Activation of Murine Macrophages by Silica Particles In Vitro Is a Process Independent of Silica-induced Cell Death Estefania Claudio, Fernando Segade, Kazimierz Wrobel, Sofia Ramos, and Pedro S. Lazo Departamento de Biologfa Funcional, Universidad de Oviedo, Oviedo, Spain We have tested the murine macrophagic cell line RAW 264.7 for its ability to undergo activation after exposure to silica particles in vitro. When exposed to silica under controlled conditions (each cell having access to about 10 silica particles), RAW 264.7 cells were able to phagocytose the particles. Concomi- tantly, there was a significant increase in tumor necrosis factor a (TNFa) mRNA accumulation and TNFa secretion. The level of TN Fa production by RAW264.7 cells increased up to 5-fold 48 h after phagocytosis of silica particles with very low cell toxicity. The phagocytic stimulus did not induce nitric oxide produc- tion. When cells were exposed to a higher number of silica particles, cell activation was attained at shorter times but a substantial number of cells were damaged at 48 h. Interferon I' (IFNI') alone induced an in- creased production of TNFa in RAW 264.7 cells, not further augmented by a subsequent exposure to silica of the If'Nv-treated cells. Other macrophage-like cell lines as well as primary peritoneal macro- phages were able to phagocytose silica particles but showed different abilities to produce and secrete TNFa once phagocytosis took place. Therefore, RAW 264.7 cells were chosen as a model for in vitro studies of the long-term response of macrophages to silica. Silicosis is a pathologic process characterized by the devel- opment of fibrotic nodules in the lung caused by cell prolifer- ation, extracellular matrix deposition, and accumulation of inflammatory cells, and whose primary origin is a long- term exposure to crystalline silica (1). The current view on the pathogenesis of silicosis and other particle-originated fibroses stresses the central role played by the macrophage, it being now widely accepted that interactions between in- haled particles of silica deposited at the alveolar level and resident macrophages produce a major alteration of the mac- rophage that is recognized as the origin of later dysfunctions. A large number of substances are then produced by the cell that are involved in other macrophage-mediated inflamma- tion and repair processes (1-3). Thus, the release of reactive oxygen (4), and neutral proteases and other enzymes (1), are responsible for the damage inflicted on adjacent lung tissues. Other agents such as polypeptide growth factors and cyto- kines induce the recruitment of cells into the growing lesion and contribute to the activation of lymphocytes, which, in turn, induce macrophages to produce new sets of factors (5). In addition, an increased production of mitogenic and/or (Received in original form March 20, 1995 and in revised form May 24, 1995) Address correspondence to: Dr. Pedro S. Lazo, Facultad de Medicina, Universidad de Oviedo, 33071 Oviedo, Spain. Abbreviations: fetal bovine serum, FBS; interferon "y, 1FN"y; lipopolysac- charide, LPS; 3-(1-4 dimethyl thiazol-2-yl)-2-5-diphenyl tetrazolium bro- mide. MTT; nitric oxide, NO; tumor necrosis factor a. TNFa; recombinant murine tumor necrosis factor a, rMuTNFa. Am. J. Respir. Cell Mol. BioI. Vol. 13. pp. 547-554, 1995 fibrogenic factors is responsible for the proliferation of type II pneumocytes (6) and fibroblasts (2), with a concomitant stimulation in collagen deposition in the lung (1). The sum of the former actions is believed to result in the development of granulomatous lesions and/or fibrotic nodules that are characteristic of the disease. The mechanisms by which silica particles are internalized and stimulate macrophages to an enhanced release of infl- ..... - matory and fibrosis-promoting mediators are still uncertam. Two mechanisms have been proposed: (J) silica particles damage macrophages, which in turn release the mediators (7-9), and (2) silica particles stimulate macrophages without cell toxicity and lead to a persistent mediator production (1, 2, 10). Evidence for the first hypothesis has been found un- der in vitro conditions. We have addressed the question of whether macrophages can be activated without cell toxicity by culturing the macrophage-like cell line RAW 264.7 with increasing concentrations of silica particles. The results presented in this report show that RAW 264.7 cells can be activated in vitro with no substantial cell death when only a few silica particles were phagocytosed by each cell, thus providing support to the hypothesis that no cell toxicity is re- quired for the release of mediators. On the other hand, when a large number of particles was available, cells initially un- derwent activation but they were eventually damaged as the number of internalized particles increased. Materials and Methods Materials Recombinant murine tumor necrosis factor a (TNFa; specific activity, 4 X 10 8 U/mg protein), interferon I'