TNF alpha gene and protein expression in alveolar macrophages in acute and chronic hyperoxia-induced lung injury.

Abstract

Alveolar-capillary membrane remodeling, including microvessel wall thickening and interstitial fibrosis, is a well-known sequela of cell proliferation in the hyperoxia-injured lung. The array of growth molecules released locally that potentially mediate this response, and their cell(s) of origin, are currently being defined. To elucidate the role of tumor necrosis factor alpha (TNF alpha), an effector molecule of cell injury and proliferation, and the role of the alveolar macrophage (AM) as its source during the acute (1 to 24 h) and chronic stages (3 to 28 days) of hyperoxia-induced injury, we have analyzed gene and protein expression in cells recovered from rat lung by bronchoalveolar lavage. In the hyperoxic lung, cell number was similar to that in normal lung (1 x 10(6)) except on day 7, when it was higher (5 x 10(6)). Virtually all cells recovered from the normal and hyperoxic lung were AMs, with the exception that on days 3 and 7 of hyperoxia these cells represented 69% and 55% of the population, respectively, and polymorphonuclear leukocytes and lymphocytes the remainder. Probe specificity was confirmed by detection of TNF alpha RNA (1.6 kb) from lung cells recovered after lipopolysaccharide (LPS) treatment (positive control) and from the hyperoxic lung (at day 3), with an extremely low level of constitutive expression detected in cells from normal lung. In cytospin preparations, TNF alpha mRNA transcripts were detected in few AMs recovered from normal lung and in most AMs after LPS treatment. In the hyperoxic lung, a signal was detected at 3 h, when approximately 25% of the population was positive. The number of hybridizing cells then increased, being highest on day 7 (day 1 approximately 30%, day 3 approximately 58%, day 7 approximately 90%, day 28 approximately 65%). No expression of TNF alpha protein was detected in AMs from normal lung; positive cells were detected in the hyperoxic lung from day 1 and thereafter. We conclude from upregulation of the TNF alpha gene in a significant number of cells, and from the increase in the number expressing biologically active protein, that AMs are an important source of this molecule both in the acute and chronic stages of hyperoxic lung injury. It is anticipated that an increased understanding of the cellular sources of mediators effecting vascular and alveolar wall remodeling in vivo will contribute to the development of strategies to inhibit the response.