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Published ahead of print on July 15, 2004, doi:10.1165/rcmb.2004-0090OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 470-477, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0090OC

Beryllium-Ferritin

Lymphocyte Proliferation and Macrophage Apoptosis in Chronic Beryllium Disease

Richard T. Sawyer, Brian J. Day, Valerie A. Fadok, Marina Chiarappa-Zucca, Lisa A. Maier, Andrew P. Fontenot, Lori Silveira and Lee S. Newman

Department of Medicine, Robert H. Hollis Laboratory of Environmental and Occupational Health Sciences, and Department of Pediatrics, National Jewish Medical and Research Center, Denver; Department of Medicine, Division of Pulmonary Science and Critical Care Medicine, and Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, Colorado; Lawrence Livermore National Laboratory, Livermore, California

Address correspondence to: Dr. R. T. Sawyer, Division of Environmental and Occupational Health Sciences, Hollis Laboratory of Environmental and Occupational Health, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: sawyerr{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A beryllium (Be)-ferritin adduct containing 270 pm of Be stimulated proliferation of bronchoalveolar lavage (BAL) lymphocytes from subjects with chronic beryllium disease (CBD) at concentrations 5–6 logs lower than the amounts of beryllium sulfate (BeSO4) needed to induce proliferation. We observed increased apoptotic CBD BAL macrophages after exposure to both BeSO4 (50 ± 6%, mean ± SEM, P < 0.05 versus unstimulated controls) and Be-ferritin (40 ± 2%), whereas only 2.0 ± 0.2% of BAL lymphocytes underwent activation-induced cell death. Be-ferritin also induced apoptosis in BAL macrophages from subjects with Be sensitization (25 ± 3%) and in the H36.12j hybrid macrophage cell line (15 ± 2%). Be-ferritin induced lung macrophage CD95 (Fas) expression and the activation of intracellular caspase-3, -8 and -9. Thus, lung macrophages take up Be-ferritin, delivering physiologically relevant levels of Be that promote Be antigen presentation and macrophage apoptosis. Be-ferritin thereby serves as a "Trojan Horse," triggering proliferation of Be-ferritin–specific CBD BAL T cells. We hypothesize that Be-ferritin exposure may result in persistent antigen exposure inducing Be-specific T cell clonal expansion and T cell helper type 1–type cytokine production and potentially explains the chronicity of CBD and its development years after environmental Be exposure has ceased.

Abbreviations: analysis of variance, ANOVA • antigen presenting cells, APC • bronchoalveolar lavage, BAL • beryllium, Be • Be lymphocyte proliferation test, BeLPT • Be sensitization, BeS • chronic Be disease, CBD • counts per minute, cpm • phagocytic index, PI • stimulation index, SI • tumor necrosis factor, TNF • triphosphate (dUTP) nick end labeling, TUNEL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The lightweight metal element beryllium (Be) causes injury to lung, skin, and other organs through direct chemical toxic effects and through its ability to induce Be antigen–specific sensitization and granulomatous lung disease (1, 2). Clinical observation suggests that some Be-exposed individuals never develop disease, whereas others develop Be sensitization (BeS) and chronic Be disease (CBD) after low levels of Be exposure (3–7). Beryllium particles created during metal machining in industry are mainly of submicron size (3, 5, 6), and can be inhaled into the lung, where they become available to complex with host molecules. Studies show that Be persists within the lungs of individuals many years after exposure has ceased (8), suggesting a failure to clear Be antigen from the lungs. This retention of Be may facilitate ongoing Be-specific immune responses.

Beryllium, with an atomic weight of 9.013 and a charge number, z = 2, has a small ionic radius, r = 0.31 nm. The ratio of the charge number to the radius is large, z/r = 6.45, and the chemical properties of Be are due to this high density of charge (9). Beryllium accepts two extra electrons and forms tetrahedral structures, principally with oxygen. Due to this high z/r ratio, Be forms complex compounds, including binding to the ubiquitous iron storage and transport protein ferritin (10). Ferritin is a high molecular weight protein (MW > 400,000 Da) composed of 24 subunits (11). Each subunit is composed of four {alpha} helices that form parallel cylinders with an external shell and a phosphorylated internal core that binds ~ 4,500 ferric atoms in a crystalline inorganic complex (11). Using the known Be chelator, sulfosalicylic acid, to remove Be metal ion from ferritin, it was demonstrated that Be tightly binds to the ferritin subunit core region, not to the protein shell, with no displacement of iron from the core (10). The tightness of Be binding occurs through the formation of covalent bonds between Be and the phosphate groups located in the subunit core region. Ferritin binds 800 g atoms of Be, suggesting that a single ferritin molecule forms a chemical adduct with small numbers of Be atoms (10).

