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Published ahead of print on January 23, 2004, doi:10.1165/rcmb.2003-0284OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 823-829, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0284OC

A Biphasic Response to Silica

I. Immunostimulation Is Restricted to the Early Stage of Silicosis in Lewis Rats

Raymond J. Langley, Roma Kalra, Neerad C. Mishra, Fletcher F. Hahn, Seddigheh Razani-Boroujerdi, Shashi P. Singh, Janet M. Benson, Juan Carlos Peña-Philippides, Edward B. Barr and Mohan L. Sopori

Immunology Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico

Address correspondence to: Mohan L. Sopori, Immunology Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108. E-mail: msopori{at}lrri.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhalation of crystalline silica may lead to acute or chronic silicosis. Although chronic silicosis is associated with increased incidence/exacerbation of autoimmune disorders, the immunologic effects of chronic silicosis are not completely understood. In an animal model of chronic silicosis, Lewis rats were exposed to filtered air or silica (1.75 µm average particle size) at an exposure concentration of 6.2 mg/m3, 6 h/d, 5 d/wk for 6 wk, and observed up to 27 wk after the exposure. Based on silica burden, lung histopathology, and immunologic changes, two distinct stages were identified in the development of chronic silicosis. Stage 1 (4–28 d after exposure) was characterized by silica deposition in various tissues, and augmented antibody and cellular immunity. Although bronchoalveolar lavage contained an increased number of activated macrophages, protein and lactate dehydrogenase levels were comparable to controls. In Stage 2 (>= 10 wk), silica was localized in epithelioid macrophages, and T cell immunity had returned to normal, but the lavage fluids contained increased protein concentration and lactate dehydrogenase activity. Moreover, lungs from silica-treated animals contained neutrophils and lymphocytes, and exhibited granulomatous changes around the silica-containing epithelioid macrophages. Thus, in the early stages of silicosis, silica activates the immune system; however, the progression of lung granulomas does not depend on a continually activated adaptive immune system.

Abbreviations: antibody-forming cell, AFC • alveolar macrophage, AM • bronchoalveolar lavage, BAL • bronchoalveolar lavage supernatant fluid, BALF • ionized Ca2+, [Ca2+]i • cell-mediated immune, CMI • lactate dehydrogenase, LDH • crystalline silica, SL • sheep red blood cells, SRBC • T cell antigen receptor, TCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Exposure to silica through coal mining, quarrying, and working with concrete can be a common occupational hazard (1, 2). Depending on the dose, inhalation of microscopic crystalline silica (SL) particles by humans and laboratory animals may lead to acute (accelerated silicosis) or chronic silicosis (2, 3). Acute silicosis is associated with an acute inflammatory response in the lung, apoptosis, and tissue destruction (4–6), whereas chronic silicosis is associated with an increased risk of autoimmune disorders and granuloma formation. In experimental models, acute silicosis is accompanied by rapid onset of lung inflammation, apoptosis, tissue destruction, and proteinosis (7). Silicosis is a debilitating and sometime fatal lung disease, characterized by progressive granulomatous and fibrogenic responses in the lung (8, 9). Chronic silicosis may be diagnosed many years after the SL exposure has stopped; thus, the disease might progress over many years after the exposure has ended (10, 11). SL particles activate macrophages and fibroblasts in the lung, leading to production of reactive oxygen species, cytokines and chemokines, inflammation, and granulomatous and fibrotic changes in the lung (2, 9, 12). SL exposures also increase the risk of tuberculosis, chronic bronchitis, chronic obstructive pulmonary disease, lung cancer, and autoimmune diseases such as rheumatoid arthritis, scleroderma, and systemic lupus erythematosus (2, 13–17).

