Abstract
Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.
Highlights
Environmental exposure to crystalline silica induces pulmonary inflammation that often leads to fibrosis [1]
Among the cytokines secreted by lung macrophages in response to silica, tumor necrosis factor ␣ (TNF␣)1 has been shown to play a critical role in the pathogenesis of silicosis, and procedures that antagonize the biological effects of TNF␣ ameliorate silica-induced pulmonary fibrosis in mice [2,3,4]
We studied the differences in TNF␣ production in two macrophage cell lines in response to in vitro silica exposure
Summary
TNF␣, tumor necrosis factor-␣; TNFR, tumor necrosis factor receptor; TRADD, TNF receptor-associated death domain protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; MEK1, MAPK/ERK kinase; IAP, inhibitor of apoptosis family of proteins; PBS, phosphate-buffered saline; TRAF1 and -2, TNF receptor-associated factors 1 and 2; LPS, lipopolysaccharide; FITC, fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent assay; CMV, cytomegalovirus; Ad, adenovirus; EGFP, enhanced green fluorescent protein. We took advantage of the difference in TNF␣ production by these macrophage cell lines to study the role that TNF␣-mediated activation of NF-B and MAPKs plays on the protection of macrophages against silica-induced apoptosis. We investigated the importance of ERK-mediated phosphorylation of p55 TNF receptor in protecting macrophages from silica-induced apoptosis
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