When ingested by macrophages, beryllium sulfate (BeSO4) causes apoptosis (12, 13). The presence of triphosphate (dUTP) nick end labeling positive (TUNEL+) nuclei in CBD lung granulomas suggested the possibility that Be might induce apoptosis in CBD bronchoalveolar lavage (BAL) cells. High concentrations of Be sulfate induce caspase-dependent apoptosis of BAL macrophages from subjects with CBD and BeS, and in mouse macrophage cell lines (12, 13). Based on these lines of evidence, we tested the hypothesis that a Be-ferritin adduct might serve as a vehicle for delivering Be to lung macrophages and thereby provide Be molecules for antigen presentation. We found that a Be-ferritin adduct triggers the activation of Be-ferritin–specific CBD BAL T cells for proliferation and also induces BAL macrophage apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Written, informed consent was obtained from each patient enrolled in this study and the protocol was approved by the Human Subject Institutional Review Board at National Jewish Medical and Research Center, Denver, Colorado. Patients with CBD (n = 8) and BeS (n = 19) were consecutively enrolled based on the availability of BAL samples. H36.12j cells were obtained from the American Type Culture Collection (ATCC, CRL 2,449). Four test reagents were prepared, including: (i) a Be-ferritin adduct reagent prepared as previously described (10), using 1 mg/ml ferritin and 0.1 M BeSO4; (ii) A ferritin protein control reagent prepared using 1 mg/ml of ferritin, but without BeSO4; (iii) a dialysis control reagent prepared using 0.1 M BeSO4, but without the ferritin; (iv) A 7Be-ferritin adduct reagent prepared using radio-7BeCl2 and 1 mg/ml of ferritin, as above (10). H36.12j cells, BAL mixed cells, isolated BAL macrophages, and isolated BAL lymphocytes were cultured under the following conditions: unstimulated (none), or exposed to 100 µM BeSO4, a 1:20 dilution of the dialysis control reagent, a 1:20 dilution of the ferritin protein reagent, 100 µM Al2(SO4)3, or a 1:20 dilution of the Be-ferritin adduct reagent.

After 24 h in culture, the amount of 7Be bound to the Be-ferritin adduct was calculated from {gamma} counting (Packard Cobra Auto-Gamma 5005; Packard Instruments, Canberra, Australia) of the 7Be-ferritin adduct reagent, and the subcellular distribution of 100 µM 7BeCl2 and of the 7Be-ferritin adduct reagent was determined in whole cells and cell cytoplasmic and nuclear extracts prepared using the Active Motif Nuclear extraction kit (Active Motif, Carlsbad, CA). The Be lymphocyte proliferation test (BeLPT) (14) was performed for the clinical evaluation of blood and BAL T cell proliferation in response to BeSO4 stimulation, as shown in Table 1, where we report the mean (± SEM) peak stimulation index (SI) for thymidine uptake in triplicate cultures as the ratio of the test sample counts per minute (cpm) to the cpm in the unstimulated (medium alone) control (14). For the evaluation of BAL T cell proliferation in response to Be-ferritin adduct exposure (Figure 1), the peak thymidine incorporation cpm for each set of untreated and treated triplicate cultures were expressed as the mean (± SEM) cpm.


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  		TABLE 1. Clinical characteristics of the study subjects with beryllium sensitization and chronic beryllium disease

 


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  		Figure 1. Thymidine uptake (mean ± SEM cpm) by CBD BAL cells (n = 5) that were unstimulated (none) or exposed to 100 µM BeSO4, the control metal-salt 100 µM Al2(SO4)3, and a 1:20 or a 1:100 dilution of the ferritin protein control reagent that contained 50 µg and 10 µg of ferritin, respectively. Test cultures were treated with a 1:20 or a 1:100 dilution of the Be-ferritin adduct reagent and contained 50 µg of ferritin adducted to 270 pmol of Be and 10 mg of ferritin adducted to 54 pmol of Be, respectively. For comparisons of Be-treated cells versus unstimulated or control-protein–treated cells: *P < 0.0001 for 100 µM BeSO4 versus none, versus the 100 µM Al2(SO4)3 control, and versus the ferritin protein control reagent at both 50 µg and 10 µg of ferritin. For comparisons of Be-ferritin adduct reagent exposed cells versus the controls: *P < 0.0026 for Be-ferritin adduct reagent at both dilutions versus none, and for both dilutions of the corresponding ferritin protein control reagent (repeated measures ANOVA).

 
Apoptosis was determined by counts of 500 cells per treatment, made from cytocentrifuge slides, to determine the percent of nuclear fragmentation, assessed by differential cell staining and by the TUNEL assay. Intracellular staining for activated caspase was performed using Carboxyfluorscein caspase-3, -8 and -9 kits (BioCarta, Carlsbad, CA) and surface marker staining for CD4, CD71, CD11b, and CD14 was performed using the corresponding isotype control antibodies (BD Biosciences, San Diego, CA). Intracellular caspase and surface marker analyses were performed using a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences). We determined the phagocytic index for untreated and Be-ferritin adduct–exposed H36.12j cells as previously described (15). For additional details, the MATERIALS AND METHODS section can be viewed in the online supplement.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Study Population
Clinical characteristics are shown in Table 1. Overall, no significant difference in age was observed between the study populations. Due to the hiring practices of the Be industry in Colorado (16), most of the subjects with BeS and CBD were male. None of the subjects with BeS, and six of eight subjects with CBD, were currently using oral glucocorticosteroids. Six of the eight subjects with CBD were former smokers, one a never-smoker, and one was a current smoker. A total of 13 of the 19 subjects with BeS were former smokers, and 6 of 19 never-smokers.