The mechanism by which SL exposure increases susceptibility to autoimmune diseases and granuloma formation is not clearly understood. Silicates may have immune adjuvant activity (13, 18), but the relationship between immune activation and development of silicosis is not clear. Previous animal studies do not distinguish between immune activation and formation of lung lesions in silicosis. Moreover, the effects of silica inhalation on adaptive immunity are largely unknown. In the present study, we investigated the immunologic and inflammatory effects of SL aerosols in a rat model of chronic silicosis. Our results indicate that although the SL exposures resulted in lower lung SL burdens than those found in some patients with silicosis (19), SL increased the inflammatory and adaptive immune responses, including T cell hyperresponsiveness, in these animals. Moreover, although the T cell hyperactivity preceded lung granuloma formation, the progression of the granulomas was not predicated on the continued activation of the adaptive immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Specific pathogen–free, male Lewis rats, 6–8 wk old, were purchased from Charles River (Raleigh, NC). During the 2-wk conditioning and 6-wk SL exposure, the animals were housed in H2000 whole-body exposure chambers (Lab Products, Inc., Maywood, NJ). After the exposure, the animals were transferred to class-100 air quality rooms in shoebox cages with hardwood chip bedding. Food and water were provided ad libitum, and animals were periodically monitored for common rat infections.

Reagents
Unless otherwise mentioned, all the reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Exposure to Crystalline Silica
During the exposures, the chambers were maintained with an airflow rate of ~ 15 cubic ft/min and at a temperature range of 22–26°C. One group of animals was exposed to SL (Min-U-Sil 5; US Silica, Mill Creek, OK) 6 h/d, 5 d/wk (Monday–Friday) for 6 wk; the second group of animals was maintained as controls and exposed to filtered air. SL was aerosolized as described by Cheng and coworkers (20). Briefly, SL was placed in a screwfeeder (Model 102, Accurate Dry Chemical Feeder; Accurate, Westbury, NY) and fed into an air jet mill (Model 101, Jet-O-Mizer; Fluid Energy Processing and Equipment Co., Hatfield, PA) that aerosolized the SL injected into a cyclone and delivered it to the inhalation exposure chamber. The cyclone removed SL particles > ~ 3 µm to reduce the size distribution of the exposure atmosphere to ~ 2 µm (mass median aerodynamic diameter). Aerosol concentration was determined gravimetrically from a filter sample collected from the animals' breathing zone. Aerosol size distribution was determined using a Lovelace Multijet Cascade Impactor (Lovelace Respiratory Research Institute, Albuquerque, NM). The overall SL concentration was 6.2 mg/m3 with a mass median aerodynamic diameter of 1.75 ± 0.05 µm. Animals were killed at 4 d, 4 wk, 10 wk, 17 wk, and 27 wk after cessation of the SL exposure.

Immunizations
Four days before the killing, animals were injected intravenously with 5 x 108 sheep red blood cells (SRBC; Colorado Serum Co., Denver, CO) to determine the antibody-forming cell (AFC) response (21).

Determination of Tissue Crystalline Silica Burden
Tissues (lungs, brains, liver, and spleen) were sent for SL determination to the Carlsbad Environmental Monitoring and Research Center (New Mexico State University, Las Cruces, NM). After the acid digestion of tissues, SL concentrations were determined by an inductively coupled plasma-mass spectrometer following EPA Method 200.8.

Lung Lavage
Rats were killed by an intraperitoneal injection (1 ml/kg body wt) of Euthasol (Great Western Animal Supply, Albuquerque, NM) containing 390 mg sodium pentobarbital and 50 mg of phenytoin. The lungs were removed, the left side of the lung was tied off at the bronchi with clips, and the right lung lobe was lavaged twice with 3-ml aliquots of sterile PBS. The lavage was centrifuged, and the cell pellet was resuspended in 1 ml of PBS. The cells were processed for FACS analysis or spun onto slides for differential cell counting. The bronchoalveolar lavage (BAL) supernatant fluid (BALF) was tested for total protein content and lactate dehydrogenase (LDH) activity.

Histopathology
After lavage, the left lung lobes were inflated for 2 h via the cannulas at a constant hydrostatic pressure of 25 cm with 10% neutral-buffered formalin, and then immersed in neutral-buffered formalin for another 48 h. After fixing, the left lung lobe was trimmed in a dorsoventral transverse direction from cranial to caudal to yield serial slices. Either the even- or the odd-numbered slices were embedded in paraffin and cut into 5-µm-thick sections. The tissue sections were stained with hematoxylin and eosin, and evaluated by light microscopy for morphologic changes. Sections were subjectively graded on a 0–4 scale: 0, no change; 1, minimal change; 2, mild change; 3, moderate change; and 4, marked change.

