Genetic loss of Gas6/Mer pathway attenuates silica-induced lung inflammation and fibrosis in mice.

Long-term inhalation of crystalline silica particles leads to silicosis characterized by pulmonary inflammation and interstitial fibrosis. The growth arrest-specific protein 6 (Gas6) and its tyrosine receptor Mer have been implicated to involve in the regulation of inflammation, innate immunity and tissue repair. However, the role of Gas6 or Mer in silica-induced lung inflammation and fibrosis has not been investigated previously. In this study, we observed a remarkable increase of Gas6 in bronchoalveolar lavage fluid (BALF) from wild-type C57BL/6 mice after silica intratracheal administration. Then, we investigated whether genetic loss of Gas6 or Mer could attenuate silica-induced lung inflammation and fibrosis. Our results showed that Gas6-/- and Mer-/- mice exhibited reduced lung inflammation response from days 7 to 84 after silica exposure. We also uncovered an overexpression of the suppressor of cytokine signaling protein 1 in silica-treated deficient mice. Moreover, Gas6 or Mer deficiency attenuated silica-induced collagen deposition by inhibiting the expression of transforming growth factor-ß. We conclude that gene absence of Gas6 or Mer is protective against silica-induced lung inflammation and fibrosis in mice. Targeting Gas6/Mer pathway may be a potential therapeutic approach to treat pulmonary fibrosis in patients with silicosis.

Ac-SDKP increases α-TAT 1 and promotes the apoptosis in lung fibroblasts and epithelial cells double-stimulated with TGF-ß1 and silica.

Crystalline silica (SiO2) particles have very strong toxicity to the lungs, and silicosis is an excessive pulmonary interstitial remodeling disease that follows persistent SiO2 injury. We showed here that DNA double strand breaks (DSBs) and apoptosis were aggravated during rat silicosis induced by SiO2 exposure. Ac-SDKP attenuates lung parenchymal distortion and collagen deposition, and decreases the expression of γH2AX, p21, and cleaved caspase-3, as well as improves the reduction of pulmonary function caused by silicosis. In vitro, we found an evolution of smooth muscle actin α (α-SMA), collagen type I (Col I) in both A549 and MRC-5 cells in response to transforming growth factor-beta 1 (TGF-ß1) + SiO2. Only A549 cells showed any reduction in the rate of apoptosis induced by the double stimulation, because of the anti-apoptotic effects of TGF-ß1. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an anti-fibrotic tetrapeptide. It also has the ability to promote the apoptosis of leukemia cells. However its role in promoting cell apoptosis in silicosis is still unknown. We here found that Ac-SDKP could induce cell apoptosis and inhibit fibrotic response in A549 and MRC-5 cells treated with TGF-ß1 + SiO2, and these effects depended on regulation of α-tubulin acetyltransferase 1 (α-TAT1). These findings suggest that Ac-SDKP may have therapeutic value in the treatment of silicotic fibrosis.

Crystalline silica alters Sulfatase-1 expression in rat lungs which influences hyper-proliferative and fibrogenic effects in human lung epithelial cells.

Lung epithelial cells are the first cell-type to come in contact with hazardous dust materials. Upon deposition, they invoke complex reactions in attempt to eradicate particles from the airways, and repair damage. The cell surface is composed of a heterogeneous network of matrix proteins and proteoglycans, which act as scaffold and control cell-signaling networks. These functions are controlled, in part, by the sulfation patterns of heparin-sulfate proteoglycans (HSPGs), which are enzymatically regulated. Although there is evidence of altered HSPG-sulfation in idiopathic pulmonary fibrosis (IPF), this is not investigated in silicosis. Our previous studies revealed down-regulation of Sulfatase-1 (SULF1) in human bronchial epithelial cells (BECs) by crystalline silica (CS). In this study, CS-induced down-regulation of SULF1, and increases in Sulfated-HSPGs, were determined in human BECs, and in rat lungs. By siRNA and plasmid transfection techniques the effects of SULF1 expression on silica-induced fibrogenic and proliferative gene expression were determined. These studies confirmed down-regulation of SULF1 and subsequent increases in sulfated-HSPGs in vitro. Moreover, short-term exposure of rats to CS resulted in similar changes in vivo. Conversely, effects were reversed after long term CS exposure of rats. SULF1 knockdown, and overexpression alleviated and exacerbated silica-induced decrease in cell viability, respectively. Furthermore, overexpression of SULF1 promoted silica-induced proliferative and fibrogenic gene expression, and collagen production. These findings demonstrate that the HSPG modification enzyme SULF1 and HSPG sulfation are altered by CS in vitro and in vivo. Furthermore, these changes may contribute to CS-induced lung pathogenicity by affecting injury tolerance, hyperproliferation, and fibrotic effects.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

[Changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer].