The BAL consists of a complex, mixed cell population. Subjects with CBD had a significantly higher total number of BAL white blood cells (43 x 106; range, 16–118, P < 0.05, repeated measures analysis of variance [ANOVA]) in comparison to subjects with BeS (25 x 106; range, 8–50). The BeS BAL cellularity and differential of subjects with BeS are approximately equal to that observed in normal control subjects who had not been exposed to Be (14, 17, 18). In contrast, the BAL cells from subjects with CBD are representative of a chronic T-cell alveolitis. The CBD BAL cellularity showed a significant increase in the absolute number of lymphocytes (25 ± 12%, mean ± SEM, P < 0.05, repeated measures ANOVA). Thus, the CBD BAL contained 10.75 x 106 lymphocytes in comparison to 3.75 x 106 lymphocytes in the BeS BAL cells. The number of alveolar macrophages present in the CBD BAL (31.8 x 106) and BeS BAL (21 x 106) cells were not significantly different. Consistent with the presence of lung disease in the CBD population and its absence in subjects with BeS, the CBD BAL contained almost three times as many lymphocytes as compared with the BeS BAL. Due to the increased numbers of white blood cells present in the CBD BAL samples, we were able to partition these cells for use in the multiple experiments described below, and thereby limit the enrollment of subjects with CBD undergoing bronchoscopy with BAL in this study.

The subjects with CBD and BeS enrolled in this study met the clinical case definitions based on the proliferation of blood and BAL T cells in the clinical BeLPT (14). The blood BeLPT SI of both subjects with BeS and subjects with CBD were significantly increased at a median values of 3.4 (range, 1.1–30) and 9.2 (range, 1.1–17), respectively. Only the subjects with CBD had significantly increased BAL BeLPT SI (median, 95; range 1.2–190; P < 0.05, repeated measures ANOVA), reflecting an increase in the number of Be-specific CD4+ effector-memory T cells only present in the lungs of patients with CBD (19) and not in the lungs of those with BeS.

Radio-Labeled 7Be-Ferritin and Calculated Translocation
Based on standard calculations using a 7Be half-life (T1/2) of 53 d, we determined that the amount of 7Be associated with a 1:20 dilution of the Be-ferritin adduct reagent contained 50 µg of ferritin and 270 pmol of 7Be (2.7 x 10–10 M 7Be + 50 µg of ferritin = Be-ferritin):

  1. (i) The cpm/count efficiency (60 min)(24 h) = dpm day –1/{lambda} = Be atoms/molecule of ferritin.
  2. (ii) The atoms of Be/molecule of ferritin (molecules of ferritin in 50 µg of protein)/Avogadro's number/volume = moles of Be.

Thus, 3,895 cpm/0.01 · (60") · (24') = 0.56 x 109 dpm · d–1/0.0131 = 4.3 x 1010/6.84 x 1013 = 0.0006 atoms of Be per ferritin (6.84 x 1013 molecules of ferritin in 50 µg) = 4 x 1010/6.02 x 1023 = 6.8 x 10–14/250 x 10–6 = 270 pmol (2.7 x 10–10 M). Throughout this study, experiments were performed in which we used two Be-containing reagents: (i) 100 µM BeSO4 and; (ii) a 1:20 dilution of the Be-ferritin adduct reagent stock solution that therefore contained 50 µg of ferritin adducted to 270 pmol of Be. Our experimental controls therefore contained corresponding 1:20 dilutions of the ferritin protein control reagent and 1:20 dilutions of the dialysis control reagent. As will be shown below, the 1:20 dilution of the Be-ferritin adduct stock reagent will be sufficient to elicit the proliferation of Be-specific T cells in culture and the apoptosis of macrophages. To avoid confusion, hereafter we refer to this dilution of the stock as the "Be-ferritin adduct reagent."

Using BAL cells from a subset of 4 subjects, we observed that after 24 h of exposure, 1 x 106 BAL cells were associated with 34.6 ± 3.7% (mean ± SEM) of the total 7BeCl2 cpm (100 µM 7BeCl2) that were added to cell cultures. Of this, 24.5 ± 5.4% of the cpm was recovered in the cytoplasmic extract, 7 ± 0.2% of the cpm were recovered in the nuclear extract. After 24 h of exposure to a 1:20 dilution of the 7Be-ferritin adduct stock reagent (50 µg of ferritin containing 270 pmol of 7Be), 1 x 106 BAL cells were associated with 25 ± 10% of the total cpm that were added to the cell cultures. Of this, 18 ± 8% of the cpm were recovered from the cytoplasmic extract and 1.7 ± 1.3% of the cpm were recovered from the nuclear extract. Thus, we were only able to recover ~ 90% of the added, cell-associated 7BeCl2 cpm, and 79% of the added cell-associated 7Be-ferritin cpm, in the cytoplasmic and nuclear extracts of BAL cells. Our inability to recover 100% of the cell-associated 7BeCl2 or 7Be-ferritin cpm was likely due to the loss of 7Be cpm during the high-speed (14,000 rpm) microcentrifugation step, in which intact nuclei are separated from the cytoplasmic extract or, alternatively, when the nuclei are lysed in NP40 detergent, liberating nuclear material that could stick to the isolation tube.