Protein and LDH Determination
A bicinchonic acid assay kit (Pierce, Rockford, IL) and an LDH assay kit (Sigma-Aldrich) were used to determine the BALF protein content and LDH activity, respectively, according to manufacturers' instructions.

Flow Cytometry
BAL cells were washed and resuspended in PBS at 1 x 106 cells/ml. One-milliliter aliquots of cells were incubated in the dark at 4°C for 1 h with 1 µg of phycoerythrin- or FITC-conjugated anti-rat CD-11a and/or CD-11b monoclonal antibodies (BD PharMingen, San Diego, CA). The cells were pelleted and washed three times with PBS. The labeled cells were analyzed by a flow cytometer (FACS Star; BD Bioscience, San Jose, CA). At least 10,000 cells were counted and analyzed by FACS Comp software (BD Bioscience).

Preparation of Spleen Cells
Spleen cell suspensions were prepared as described by Sopori and colleagues (22). Briefly, spleens were pressed through stainless-steel mesh, and red blood cells were lysed by treatment with cold NH4Cl solution. After the centrifugation, cells were washed twice with cold PBS and counted on a hemocytometer in 0.1% eosin in PBS.

Antibody-Forming Cell Assay
The primary direct AFC response was determined by the method of Cunningham and Szenberg (23). Briefly, spleen cells (2 x 105) were mixed with 2% SRBC in PBS and 25 µl of guinea pig complement (Cederlane, Hornby, ON, Canada) preabsorbed on SRBC in a final volume of 250 µl of complete medium (RPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, 50 µM 2-mercaptoethanol, and 10 µg/ml gentamicin). Aliquots were distributed in duplicate on Cunningham slides and incubated for 45 min at 37°C. The AFC plaques were counted and normalized to 1 x 106 spleen cells.

T Cell Proliferation Assay
The proliferative response of spleen cells to the T cell mitogen Con A was measured as described (24). Briefly, 2 x 105 spleen cells were cultured in 0.2 ml of complete medium in the presence of various concentrations of Con A in microtiter plate wells. The cultures were incubated at 37°C in the presence of 5% CO2, and cells were harvested after 3 d by a Skatron cell harvester (Skatron Instruments, Inc., Sterling, VA). Proliferation was assayed by pulsing the culture wells with 0.5 µCi of [3H] Tdr (ICN, Irvine, CA) for 18 h before harvesting.

Assay for Intracellular Ionized Ca2+
Spleen cells were loaded with acetoxymethyl ester of indo-1 (indo-1; Molecular Probes, Eugene, OR) under conditions as described (25). Briefly, 5 x 106 cells/ml were incubated at 37°C with 5 µM indo-1 in loading medium (PBS containing 1% fetal calf serum). After washing, cells were suspended in loading medium, incubated at 37°C for 15 min in a 5% CO2 atmosphere, and kept on ice in the dark until the assay. Before each measurement, a 2-ml aliquot of the cell suspension was centrifuged, and cells were resuspended in 2 ml PBS containing 1 mM Ca2+. Ionized Ca2+ ([Ca2+]i) of the cells was determined by spectrophotometry in a PTI Deltascan fluorometer (Photon Technology International, South Brunswick, NJ) at 37°C with constant gentle stirring. Cell fluorescence was recorded at 60 s before the addition of 10 µg/ml of anti-CD3 or anti-{alpha}ß–T cell antigen receptor (TCR) mAb followed 20 s later by 28 µg of the second antibody (goat anti-mouse). The excitation wavelength for indo-1 is 355 nm, and emissions were measured at 410 and 485 nm. Background fluorescence at each wavelength, determined from unlabeled cells, was subtracted from the fluorescence of indo-1–loaded cells. [Ca2+]i was calculated from the ratio of indo-1 fluorescence at the two wavelengths and the dissociation constant, assuming that it was 250 nM (26). The ratio of maximum to minimum indo-1 fluorescence was determined by lysing the cells with 100 µg/ml of digitonin in the presence of Ca2+, then quenching the dye by removing Ca2+ with 10 mM EGTA at pH 8.5.