OBJECTIVE: To investigate the changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer. METHODS: Serum samples of patients with silicosis, tuberculosis, and lung cancer were collected. The variation trends of the expression of granzyme A, cathepsin G, apolipoprotein A, and interferon-ß (IFN-ß) were analyzed using enzyme-linked immunosorbent assay. RESULTS: The concentration of apolipoprotein A of the silicosis group was 200 µg/ml, significantly higher than those of the tuberculosis and lung cancer groups (P < 0.05), and the lung cancer group had a significantly higher concentration of apolipoprotein A compared with the tuberculosis group (P < 0.05). The silicosis group had significantly higher expression of cathepsin G compared with the tuberculosis and lung cancer groups (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in the concentration of cathepsin G (P > 0.05). The tuberculosis group had a significantly higher concentration of granzyme A than the silicosis and lung cancer groups (P < 0.05), and the silicosis group and lung cancer group had similar protein concentration trends (P > 0.05). The tuberculosis group and lung cancer group had significantly higher concentration of IFN-ß compared with the silicosis group (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in IFN-ß concentration (P > 0.05). CONCLUSION: This study may offer diagnostic markers for the clinical diagnosis of silicosis, tuberculosis, and lung cancer, and could provide a basis for the research, as well as potential molecular targets for the diagnosis and treatment of these diseases.

Serum heme oxygenase-1 as a marker of lung function decline in patients with chronic silicosis.

OBJECTIVE: To identify predictive factors of excess decline in forced expiratory volume in one second (FEV1) in patients with chronic silicosis. METHODS: Forty-six male patients enrolled in 2004 were screened and received pulmonary function tests. RESULTS: Among the 33 included patients, 12 were categorized as rapid decliners (reduction in FEV1 > 60 mL/yr). The mean level of serum heme oxygenase-1 (HO-1), a marker of oxidative stress, was significantly lower in rapid decliners than in normal decliners (P = 0.002). Logistic regression analysis revealed that serum HO-1 was a factor affecting clinically important decline in FEV1 (odds ratio = 0.52; 95% confidence interval, 0.31 to 0.88) independent of the effects of age, height, weight, smoking, exposure status, and C-reactive protein. CONCLUSIONS: Serum HO-1 may be a predictor of lung function decline in silicosis patients.

Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis.

In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease.

Silica-induced TNF-alpha and TGF-beta1 expression in RAW264.7 cells are dependent on Src-ERK/AP-1 pathways.

The cytokines secreted by lung macrophages have been shown to play a critical role in the pathogenesis of silicosis, tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) are prominent cytokines in silicosis, but the underlying mechanism remains to be determined. The aim of the present study was to investigate the roles of Src-mitogen-activated protein kinase (MAPKs)/activator protein-1 (AP-1) signaling pathways in silica-induced TNF-alpha and TGF-beta1 expression in macrophage cells (RAW264.7). It was found that silica activated Src, p38 kinase, and extracellular signal-regulated kinase (ERK) in RAW264.7 cells. The induction of TNF-alpha and TGF-beta1 by silica was suppressed by Src inhibitor (PP1), ERK inhibitor (PD98059), but not by p38 kinase inhibitor (SB203580). Dominant negative mutant c-Jun (TAM67) inhibited silica-induced AP-1 DNA binding activity and downregulated the TNF-alpha and TGF-beta1 expression. In addition, PD98059 but not SB203580 inhibited the AP-1 DNA binding activity induced by silica. Based on these findings, it was conclude that Src-ERK/AP-1 signaling pathways are involved in the TNF-alpha and TGF-beta1 expression induced by silica in macrophages.

Cloning of a rat lung fibrogenic factor.

In a previous study a specific single polypeptide has been purified and characterized that it was capable of promoting human embryonic lung 2BS fibroblasts proliferation in vitro, whose N-terminal 15 amino acid have high sequence homology with members of the mammalian chitinase-like protein family. Here the cloning of the gene is reported. Its cDNA contains an open reading frame 1421 bp long and encodes a protein with a characteristic N-terminal 21 amino acid endoplasmic reticulum signal peptide and the putative protein is highly homologous to acidic mammalian chitinase (AMCase) precursor of mouse and human. Recombinant proteins demonstrate chitinolytic activity, therefore the gene is termed as rat AMCase. Sequence analysis indicates that the gene spanned a 46.2 kb region in rat chromosome 2. Its expression in several tissues other than alveolar macrophages suggests that it might play multiple biological roles in vivo. Our findings will facilitate studies on its roles in physiological and pathological processes.