Be-Ferritin Induces Be-Specific CBD BAL T Cell Proliferation
Using a subset of our subjects with CBD (n = 5), we tested the ability of Be-ferritin to induce the proliferation of Be-specific CBD BAL T cells (Figure 1). The mean peak (± SEM) cpm was 12,958 ± 3,766 cpm after exposure to 100 µM BeSO4 (P < 0.05 versus the unstimulated control, repeated measures ANOVA). Exposure to a 1:20 dilution of the stock Be-ferritin adduct reagent increased thymidine uptake to 2,103 ± 615 cpm (P < 0.05 versus the unstimulated control and P = 0.0026 versus cells treated with a 1:20 dilution of the ferritin protein control reagent, repeated measures ANOVA). Similar results were obtained using a 1:100 dilution of the Be-ferritin adduct reagent. Based on our calculations above, we estimate that a 1:100 dilution of the stock Be-ferritin adduct control solution contained ~ 54 pmol of the Be adducted to 10 µg of ferritin. Exposure to 100 µM Al2(SO4)3, the ferritin protein control reagent (a 1:20 dilution of the 1 mg/ml ferritin protein control stock reagent), or to the dialysis control reagent (a 1:20 dilution of the stock reagent) (data not shown) did not stimulate CBD BAL T cell proliferation, showing that CBD BAL lymphocytes proliferative responses were Be-ferritin adduct reagent specific.

Be-Ferritin–Induced Macrophage Apoptosis
We developed a simple, reliable apoptosis assay, using differential cell staining to identify cells with fragmented nuclei. To validate the nuclear fragmentation assay, we compared the ability to detect damaged nuclei by quantitative counting of nuclear fragmentation versus TUNEL staining (Figure E1 in the online supplement). CBD BAL cells (n = 8) were exposed to 100 µM BeSO4. From counts of 500 cells per patient, we determined the percent of cells that were TUNEL+ and the percent of cells that had fragmented nuclei demonstrated by staining with the protocol hema 3 differential stain. Unstimulated control CBD BAL cells were < 3% TUNEL+, or had fragmented nuclei. After 24 h of Be exposure, 19 ± 6% (mean ± SEM, P < 0.05 versus the unstimulated control, repeated measures ANOVA) of the CBD BAL cells were TUNEL+, whereas 41.7 ± 7% (P < 0.05 versus the unstimulated control) of the CBD BAL cells had fragmented nuclei based on differential staining. Thus, the TUNEL assay and the determination of nuclear fragmentation by differential staining were sufficient to detect Be-induced apoptosis in CBD BAL cells.

Using the nuclear fragmentation assay, we determined if Be-ferritin could induce apoptosis in CBD (n = 5) and BeS (n = 15) BAL cells and H36.12j macrophages (Figure 2). After 24 h, 33 ± 5% of CBD BAL cells and 24.2% ± 3% of BeS BAL cells exposed to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin, had apoptotic nuclei (P = 0.0023 and P < 0.0001, respectively, based on a comparison to the corresponding ferritin alone–exposed controls, repeated measures ANOVA). After exposure to the 100 µM BeSO4 nuclear fragmentation increased to 55 ± 8% in CBD BAL–positive control cells (P = 0.0002 versus the unstimulated control), and to 41 ± 7% in the BeS BAL–positive control (P < 0.0001 versus the unstimulated control). Exposure to 100 µM Al2(SO4)3, a 1:20 dilution of the dialysis control reagent or to a 1:20 dilution of the ferritin reagent control containing 50 µg of ferritin, did not significantly increase nuclear fragmentation in CBD and BeS BAL cells in comparison to the unstimulated control.



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  		Figure 2. The percent (mean ± SEM) of (a) CBD BAL (n = 5), (b) BeS BAL (n = 15), and (c) H36.12j cells (n = 5) with nuclear fragmentation that were unstimulated or exposed for 24 h to 100 µM Al2(SO4)3, a 1:20 dilution of the "dialysis control," 100 µM BeSO4, 50 µg of the ferritin protein control reagent, or the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin. *For the 100 µM BeSO4-exposed CBD BAL and BeS BAL cells, P = 0.0002 and P < 0.0001 versus the unstimulated (none) controls, respectively. For Be-ferritin adduct reagent–exposed CBD BAL and BeS BAL cells, P = 0.0023 and P < 0.0001 versus the ferritin reagent control–exposed cells, respectively and P < 0.05 for both the 100 µM BeSO4 and Be-ferritin adduct reagent–exposed H36.12j cells versus their corresponding controls (repeated measures ANOVA).