Statistical Analysis
Data were analyzed for statistical significance by Prism Software 3.0 (Graphpad Software, Inc., San Diego, CA) using a student's t test or by two-way ANOVA. Values were considered significant at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhaled Crystalline Silica Is Detected in the Lung, Spleen, and Brain
To determine whether inhaled SL remained primarily in the lung or was transported elsewhere in the body, SL contents of the lung, brain, spleen, and liver were quantitated at 4 d and 10 wk after SL exposure by inductively coupled plasma-mass spectrometry. As expected, SL was present in the lungs of SL-exposed rats on Day 4 (Table 1). The SL concentrations in the lungs of these animals were noticeably lower than those reported from human silicosis patients (27). Interestingly, a significant amount of SL was also present in the animals' spleens and brains 4 d after the last exposure. As with other particles (27), it is likely that SL-containing lung macrophages transported the SL to other lymphoid tissues such as the spleen. Although a significant number of SL particles were detected by polarized microscopy in epithelioid macrophages in the lung and mediastinal lymph nodes, most SL was cleared from tissues within 10 wk after the SL exposure (Table 1). Therefore, low concentrations of small (< 2 µm) SL particles might be cleared relatively quickly from the tissues; however, a small but significant number of SL particles are retained in the lung within epithelioid macrophages, and this initiates the granulomatous process.


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  		TABLE 1. Tissue silica burden (ng/mg) at the end (0 time) and 10 wk after silica treatment

 
Crystalline Silica Affects Lung Histology
Despite the presence of SL in alveolar macrophages (AMs) at 4 d and 4 wk after SL exposure, lungs did not show any significant overt histopathologic changes; however, SL-exposed animals had focal accumulation of activated (enlarged and vacuolated) AMs in the lung (data not shown). Moreover, histopathology indicated focal accumulations of lymphocytes in alveolar septae (Figure 1B); these cells are not accessible to flushing during BAL collection and might represent the early foci for granuloma formation. At 10 wk and beyond, SL exposure led to definite inflammatory changes in the lung, including increased focal accumulation of AMs, infiltration of PMNs and lymphocytes, and the presence of epithelioid macrophages in the bronchus-associated lymphoid tissue (Figures 1C and 1D). Microscopic analysis of BAL showed that whereas 4 d after SL exposure macrophages represented > 95% of BAL cells, by 10 wk after exposure significant numbers of neutrophils and lymphocytes were present (not shown). These results were further confirmed by flow cytometry, showing the presence of neutrophils (CD11a–CD11b+), lymphocytes (CD11a+-CD11b–), and macrophages (CD11a+-CD11b+) in the BAL of SL-treated animals at 10 wk after SL exposure (not shown). Lung granulomas started to form in ~ 40% of the animals at Week 17 and were observed in all animals at 27 wk after SL exposure. These results suggest that between 0–4 wk after SL exposure, lungs contained SL, increased numbers of activated macrophages in the BAL, and focal accumulations of macrophages and lymphocytes in the alveolar septi, perhaps, representing early granulomatous response. Infiltration of large numbers of inflammatory cells (PMNs and lymphocytes) was seen after 4 wk and overt lung granuloma formations only after 10 wk of SL exposure. Interestingly, the presence of epithelioid macrophages characterized the early granulomas. Thus, SL-induced granulomas might begin with lymphocytic infiltration and development of epithelioid macrophages. Although most SL was cleared from the lung within 10 wk after SL exposure, a small but significant number of SL particles were detectable by polarized microscopy in the lung, indicating that although the body can clear SL from the lung, it may only take a small number of SL-containing epitheliod macrophages to initiate granuloma formation in the lung.



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  		Figure 1. Kinetics of granuloma formation in response to SL exposure. Lung tissue sections were obtained from (A) control and SL-exposed animals at (B) 4 d, (C) 17 wk, and (D) 27 wk after SL exposure. Sections were stained with hematoxylin and eosin. Note the extensive accumulation of leukocytes and early granuloma formation (arrow) at 17 wk (C), which is well established at 27 wk (D). Representative data from five animals/group.

 
Crystalline Silica Increases BAL LDH and Protein Levels in Later Stages of Silicosis
Silicosis starts with lung inflammation (alveolitis and tissue destruction) as a consequence of SL-induced macrophage activation and the release of inflammatory mediators (9, 28). Cellular damage or death from inflammation is associated with the increased release of protein and LDH. Table 2 shows that LDH activity and BALF protein content were significantly elevated at 17 wk after SL exposure. Thus, although SL exposure at 10 wk moderately increased the infiltration of PMN and lymphocytes in the lung, increased lung destruction (LDH activity and protein in BALF) is associated with increased leukocytic infiltration and granuloma formation (Table 2).