Cysteine cathepsins and caspases in silicosis.

Silicosis is an occupational pneumoconiosis caused by inhalation of crystalline silica. It leads to the formation of fibrohyalin nodes that result in progressive fibrosis. Alternatively, emphysema may occur, with abnormal destruction of collagen fibres in the advanced stages. Although the pathophysiological mechanisms remain unclear, it has been established that the lung responds to silica by massive enrollment of alveolar macrophages, triggering an inflammatory cascade of reactions. An imbalance in the expression of lung proteases and their inhibitors is implicated in extracellular matrix remodelling and basement membrane disruption. Moreover, exposure to silica can initiate apoptotic cell death of macrophages. This review summarises the current knowledge on cysteine cathepsins that have been ignored so far during silicosis and outlines the recent progress on cellular pathways leading to silica-induced caspase activation, which have been partly delineated.

Resistance to acute silicosis in senescent rats: role of alveolar macrophages.

We have previously demonstrated in alveolar macrophages that aging is associated with a decline in lipopolysaccharide-induced tumor necrosis factor-alpha production. The purpose of the present study was to investigate the immunotoxicological consequences of this defective activation in an experimental model of acute silicosis. Young (3 months old) and old (>18 months old) rats were intratracheally instilled with silica or saline as control. In young animals, as expected, silica induced a significant increase in bronchoalveolar lavage fluid of tumor necrosis factor-alpha, lactate dehydrogenase, and cell numbers, which correlated with increased collagen deposition and silicotic nodule formations. On the contrary, in old rats, no changes in bronchoalveolar lavage fluid or lung parameters were observed, indicating that senescent rats are resistant to the acute effects of silica. These in vivo results were confirmed in vitro, where silica-induced tumor necrosis factor-alpha release was drastically reduced in alveolar macrophages obtained from old animals. This could be explained with a defective protein kinase C betaII translocation in aged macrophages, due to decreased expression of its anchoring protein RACK-1. Furthermore, a decrease in FAS-L expression and silica-induced apoptosis in old macrophages was observed, supporting the idea that age-associated alterations in signal transduction pathways contribute to decreased sensitivity to silica-induced acute lung fibrosis in old animals.

Expression of matrix metalloproteinase, tissue inhibitors of metalloproteinase and adhesion molecules in silicotic mice with lung tumor metastasis.

The number of metastatic foci in silicotic mice is approximately 1.5-fold that in normal mice and in mice treated with TiO2 as inert particles. Expression of matrix metalloproteinases (MMPs) tissue inhibitors of metalloproteinases (TIMPs) and selectins was investigated in silicotic mice with lung tumor metastasis. Expression of MMP-9 and P-selectin mRNA, but not MMP-2 and E-selectin, increased significantly, showing decreases of the ratio of expression in TIMPs/MMP-9 in tumor-bearing silicotic mice compared with the tumor-bearing normal mice and mice treated with TiO2. Pretreatment with anti-P-selectin antibody inhibited number of metastatic foci significantly in silicotic mice, while pretreatment of animals with anti MMP-9 antibody showed slight decrease of metastatic foci. This evidence indicated that up-regulation of P-selectin expression contributed to enhanced rate of tumor metastasis in lung with silicosis.

Long term iNOS expression in thoracic lymph nodes of silicotic rats.

Beside the lung, thoracic lymph nodes are most affected during silicosis. The mechanisms leading to enlargement of the lymph nodes and partial activation of lymph node cells are still unclear. The present study demonstrates an increase in iNOS mRNA expression in the lung draining lymph nodes of rats at 1, 2, and 8 months following silica exposure. Histopathological analysis revealed that iNOS protein was exclusively expressed by macrophages located within the granulomatous areas of the enlarged lymph nodes. In contrast, no differences in mRNA expression and number of iNOS-positive cells were found in the lungs of silica-exposed and non-exposed rats. In vitro experiments showed that silica particles alone did not induce NO release in primary alveolar macrophages (AMs) or the alveolar macrophage cell line NR8383. However, the addition of interferon (IFN)-gamma led to a significant nitric oxide production by primary AMs. NR8383 cells responded only when a combination of IFN-gamma and silica particles was applied. These results indicate that the macrophage activator IFN-gamma, which has already been shown to be expressed at elevated levels by lymphocytes of the silicotic lymph nodes, may be responsible for the long-lasting iNOS expression in thoracic lymph nodes. Our observations support the hypothesis that the mutual activation of lymphocytes and macrophages is a central process in the development of chronic silicosis.