 
In H36.12j cells (n = 5), exposure to 100 µM BeSO4 induced a significant increase in the percentage of cells with fragmented nuclei (5 ± 1% versus the unstimulated control = 0.3 ± 0.2%; P < 0.05, repeated measures ANOVA). After 24 h of exposure to the Be-ferritin adduct reagent that contained 270 pmol Be adducted to 50 µg of ferritin, 16 ± 2% of the H36.12j cells had fragmented nuclei versus 0.5 ± 0.2% nuclear fragmentation in H36.12j cells treated with a 1:20 dilution of the ferritin reagent control that contained 50 µg of ferritin (P < 0.05).

Be-Ferritin Induced Apoptosis Is Selective
We determined which class of cells in the BAL mixed cell population might be susceptible or resistant to Be-induced apoptosis. CBD BAL cells are an exudate, consisting principally of macrophages and Be-specific CD4+ T cells that display a T cell helper type 1–type phenotype (19). We determined whether the Be-ferritin adduct reagent could induce nuclear fragmentation in isolated CBD BAL macrophages and isolated CBD BAL lymphocytes (20). However, Be-exposed CBD and BeS BAL macrophages lose the expression of typical myeloid differentiation markers, such as CD14 (12). In a subset of four subjects with CBD, we determined by flow cytometry that surface expression of the transferrin receptor CD71 was stable 24 h after Be-exposure. CBD BAL macrophage populations were identified by forward and 90° light scatter patterns, and back-gating of the isotype-stained controls and CD71+ macrophages was performed to exclude CD4+ lymphocytes and macrophages.

After exposure to 100 µM BeSO4, 58.3 ± 9.8% (mean ± SEM) of the CBD BAL macrophages were CD71+ and 53.5 ± 13% (P = NS) of the unstimulated CBD BAL macrophages were CD71+. A comparison of CD71 coexpression with macrophage and lymphocyte surface differentiation markers shows that < 1% of this subject's unstimulated CD4+ CBD BAL T cells coexpressed CD71+, whereas 91% of the CD71+ CBD BAL macrophages coexpressed CD11b+ (iC3b complement receptor) or CD14+ (macrophage lineage differentiation marker). After 24 h of exposure to 100 µM BeSO4, < 1% of the CD4+ CBD BAL T cells coexpressed CD71+; however, high levels of CD71 expression were maintained on CBD BAL macrophages. Less than 1% of these CD71+ Be-exposed cells coexpressed either CD11b+ or CD14+. Thus, separation of CBD BAL cells by adherence (20) yielded a nonadherent population that was enriched for small CD4+ T cells that were 90 ± 2% nonspecific esterase–negative, and that did not express CD71, CD14, or CD11b. The adherent cells were 90 ± 2% nonspecific esterase–positive macrophages that maintain surface CD71+ expression after Be exposure in vitro.

We compared nuclear fragmentation in CBD BAL mixed cells to that present in isolated CBD BAL macrophage and lymphocyte populations (20). After 24 h of exposure to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin (Figure E2a in the online supplement), 54 ± 14% of the CBD BAL mixed cell population (n = 6, macrophages + lymphocytes) had fragmented nuclei (P < 0.05 versus the unstimulated control, nonparametric repeated measures ANOVA). Isolated CD71+ CBD BAL macrophages showed nuclear fragmentation (83 ± 7% cells; P < 0.05 versus the unstimulated control), whereas isolated CD4+ CBD BAL T cells did not (2 ± 0.2% cells; P = NS versus the unstimulated control). A representative photomicrograph shows that the Be-ferritin adduct reagent induced cytoplasmic membrane blebbing and nuclear fragmentation in isolated CBD BAL macrophages (Figure E2b in the online supplement). In comparison, isolated CBD BAL lymphocytes did not show changes in cytoplasmic membranes and had intact nuclei after exposure to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin (Figure E2c in the online supplement). Similar results were observed with BeSO4.

Be-Ferritin Activates Intracellular Macrophage, but Not Lymphocyte, Caspase
We used flow cytometry to determine whether exposure to the Be-ferritin adduct reagent or to BeSO4 could activate intracellular caspases-3, -8, and -9 in CBD BAL macrophages and lymphocytes. Approximately 15–20% of the BAL macrophages from subjects with CBD (Figure 3) stained positively for intracellular expression of activated caspase-3, -8, and -9 after 24 h of 100 µM BeSO4 exposure. Similar staining patterns were observed by flow cytometry in CBD BAL macrophages after 24 h of exposure to the Be-ferritin adduct reagent containing 270 pmol of Be adducted to 50 µg of ferritin. In comparison, we observed that < 1% of the BAL lymphocytes from subjects with CBD expressed caspase-3, -8, or -9 after exposure to either BeSO4 or the Be-ferritin adduct reagent. Intracellular expression of caspase-3, -8, and -9 were upregulated in 10–15% of BeS BAL macrophages and 24–40% of H36.12j cells that were Be-exposed as above. Less than 1% of the Be-exposed BeS BAL lymphocytes were positive for intracellular expression of caspase-3, -8, or -9.



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  		Figure 3. The activation of intracellular caspase-3, -8, and -9 in the macrophages and T cells isolated from the BAL of a CBD subject 24 h after exposure to 100 µM BeSO4 or to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin. The percent of positive cells is shown in the lower right quadrant of each panel.