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  		TABLE 2. Silica increases protein and LDH content of BALF at later stages*||

 
Crystalline Silica Activates the Immune Response in Early Stages of Silicosis
Crystalline silica has adjuvant-like properties (18, 29), but the mechanism of this action is unclear. To determine whether SL stimulates the immune system, we evaluated whether SL affected the antibody response of spleen cells. Because the effects of various xenobiotics on the antibody response in the lung are similar to those in the spleen (30–32) and the mediastinal lymph nodes contained a lot of SL, we ascertained the effects of SL exposure on the antibody response of spleen cells. Animals were immunized with the T-dependent antigen, SRBC, and killed 4 d later. Figure 2A shows that compared with the control, at 4 d after cessation of SL exposure, SL significantly increased the number of anti-SRBC AFCs in the spleen. Similar increases in the AFC response were observed at 4 wk; however, at 7 wk after SL exposure the increase in the AFC responses did not reach statistical significance, and the response was comparable to that of controls at 10 wk after the exposure (Figure 2B). Thus, SL can stimulate the antibody response only in the early phase of silicosis (i.e., before the appearance of granulomas).



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  		Figure 2. The AFC response is increased in the early phase of silicosis. Rats were immunized with SRBC 4 d before killing. Spleen cells were obtained from control (CON) and silica-exposed animals at 4 d (A) and 10 wk (B) after silica exposure. Data represent mean ± SEM of anti-SRBC AFC from five rats/group. **Statistically significant (P = 0.003).

 
T cell proliferation in response to Con A is an index of the cell-mediated immune (CMI) response. To determine whether SL affected the CMI response, spleen cells from control and SL-exposed animals were cultured in the presence and absence of various concentrations of Con A, and proliferation of T cells was determined by the incorporation of [3H] Tdr into the cells. Figure 3A shows that on Day 4 after SL exposure, Con A–induced T cell proliferation was significantly higher in SL-treated than in control splenocytes; however, T cell proliferation returned to control levels at 10 wk after SL exposure (Figure 3B). These data suggest that as with the AFC response, SL activates the CMI response in the early phase, but the response returns to normal within 10 wk of SL exposure.



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  		Figure 3. The Con A response is increased in the early phase of SL exposure. Spleen cells from control (open bars) and silica-exposed (filled bars) rats were obtained at 4 d (A) and 10 wk (B) after silica exposure. Cells of individual animals were cultured in the presence of indicated concentrations of Con A for 4 d. Cells were labeled with [3H] Tdr 18 h before harvesting. Data represent the values ± SEM from five animals/groups. Figure 3A is statistically significant (P <= 0.0001) by two-way ANOVA.

 
Ligation of the TCR with anti-TCR antibodies increases [Ca2+]i and simulates the antigen-mediated activation of T cells (33). To determine whether an increased CMI response in the early phase of SL exposure was associated with changes in the TCR-mediated signaling, spleen cells were incubated with anti-CD3 mAb. Results representative of four experiments show that the ability of spleen cells to raise [Ca2+]i in response to anti-CD3 was significantly elevated in animals on Day 4 after SL exposure (Figure 4). A similar increase in [Ca2+]i was also observed at 4 wk but not at 10 wk after SL exposure (not shown). These data suggest T cells exhibit hyperresponsiveness to antigens and mitogens in the early stages of silicosis, and might reflect changes in the antigen-mediated T cell signaling.



