated in other model systems, and the current studies implicate
their activation in silica-induced apoptosis. An increase in the level
of p20 Ich-1L and degradation of
cpp32
were detected in silica-treated human AM within 1 h (Figs. 5
and 8), indicating that both of these proteases are rapidly activated
by silica treatment. Furthermore, inhibition of cpp32
by DEVD-FMK
resulted in a corresponding inhibition of apoptosis in silica-treated
human AM (Fig. 6). Therefore, it can be concluded that silica induces
apoptosis in human AM by stimulating members of the ICE family of
cysteine proteases, namely ICE,
Ich-1L, and cpp32
.
Consequently, this pathway may be an important apoptotic mechanism used
by other fibrogenic agents, such as asbestos.
Although it is well established that the ICE proteases play a role in
apoptosis, the individual roles are not clear. It is still not known
whether one or all of these proteins may be required in the apoptotic
process. In silica-induced apoptosis, at least three members of this
family were activated. If these events occur in parallel, it might
indicate a redundancy of function in these proteases. However, it has
been demonstrated that cpp32 and
Ich-1L can be cleaved by ICE, thus
suggesting that this pathway may be sequential. Consistent with this
proposal, pretreatment of cells with the ICE inhibitor blocked the
formation of the p20 form of Ich-1L in silica-treated cells
(Fig. 5), implying that Ich-1L may
be downstream of ICE. Conversely, the inhibitor used may not be
specific for ICE and may inhibit
Ich-1L itself. In that regard, zVAD-FMK has been suggested to inhibit both ICE and cpp32 (28). It
could also be speculated that this rapid and wide response of the ICE
proteases to silica treatment may overwhelm the survival mechanism of
the cell thus totally committing the AM to die by apoptosis.
Many apoptotic agents induce cell death by interacting with surface receptors stimulating one or more intracellular signaling pathways. Previous studies in our laboratory have reported the involvement of the scavenger receptor (SR) in silica-induced apoptosis (18). The SR is a membrane receptor involved in the uptake of a broad array of negatively charged ligands and is speculated to play a role in atherosclerosis (2, 11, 12). Additionally, it has been proposed to be involved in the uptake of silica by the AM (18, 21). However, the intracellular signal transduction pathways stimulated by ligand-SR interaction have not been elucidated. One of the better characterized mechanisms by which ligand-receptor interaction leads to apoptosis is the tumor necrosis factor (TNF) and Fas pathways. It has been reported that the Fas and TNF receptors associate with intracellular proteins Fas-associated death domain and TNF receptor-associated death domain, respectively, which trigger a death stimulating pathway, ultimately resulting in activation of ICE proteases leading to apoptosis (3, 17). It is possible, therefore, that binding of silica to the SR results in the activation of ICE proteases by a similar mechanism; however, a direct relationship between the SR and the cysteine proteases needs to be demonstrated.
Generally, apoptosis is a normal physiological process; however,
extensive injury to selective cell populations has been observed in
certain disease conditions. Preferential cell death of a specific cell
population could provide a mechanistic linkage between apoptosis and
fibrosis. Studies have demonstrated that the AM is a heterogenous population of several functionally distinct subpopulations
(32-34), mostly comprising of a large immunosuppressive
(RFD1+RFD7+) and a relatively smaller immune inducer (RFD1+
RFD7) population (16, 20). It is generally believed that there
is dynamic balance between the suppressive and inductive AM populations
and that a perturbation in this ratio due to targeted cell death may
result in altered lung homeostasis; conversely, the ratio of these
subpopulations could be altered by disease and by therapeutic regimes
(33). Taking into consideration these two AM subpopulations and
applying our in vitro results to the in vivo situation, it is possible that chronic exposure to crystalline silica may lead to the depletion of the predominant suppressor AM population by apoptosis, resulting in
a predominance of the inducer AM population. The combined events of a
change in the relative ratio of the suppressor to inducer AM
populations and the consequent decrease or absence of the immune suppressive factors released by the suppressor population may result in
an inflammatory response, ultimately causing fibrosis.
In summary, these studies demonstrate the role of multiple members of the ICE family of proteases in silica-induced apoptosis. If apoptosis is a critical step in the fibrogenic process then a better understanding of the regulation of these proteases may lead to important therapeutic intervention.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants ES-04804 Clinical Research Center M01-RR-02558.
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FOOTNOTES |
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Address for reprint requests: A. Holian, Dept. of Internal Medicine, University of Texas Medical School, 6431 Fannin, Rm. 1.276, Houston, TX 77030.
Received 10 February 1997; accepted in final form 25 June 1997.
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