Crystalline and amorphous silica differentially regulate the cyclooxygenase-prostaglandin pathway in pulmonary fibroblasts: implications for pulmonary fibrosis

Abstract

Inhalation of crystalline (CS) and amorphous silica (AS) results in human pulmonary inflammation. However, silicosis develops only following CS exposure, and the pathogenic mechanisms are poorly understood. This report describes the differential abilities of CS and AS to directly upregulate the early inflammatory mediator COX-2, the recently identified prostaglandin E (PGE) synthase and the downstream mediator PGE2 in primary human lung fibroblasts. Increased cyclooxygenase (COX)-2 gene transcription and protein production were demonstrated by ribonuclease protection assay, Western blot analysis, and immunocytochemistry. In each case the ability of AS to induce COX-2 exceeded that of CS. Similarly, downstream of COX-2, production of the antifibrotic prostaglandin PGE2 was induced in a dose-dependent fashion, but AS was significantly more potent (maximal production: CS = 4,710 pg/ml and AS = 7,651 pg/ml). These increases in COX-2 and PGE2 were preceded by induction of the PGE2 synthase protein, demonstrating the potential role of this novel molecule in silica-mediated inflammation. There was specificity of induction of prostaglandins, as PGF2alpha, but not PGD2, was induced. Using specific COX-2 inhibitors, we showed increased PG production to be dependent on the COX-2 enzyme. Furthermore, stimulation of fibroblasts was particle specific, as silica but not carbon black resulted in fibroblast activation. These results demonstrate that silica can directly stimulate human lung fibroblasts to produce key inflammatory enzymes and prostaglandins. Moreover, they suggest a mechanism to explain the differing fibrogenic potential of CS and AS. The molecules COX-2, PGE synthase, and PGE2 are identified as effectors in silicosis.

Figure 1.

Silica induces COX-2 but not COX-1 protein in human lung fibroblasts. Fibroblasts in culture were treated for 24 h with the indicated concentrations of amorphous (AS) or crystalline (CS) silica. Carbon black (CB, 100 μg/ml) was included as a negative control. Total cell protein was analyzed by Western blot for expression of COX-2, COX-1 and actin (to confirm equal protein loading). Amorphous silica induces COX-2 slightly at 2.5 μg/ml and strongly at 10 μg/ml. 10-fold higher concentrations of crystalline silica are required to produce similar elevations in COX-2 protein. Neither form of silica alters the constitutive expression of COX-1.

Figure 2.

COX-2 protein expression is induced by silica, but not carbon black (CB), in human lung fibroblasts. After 24h of treatment with 10 μg/ml amorphous silica (AS), 100 μg/ml crystalline silica (CS), and 100 μg/ml carbon black (CB), immunocytochemistry was performed for COX-2 protein. Both CS and AS induce COX-2 expression as seen by the deep red cytoplasmic staining. Whereas CB, a relatively inert particle does not. Pictures were taken with a 40x objective.

Figure 3.

Amorphous and crystalline silica induce COX-2 but not COX-1 mRNA in human lung fibroblasts. Total RNA was extracted from fibroblasts after 6 h exposure to selected agents. The RNA was then analyzed by RPA. Lane 1, undigested probe; lane 2, digested probe; lane 3, untreated fibroblasts; lane 4, fibroblasts treated with 100 μg/ml crystalline silica (CS); lane 5, fibroblasts treated with 10 μg/ml amorphous silica (AS). A. Neither form of silica induces COX-1 gene expression. B. Both CS and AS induce COX-2, with AS at least 10 times more potent than CS.

Figure 4.

PGE₂ production is induced in a dose dependent fashion by silica. Fibroblasts were exposed to crystalline (CS) or amorphous (AS) silica at the indicated concentrations; the cell supernatants were harvested after 48 h. and analyzed for PGE₂ using an enzyme immunoassay (EIA). MC; media control. The increases in PGE₂ production are statistically significant (*p<0.01, **p<0.001). Data represent mean ± SEM of 6 samples per condition. Note the different scales on the y-axis.

Figure 5.

Silica induced PGE₂ production is dependent upon increased COX-2 expression. Suspensions of crystalline (CS) and amorphous silica (AS) and either a non-selective COX inhibitor, indomethacin (20 μM) or a selective COX-2 inhibitor, SC58125 (5 μM) were applied to fibroblasts in culture. IL-1β (10 ng/ml), a cytokine that is known to stimulate COX-2, is used as a positive control. Cell supernatants were harvested after 48 h and PGE₂ levels measured using a specific enzyme immunoassay (EIA). Both indomethacin and SC58125 independently attenuated the increase in PGE₂ seen with CS (*p<0.005) and AS (**p<0.001). Data represent mean ± SEM of 6 samples per condition.

Figure 6.

Crystalline and amorphous silica induce mPGE synthase in primary human lung fibroblasts. Normal pulmonary fibroblasts in culture were exposed to 100 μg/ml crystalline silica (CS), 10 μg/ml amorphous silica (AS), or IL-1β (10 ng/ml) as a positive control. Cell lysates were harvested at 24, 48 and 72 h, and Western blot analysis for mPGES was performed. MC, media control.

Figure 7.

PGF₂α production is stimulated by crystalline silica (CS) and amorphous silica (AS). Fibroblasts were exposed to indicated doses of CS and AS. MC, untreated media control. The cell supernatants were harvested after 48 h and analyzed for PGF₂α using enzyme immunoassays. The increase in PGF₂α production is statistically significant (*p<0.01).