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  		Figure 4. SL exposure elevates [Ca2+]I in response to TCR ligation. Spleen cells from control (CON) and 4 d after silica-exposure were labeled with indo-1 and treated with mouse anti-rat {alpha}ß-TCR monoclonal antibody followed after 20 s by goat anti-mouse IgG as the second antibody (2nd IgG). Data are representative of the Ca2+ response from five animals/group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies presented herein used a rat model of chronic silicosis to determine the effects of SL inhalation on the immune response and the kinetics of granuloma formation in the lung. Our data provide the first evidence that activation of SL induces changes in the adaptive immune response, and that these changes precede the formation of granulomas in the lung. Exposure to high concentrations of SL leads to acute or accelerated silicosis that is associated with acute inflammation, apoptosis, and tissue destruction (8, 9, 34). On the other hand, epidemiologic studies suggest that a large time gap exists between SL exposure and diagnosis of chronic silicosis, and that once initiated, the disease might progress in the absence of continued SL exposure (10, 11). Because most animal studies have used intratracheal instillation or inhalation of large doses of SL (i.e., models of acute silicosis), temporal changes in the biological responses to SL might have been difficult to identify. In our studies, the lung SL burden of rats was lower (413 ± 59 ng/mg tissue) than the lung SL burden of some gold miners with silicosis (2,240 ± 410 ng/mg tissue) (18). Thus, these SL concentrations are well within realistic human occupational exposures. With this silicosis model, we have shown that SL causes T cell hyperresponsiveness and increased antibody production, which precede overt changes in lung pathology. A similar inhalation protocol was used by Porter and coworkers (35, 36), who exposed rats to 15 mg/m3 of SL for 6 d/wk for 116 d and observed moderate increases in BAL PMN numbers from Day 5 of SL exposure; however, dramatic increases in PMN levels were observed only after Day 41. Given that normal BALF contains very few PMN numbers, a 3- to 5-fold increase in PMN numbers during the early phase of SL exposure is not a large increase. Moreover, the exposure concentration of SL used by Porter and colleagues (35, 36) was about 3-fold higher than that used in the present study. Similarly, increases in the LDH, protein, and tumor necrosis factor-{alpha} content of BALF also became highly noticeable after 40 d of SL exposure, and significant changes in fibrosis were not seen until 79 d of SL exposure (35). Thus, differences in the kinetics of SL-induced histopathology between our study and those of Porter and coworkers (35, 36) might reflect differences in the lung SL burden. Indeed, recent experiments by Porter and coworkers (37) demonstrated that unlike high doses of SL, which caused lung damage as seen by LDH activity and albumin content in BALF, low doses produced lung inflammation without significant lung damage. Using varying doses of SL (0.1–10 mg/m3), Henderson and colleagues (38) showed that a 4-wk exposure to SL increased PMN accumulation and protein and LDH content in BAL that correlated positively with the SL dose and the time after SL inhalation. These observations are similar to our findings, suggesting that unlike acute silicosis, major increases in the lung inflammation occur several months after SL exposure.

In addition to the lung, significant amounts of SL were also observed in the spleen and brains of SL-exposed animals immediately after the end of exposure. Although transport of SL-containing macrophages between the lung and lymphoid tissues (26) could explain the presence of SL in the spleen, the mechanism of SL transport across the blood–brain barrier and its biological significance remain unclear. It has been suggested that SL is retained for years in the lung (39). Interestingly, however, most SL was cleared from the tissues at 10 wk after the exposure; nevertheless, a significant number of SL particles were retained in the lung, particularly in epithelioid macrophages. Honnons and Porcher (40) also observed the clearance of SL over time after inhalation of large doses (100 mg/m3/d for 4 wk). The presence of SL in the brain is somewhat intriguing; however, particles such as talc (41) and nano-sized silicon particles (42) have accumulated in the brain. It is possible that systemic presence of SL through mechanisms such as induction of cytokines (36) compromises the blood–brain barrier that facilitates its entry into the brain (43).

Among the notable early histopathologic changes associated with granulomas formation was the accumulation of lymphocytes and macrophages in the alveolar septae. The macrophage cytoplasm was strongly acidophilic (stained red with eosin) and exhibited epithelioid morphology. Epithelioid macrophages have been present in tuberculosis-associated granulomas (44). The focal accumulations of epithelioid macrophages and lymphocytes within alveolar septae might represent the sites for granuloma formations. The observation that early cell accumulations are not in the alveolar lumen explains their inability to appear in the BALF.