 
Be-Ferritin Induces CD95 Upregulation in CBD BAL Macrophages and H36.12j Mouse Macrophages
The activation of intracellular caspase-8 suggested the possibility that Be-stimulation might induce apoptosis by up-regulating expression of a surface death receptor. In a subset of subjects with CBD (n = 3), we used flow cytometry to determine if unstimulated CBD BAL cells express CD95 (Fas, Apo-1) after Be-exposure. CBD BAL cells were back-gated to exclude CD4+ T cells (19) and to identify BAL macrophages in the unstimulated control. After 24 h exposure to 100 µM BeSO4, a median 30% (range, 28–41%) of CBD BAL macrophages were CD95+ (P < 0.05 versus the unstimulated control, median, 12%; range 9–16%, repeated measures ANOVA). After 24 h exposure to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin, a median 38% (range, 15–38%) of CBD BAL macrophages were CD95+ (P < 0.05 versus the ferritin reagent control-stimulated cells; median 3%; range, 1.3–3%). Typical results are shown in Figure 4, in which 32 and 40% of the BAL macrophages from a subject with CBD upregulated CD95+ expression after both BeSO4 and Be-ferritin adduct reagent exposure, respectively. Similar observations were made for H36.12j cells (data not shown) 24 h after exposure to 100 µM BeSO4 or to the Be-ferritin adduct reagent containing 270 pmol of Be adducted to 50 µg of ferritin.



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  		Figure 4. Flow cytometry showing the upregulation of CD95+ on the surface of BAL macrophages from a subject with CBD after 24 h of exposure to 100 µM BeSO4 or to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin. The histograms show the distribution of CD95+ cells on unstimulated cells (none) and Be-exposed cells. The percent of CD95+ cells is shown in the upper right quadrant of each panel.

 
Be-Ferritin Induces Apoptotic 12j Cell Phagocytosis
To test the hypothesis that apoptotic macrophages might be removed from culture due to their phagocytic uptake by neighboring, nonapoptotic macrophages, we determined the phagocytic index (PI) at 24 h for H36.12j cells (n = 3) exposed to 100 µM BeSO4 or to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin. Figure 5a shows the phagocytic uptake of differentially stained apoptotic bodies by intact H36.12j macrophages, as demonstrated by a central stained apoptotic nucleus surrounded by a "halo" of the apoptotic cell's cytoplasm. Phagocytized cellular and nuclear debris did not show this typical "halo." Figure 5b shows confirmatory staining of an apoptotic body TUNEL+ nucleus that was phagocytized by an intact H36.12j macrophage with a TUNEL– nucleus. By differential staining (Figure 5c), we observed a significant increase in the phagocytosis of BeSO4-induced apoptotic macrophages (PI = 1.5 ± 0.4, mean ± SEM; P = 0.0015 versus the unstimulated control [0.3 ± 0.4], repeated measures ANOVA), and of Be-ferritin adduct reagent exposed apoptotic macrophages (PI = 5.8 ± 0.3; P = 0.0001 versus the ferritin-alone–stimulated control [0.2 ± 0.4]). To confirm the phagocytic uptake of apoptotic bodies, as determined by the differential staining assay above, we determined the PI based on the presence of phagocytized apoptotic bodies with TUNEL+ nuclei inside H36.12j cells that contained TUNEL–, intact nuclei. TUNEL staining showed that the PI was equal to 6.4 ± 0.5 (mean ± SEM, n = 3) for phagocytized apoptotic bodies with TUNEL+ nuclei after 24 h exposure to 100 µM BeSO4 (P < 0.05 versus the unstimulated control; PI = 0.7 ± 0.4) while the PI was 5.9 ± 0.4 for phagocytized apoptotic bodies with TUNEL+ nuclei after 24 h exposure to 270 pmol of Be adducted to 50 µg of ferritin (P < 0.05 versus the control that was exposed to 50 µg of ferritin reagent).



