Pleural Macrophages Differentially Alter Pleural Mesothelial Cell Glycosaminoglycan Production

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Abstract
Glycosaminoglycans are produced in abundance by the pleural mesothelium and likely participate in the inflammatory response to pleural injury. Because intrapleural tetracycline (TCN) results in pleural macrophage influx and pleural fibrosis, this study attempted to define the role of pleural macrophage products on mesothelial glycosaminoglycan (GAG) production. Pleural macrophages were isolated 72 h after intrapleural TCN or intrapleural carrageenan (CAR), a substance that recruits pleural macrophages without producing pleural fibrosis. Macrophage cultured for 24 h produced a conditioned medium that was added to pleural mesothelial cell culture containing [3H]-glucosamine and was compared to control cultures treated with RPMI culture media alone or with the addition of TCN or CAR. After 72 h, GAGs were isolated by pronase digestion, cetyl pyridinium precipitation, and MgCl2 and ethanol extraction. The majority of GAGs were found in the culture media as compared to the combined mesothelial cell and basement membrane fractions of control mesothelial cells (883 ± 33 vs. 216 ± 16, cpm, counts per minute), TCN-treated (792 ± 48 vs. 204 ± 18 cpm), CAR-treated (849 ± 45 vs. 223 ± 13 cpm), and macrophage-conditioned media-treated mesothelial cells (TCN macrophage-conditioned media: 1420 ± 42 vs. 356 ± 11 cpm; CAR macrophage-conditioned media: 1241 ± 38 vs. 339 ± 10 cpm) (all p >.05). Media samples were enzymatically digested and individual GAG species were separated by Sephadex G-50 column chromatography. TCN macrophage-conditioned media induced more GAG production by the mesothelial cell into the cell media (1420 ± 42 cpm) than CAR macrophage-conditioned media (1241 ± 38 cpm) (p >.05), which was predominantly a difference in hyaluronate production (342 ± 53 cpm vs. 186 ± 7 cpm) (p >.05). The results show that pleural macrophages modulate mesothelial GAG production during tetracycline pleural injury. Increases in mesothelial cell hyaluronate production may be important in the fibrotic response to chemical pleural injury.