Crystalline silica-exposed animals have significantly more SRBC-specific AFC in the spleen than control animals at 4 d after exposure, indicating that SL activates the immune system during the early phases of exposure. The presence of SL in mediastinal lymph nodes made the direct analysis of the AFC response in these tissues difficult, but data from various studies have shown an excellent correlation between the antibody responses in the spleen and lungs (21, 30–32). Although SRBC is a T cell–dependent antigen, the antibody production against this antigen requires B cells and antigen-presenting cells (e.g., macrophages, dendritic cells) in addition to T cells. Therefore, the anti-SRBC AFC results do not identify the immune cell type(s) affected by SL exposure. Results from Con A–induced T cell proliferation data strongly suggest that SL heightens T cell responses and may contribute to its adjuvant effects (29). By similar criteria, significant immune activation was also observed at 4 wk and, to a lesser extent (P > 0.05), at 7 wk after the SL exposure (data not shown). These changes in T cell reactivity were not associated with any significant change in the subset distribution of lymphocytes in the spleen (i.e., the proportion of CD4+ T cells, CD8+ T cells, and B cells that remained essentially unaltered by SL exposure). Although these data per se do not explain the increased incidence of autoimmune diseases in SL-exposed subjects, they do suggest that SL activates both adaptive and inflammatory responses; in patients with susceptibility factors for autoimmunity, SL might trigger and/or exacerbate the disease process.

One of the early-recognized events of TCR-dependent T cell activation is the increase in the cytoplasmic [Ca2+]i level (33). Our results show that anti-CD3–induced elevation in [Ca2+]i is exaggerated in T cells at 4 d and 4 wk after SL-exposure. Although the mechanism by which SL increases the antigen-induced Ca2+ response is not known, it is clear that SL affects the TCR-mediated signaling in T cells. The Ca2+ response returned to control levels within 10 wk of SL exposure, as with other indices of immune activation.

Interestingly, although epithelioid macrophages and some lymphocytes were detected within the alveolar septae at 4 wk after SL exposure, histopathology and FACS analysis of BAL did not reveal significant neutrophil or lymphocyte infiltration. Moreover, BAL protein and LDH activity of SL-exposed animals were not significantly different from controls until after 10 wk of SL exposure, indicating no significant cell death at this stage of silicosis. This observation is different from that in acute silicosis, where acute inflammation is associated with apoptosis and tissue destruction in the lung (4–6). In fact, our preliminary results strongly indicate that in this model of chronic silicosis, SL induces strong anti-apoptotic phenotype in BAL cells (R. J. Langley and coworkers, unpublished observation). Anti-apoptotic responses also characterize initiation of other granulomatous changes (45, 46). Approximately 10 wk after SL exposure, significant changes in the lung, including infiltration of lymphocytes and neutrophils, strong focal aggregation of macrophages, the beginning of septal fibrosis, and alveolar epithelial hyperplasia without significant granuloma formation, were noticeable. Activation of macrophages has been postulated to be critical for the histopathology of silicosis (34, 47). Although macrophages were activated (enlarged and vacuolated) in the early stages of silicosis, the granulomatous changes were apparent only after the infiltration of large numbers of lymphocytes and neutrophils into the lung. Thus, the macrophage activation may be pivotal, but is not sufficient to cause SL-induced granulomatous changes in the lung.

Changes in the lung pathology between 10 and 17 wk after SL exposure were similar to earlier reports (48, 49). Our results clearly define two stages in the evolution of silicosis. Stage 1 encompasses the activation of AM and enhanced humoral and cell-mediated immunities. In Stage 2, although the adaptive immune response returns to normal, lungs show septal fibrosis, lymphocyte/PMN infiltration, granuloma formation, and anti-apoptotic phenotype of BAL cells.


   Acknowledgments
 
This study was supported in part by the U.S. Army Medical Research and Materiel Command under Contract DAMD17-00-1-0073. The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official U.S. Department of the Army position, policy, or decision unless so designated by other documentation. In conducting research using animals, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals," prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 86-23, Revised 1985). The Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Funding for this work also came from NIH grants DA04208-12 and DA04208S-7 and from the Lovelace Respiratory Research Institute. The authors thank the technical staff of the LRRI Aerosol and Respiratory Dosimetry Program for fabricating the silica exposure system and exposing the animals. They also thank Ms. Theresa Espindola and Ms. Yoneko Knighton for technical assistance, and Ms. Paula Bradley for editorial assistance.

Received in original form July 29, 2003

Received in final form December 18, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Thoracic Society Committee of the Scientific Assembly on Environmental and Occupational Health. 1997. Adverse effects of crystalline silica exposure. Am. J. Respir. Crit. Care Med. 155:761–765.[Medline]
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