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  		Figure 5. (a) A photomicrograph showing the phagocytosis of apoptotic bodies (arrows) by H36.12j cells after 24 h of exposure to the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin (original 200x). (b) Examples of: a Be-ferritin adduct reagent exposed intracellular H36.12j apoptotic body's TUNEL+ nucleus (white arrow 1) phagocytized by an H36.12j cell with an intact TUNEL– nucleus; H36.12j cells with intact TUNEL– nuclei (arrows 2); and an H36.12j cells with a TUNEL+ nucleus that has not been phagocytized (arrow 3) (original 200x). (c) The phagocytic index (PI = % of cells positive for apoptotic bodies X [number of apoptotic bodies/number of macrophages with apoptotic bodies]) for unstimulated H36.12j cells (n = 3) and cells exposed to 100 µM BeSO4, 50 µg of the ferritin reagent control, or the Be-ferritin adduct reagent that contained 270 pmol of Be adducted to 50 µg of ferritin. *P = 0.0015 for 100 µM BeSO4–exposed versus the unstimulated control and P = 0.001 for Be-ferritin adduct reagent–exposed cells versus the ferritin reagent control (repeated measures ANOVA).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data demonstrate a differential effect of Be-ferritin (containing 270 pmol of Be) on human lung macrophages and T cells, inducing apoptosis in the lung macrophage population that was Be metal–specific and independent of lung granulomatous inflammation, while triggering T cell proliferation in CBD. We hypothesize that this dual effect may help explain the persistence of Be antigen and a failure to delete Be-reactive T cell clones in the lungs of patients with CBD. CBD BAL macrophage and H36.12j cell apoptosis was highly induced by small amounts of Be in association with Be-ferritin. This small amount of Be adducted to ferritin induced the proliferation Be-ferritin–specific CBD BAL T cells that failed to undergo apoptosis upon antigen presentation. Our observation that 7Be, as 7BeCl2 or as a 7Be-ferritin adduct reagent, trafficks into the cell nucleus suggests that Be-ferritin is thereby able to serve as a "Trojan Horse," delivering low levels (270 pmol in this study) of Be inside macrophages via putative ferritin-receptor uptake. Failure of the dialysis control reagent to elicit lymphocyte proliferation in our study shows that the Be-ferritin dialysis method was sufficient to remove BeSO4, at least to levels below the 54–270 pmol of Be adducted to the Be-ferritin reagent that were able to induce lymphocyte proliferation. Failure of the ferritin protein control reagent to elicit proliferation shows the specificity of CBD BAL lymphocyte proliferative responses to the Be-ferritin adduct reagent.

In all contemporary in vitro studies of Be-induced apoptosis and Be-specific CD4+ T cell proliferation (12–14, 19) BeSO4 concentrations of 10 µM to 100 µM have been needed to induce clonal expansion of Be-specific T cells, a seemingly high concentration relative to environmental exposure levels (3–7). Previous study shows that the ability of Be-specific CBD BAL T cells to proliferate in vitro is not significant using concentrations < 1 µM BeSO4 (21). Ferritin is a ubiquitous iron transport and storage protein that is readily available to form Be adducts upon inhalation of Be particles. Adult serum contains 39–149 µg/ml of ferritin. The lung's epithelial lining fluid contains a variable amount of ferritin that is excreted by alveolar macrophages, epithelial cells, and emigrant neutrophils (22). The cellular receptor for ferritin is unknown (11). Based on the binding of carrier-free 7Be to ferritin, we found that 50 µg of the Be-ferritin adduct reagent contained 270 pmol of Be. Thus, a striking observation in this study was that Be-ferritin induced both of these cellular responses at Be concentrations that were 5–6 logs less Be than the amount of BeSO4 needed to induce these same responses in standard Be lymphocyte proliferation assays.

The subjects with CBD enrolled in this study show features associated with this disease, including the presence of noncaseating lung granulomas, increased BAL lymphocytosis, and a positive clinical BeLPT proliferation response in their blood and BAL cells, whereas subjects with BeS had normal lung histology, normal BAL cell constituents, and positive clinical BeLPT proliferation responses limited to the blood (14, 17, 18). Independent from the absence or presence of lung granulomatous inflammation, we observed dramatic morphologic changes in both BeS BAL and CBD BAL macrophages after Be-ferritin exposure, including cytoplasmic blebbing, nuclear condensation, high levels of nuclear damage and fragmentation detected by TUNEL staining, and simple differential staining. Six of eight subjects with CBD were current glucocorticosteroid users; however, steroid use has not been found to be associated with either Be-stimulated BeS or CBD BAL macrophage apoptosis (12). We did not observe an increase in the frequency of apoptosis in freshly obtained BAL cells from these six subjects. A recent study by Tomita and colleagues (23) shows that alveolar macrophages and bronchial epithelial cells from smokers have reduced apoptosis as compared with those from normal subjects and subjects with asthma, whereas our study shows that BeSO4 and the Be-ferritin adduct reagent were able to induce high levels of apoptosis in BeS BAL and CBD BAL macrophages, regardless of the subject's smoking status, suggesting that smoking did not alter the susceptibility of lung macrophages to Be-induced apoptosis.

Based on our data showing the cellular uptake of 7Be-ferritin, the observed pattern of caspase activation, and the upregulation of CD95 (Fas) expression, we hypothesize that Be-ferritin endocytosis may trigger the upregulation of surface CD95 (Fas) on BAL macrophages, albeit by an unknown mechanism. The ligation of CD95 by CD95L, from any cellular source, could induce caspase-8 activation with the subsequent activation of Bid, which in turn damages mitochondria releasing cytochrome c and activates caspase-9. Activated caspase-9 cleaves the inhibitor from caspase-activated DNase that nicks (TUNEL+) DNA, triggering nuclear fragmentation, as observed in this study, and DNA destruction. While some of the components of this hypothetical pathway have not been completely identified, it is reasonable to suggest that Be-induced macrophage apoptosis is mediated by CD95 and its well characterized downstream cascade of events that induce the nuclear damage observed in here. Our study does not rule out a possible role for other tumor necrosis factor (TNF) superfamily death receptors in Be-ferritin induced apoptosis. For example, previous studies show that Be-stimulation upregulates the intracellular expression and release of TNF-{alpha} protein by CBD peripheral blood mononuclear cells and CBD BAL cells (19, 24, 25), and H36.12j cells (26). CBD BAL macrophages, as well as H36.12j cells, constitutively express TNF-{alpha} (24–26). Ligation of TNF-R1, either by constitutively produced or Be-stimulated TNF-{alpha}, could serve as a second death receptor signal for Be-induced macrophage apoptosis. Future research will be needed to determine the contribution of these pathways.