[Effect of tea polypheonls and ascorbic acid on the activity of antioxidant enzymes in rats with experimental silicosis].

To explore the effect of tea polypheonls (TP) combined with ascorbic acid on the activity of antioxidant enzymes of rats induced by silica, the activities of nitric oxide synthase (NOS), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were determined. Compared with the normal saline control group, the activities of SOD and NOS of the silica-induced group increased (P < 0.05), but the activity of GSH-PX was gradually decreased. The activities of SOD and NOS decreased and GSH-PX increased in the intervention group. It is concluded that TP combined with ascorbic acid can alleviate the abnormal change of antioxidant enzymes in rats induced by silica.

Tumor necrosis factor receptor deficiency alters matrix metalloproteinase 13/tissue inhibitor of metalloproteinase 1 expression in murine silicosis.

Murine exposure to silica is associated with enhanced tumor necrosis factor alpha (TNF-alpha) expression and matrix deposition. The regulation of TNF is mediated through TNF receptor (TNFR) activation of transcription factors. In the present work we have studied the importance of the individual TNFR in silica-induced lung inflammation and matrix deposition in mice. We studied RNA expression of TNF, alpha1(I) collagen, interstitial collagenase (MMP-13), and its inhibitor (TIMP-1) in the lungs of silica-treated mice. Furthermore, we correlated MMP-13/TIMP-1 RNA abundance with activation of the transcription factors AP-1 and NF-kappaB in the lungs of C57BL/6 mice, and of mice deficient in one of the two types of TNFR (p55(-/-) or p75(-/-)), exposed to silica (0.2 g/kg) or saline by intratracheal instillation. Animals were killed 28 d after exposure and lung hydroxyproline (HP), TNF, alpha1(I) collagen, MMP-13, and TIMP-1 RNA abundance was measured. AP-1 and NF-kappaB activation was studied by gel-shift assays. Compared with C57BL/6 mice, p55(-/-) and p75(-/-) mice significantly (*p < 0.05) decreased lung HP accumulation in response to silica. All murine strains enhanced TNF and alpha1(I) collagen mRNA in response to silica. Enhanced (p < 0.05) MMP-13 RNA expression was also observed in all murine strains in response to silica. Enhanced (p < 0.05) TIMP-1 RNA expression was observed in C57BL/6 mice, but not in p55(-/-) or p75(-/-) mice, in response to silica. NF-kappaB activation was observed in all murine strains, whereas AP-1 activation was observed only in C57BL/6 mice after silica treatment. These data suggest that TNFR deletion modifies MMP-13/ TIMP-1 expression in favor of matrix degradation.

Activation of telomerase by silica in rat lung.

By measuring the activity of telomerase in a silica-instilled rat lung, the study found a significant increase in telomerase activity compared to that of the control. Pneumoconiosis displays the characteristics of fibroblast-proliferation and accumulation of collagen, which finally causes the pathologic changes of irreversible and progressive fibrosis of the lung. On the basis of the hypothesis that cellular proliferation may trigger telomerase-activity, the experiment was carried out with telomerase-activation in silicosis. Silica-instilled rat lungs showed increased activity of telomerase, which was measured by TRAP (telomeric repeat amplification protocol) assay, at the time of the 1st, 5th and 8th week after intratracheal instillation of silica in vivo. However, no activity was shown in silica-co-cultured fibroblast in vitro. By summarizing these results, the activity of telomerase is thought to be a very sensitive marker for the evaluation of pathogenicity, showing cellular immortalization in an experimental silicosis model.

Matrix metalloproteinases 2, 9, and 13, and tissue inhibitors of metalloproteinases 1 and 2 in experimental lung silicosis.