Studies on the role of apoptosis in granulomatous lung disease are limited. Data on sarcoidosis (27–29) suggest that apoptosis in sarcoidosis granulomas could be beneficial by limiting the presentation of antigen to apoptotic CD4+ T cells. While T cell apoptosis might be beneficial in sarcoidosis, our data show that Be-ferritin–exposed CBD BAL lymphocytes did not undergo apoptosis. Granuloma formation and maintenance are believed to depend in part on the persistence of antigen (1, 2, 8), coupled to chronic stimulation of proinflammatory cytokines, such as TNF-{alpha} (1, 25, 26). Our studies show that Be-induced apoptotic H36.12j cells are cleared by phagocytic uptake. Early elimination of Be-ferritin–induced apoptotic human lung macrophages could hypothetically serve to limit Be exposure and maintain manageable levels of pulmonary inflammation. A recent study by Schaible and colleagues (30) showed that mycobacteria-induced macrophage apoptosis results in the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen presenting cells (APCs) that in turn engulf these extracellular vesicles and cross-present major histocompatibility complex class I–restricted antigens to T cells from mycobacteria-sensitized donors. By serving as a "Trojan Horse," Be-ferritin introduces very low physiologic levels of Be into human lung macrophages in a way that triggers both apoptosis and antigen presentation. By inducing BAL macrophage apoptosis over long periods of time in the lung, Be-ferritin could serve to trap Be within BAL macrophages in a cycle of Be-ferritin uptake, apoptosis, and Be-ferritin rerelease. We suggest the possibility that, in a manner similar to that described for mycobacteria-induced macrophage apoptosis (30), the phagocytosis of Be-induced apoptotic bodies, as shown in this study, or apoptotic cell vesicles could introduce low levels of Be-ferritin into APCs that survive this level of Be-exposure. At present, our study does not rule in or rule out a role for exogenous antigen processing by APCs after Be-ferritin adduct uptake. However, our data show that Be-ferritin adduct exposure induces the proliferation of Be-ferritin–specific CBD BAL lymphocyte; thus, it is reasonable to suggest that this hypothetical model may help explain why very low environmental Be exposures cause Be sensitization and CBD (3–7). This hypothetical interpretation merits some degree of caution based on our initial and limited observation that both BeS and CBD BAL macrophages undergo Be-ferritin adduct–induced apoptosis. If both cell types respond equally to Be-ferritin adduct exposure, a simple and alternate interpretation is that these events alone do not explain why BeS patients progress to CBD.

The observation that apoptosis of activated T cells, mediated by caspase activation and by reactive oxygen species, may eliminate antigen-specific T cell clones and thus downregulate adaptive immunity has implications for the immunopathogenesis of CBD (31, 32). Our data show that CBD BAL T lymphocytes are resistant to Be-induced apoptosis. Quiescent lymphocytes do not express CD71, receptors for transferrin (33), and we observed that freshly isolated CBD BAL CD4+ T cells were CD71-negative. We hypothesize that quiescent lymphocytes might also lack expression of the putative ferritin receptor, and could thereby resist Be ferritin–induced apoptosis. Thus, even after exposure to low levels of Be adducted to Be-ferritin, Be antigen–specific T cell clones could persist and orchestrate continued granulomatous inflammation. Support for T cell clonal persistence comes from our previous longitudinal studies, showing that Be-specific T cell clones are retained in the CBD lungs for at least 3 yr after their initial identification (34).

Our data suggest that, hypothetically, the effects of Be could be a consequence of its ability to bind ferritin, enter alveolar macrophages at physiologic concentrations, trigger macrophage apoptosis and Be retention, and induce T-cell proliferation without causing T-cell clonal deletion. These results, when taken in context with the evidence for robust cytokine production by Be-responsive macrophages and T cells (19, 24–26), presents a compelling case for why CBD is a persistent disease that can develop and worsen many years after occupational exposures have ceased.


   Acknowledgments
 
The authors thank Mary Solida, R.N., and Linda Staehler, R.N., for their patient care, Dr. John Kappler, Dr. Jay Westcott, Alexas Jonth, and Michele Baush for their technical assistance. They are indebted to their patients, who make this and other Be-related research possible. This study was supported by RO1 ES-06358, PO1 ES11810, RO1 ES012504, RO1 HL62410, K08 HL03887, and MO1 RR00051 from the National Institutes of Health.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: R.T.S. has no declared conflicts of interest; B.J.D. has no declared conflicts of interest; V.A.F. has no declared conflicts of interest; M.C-Z. has no declared conflicts of interest; L.A.M. has no declared conflicts of interest; A.P.F. has no declared conflicts of interest; L.S. has no declared conflicts of interest; and L.S.N. has no declared conflicts of interest.

Received in original form March 10, 2004

Received in final form July 5, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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