Exposure to silica induces granulomatous lung inflammation evolving to fibrosis through yet unclear pathogenic mechanisms. We examined the expression of extracellular matrix remodeling molecules: collagenase 3, gelatinases A and B, and TIMP-1 and TIMP-2 in experimental lung silicosis. Rats were instilled with 50 mg of silica and sacrificed after 15 and 60 d. At 60 d a significant increase in lung collagen content was found (170.2 +/- 34.4 versus 88.2 +/- 20.8 microgram/mg in controls, p = 0.01). Gelatin zymography of bronchoalveolar lavage fluid (BALF) from 15 and 60 d revealed bands of progelatinase A and progelatinase B, and lung tissue zymograms showed in addition, the active gelatinase A form at 15 d. By in situ hybridization and immunohistochemistry, early silicotic granulomas exhibited intense staining for all matrix metalloproteinases (MMPs) and TIMPs assayed. Labeling was restricted inside granulomas and surrounding areas. Late silicotic granulomas at 60 d showed lower MMP expression than did early lesions, and in highly fibrotic nodules scarce signal was usually found. TIMP-1 and TIMP-2 showed a moderate reduction in 60-d silicotic nodules. These findings suggest that an imbalance in the expression of MMPs and TIMPs may be implicated in extracellular matrix remodeling and basement membrane disruption during experimental lung silicosis.

Collagenase-3 induction in rat lung fibroblasts requires the combined effects of tumor necrosis factor-alpha and 12-lipoxygenase metabolites: a model of macrophage-induced, fibroblast-driven extracellular matrix remodeling during inflammatory lung injury.

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-alpha (TNF-alpha) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-alpha and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Serum lactate dehydrogenase and its isoenzyme pattern in ex-coalminers.

Serum lactate dehydrogenase (LDH) activity, a marker of cell damage, is increased in several pulmonary disorders, especially when fibrosis is involved. In rats exposed to silica, high levels of LDH activity were found. A rise of serum LDH3 has been associated with lung tissue injury. The aim of this study was to investigate the serum LDH isoenzyme pattern after coal-dust exposure and the possible relation to pulmonary function tests. Ex-coalminers (n = 201), with a history of coal-dust exposure more than 20 yr ago, were admitted to the authors' hospital for a medical check-up and were included in the study. The serum LDH activity was found to be elevated in 79.1% of the ex-coalminers (634 +/- 245 U I-1). Moreover, in 196 of the 201 cases (97.5%), a high LDH3 level (31 +/- 4%) was demonstrated. A moderate negative relation was found between the forced expiratory volume in 1 s (FEV1) and the LDH activity (r = -0.26; P < 0.001), as well as between FEV1 and LDH3 activity (r = -0.23; P < 0.001), even in the subgroup (n = 42) with a normal LDH. All other liver function tests were within normal limits. These results suggest that coal-dust, even many years after the actual exposure, causes an increase in the total serum LDH activity and changes in the LDH-isoenzyme pattern, mainly characterized by a high LDH3 activity.

Serum angiotensin-converting enzyme activity, concentration, and specific activity in granulomatous interstitial lung disease, tuberculosis, and COPD.

Angiotensin-converting enzyme (ACE) activity in serum is used as an aid to the diagnosis and follow-up of patients with sarcoidosis. A theoretical limitation of measurements of activity is that these may be affected by the presence of pharmacologic or endogenous inhibitors of ACE. Immunoassays of ACE concentration avoid this problem and, when combined with tests of ACE activity, permit calculation of specific activity of ACE. In this study, we set out to develop a sensitive radioimmunoassay for ACE to compare results obtained with this method with results of ACE activity and calculated ACE specific activity in patients suffering from a variety of lung diseases. In a group of control subjects (n = 32), the ACE concentration was 453.7 +/- 159.8 (SD) ng/mL; 95% confidence interval (CI), 398.34 to 509.06, but levels were significantly elevated in sarcoidosis (979.3 +/- 558.6 ng/mL; 95% CI, 827.5 to 1,131.1; n = 51; p < 0.001 vs control subjects), silicosis (646.5 +/- 239.1 ng/mL; 95% CI, 544.2 to 748.8; n = 21; p < 0.01), and miliary tuberculosis (647.0 +/- 217.1 ng/mL; 95% CI, 551.9 to 742.1; n = 29; p < 0.01). The levels were normal in COPD, interstitial pulmonary fibrosis, and active cavitary pulmonary tuberculosis. The overall correlation between ACE activity and concentration measurements was strong (r = 0.93). No evidence of endogenous ACE inhibition was observed in any of the disease categories studied except in COPD where an elevation of ACE specific activity was observed, raising the possibility that in this condition different isozymes of ACE with higher specific activity might be released.