Expression of elastase activity by human monocyte-macrophages is modulated by cellular cholesterol content, inflammatory mediators, and phorbol myristate acetate.

Abstract

We investigated the effects of a number of stimulatory agents on the production of both cell-associated and extracellular elastase-type enzymes on human monocyte-macrophages in vitro and of the modulation of such effects by modification of cellular cholesterol content. The stimulatory agents included phorbol myristate acetate (PMA) and the inflammatory mediators, lipopolysaccharide (LPS), opsonized zymosan (OZ), and platelet activating factor (PAF). Using the synthetic substrate, N-succinyl-trialanyl-paranitroanilide (SANA), we detected cell-associated elastase-like activity in monocyte-derived macrophages. Such activity increased markedly with cell maturation over the period from 5 to 15 days of adherence culture. While PAF (10 micrograms/ml) and LPS (10 micrograms/ml) were without effect on cell-associated elastase-like activity in macrophages, PMA (100 ng/ml) and OZ (1 mg/ml) markedly stimulated such activity in cells cultured for 15 days. Furthermore, a fivefold increase in the cell-associated elastase-like activity of macrophages occurred upon cholesterol loading of the cells with acetylated low density lipoprotein (AcLDL). By contrast, this activity was markedly diminished upon depletion of cellular cholesterol content after incubation with high density lipoprotein (HDL3). Latent elastinolytic activity in the culture medium was detected by use of a radioactive substrate, insoluble 3H-elastin, after initial tryptic treatment of the medium. Such latent elastase activity was secreted only by activated macrophages; the relative potency of stimulation was: PMA greater than LPS = PAF greater than OZ. Increase in cellular cholesterol content alone markedly enhanced the secretion of elastase (from undetectable levels to 28 ng of 3H-elastin degraded/hr/micrograms DNA). In all cases, both the cell-associated and secreted latent elastinolytic activities were due to metalloproteases, in view of their 90% inhibition by 2 mM EDTA. Cholesterol-loaded macrophages, which displayed an approximately 40-fold increase in total cholesterol content as compared to control cells, remained sensitive to the action of activators of OZ and PMA, while LPS and PAF exerted only weak effects. Our data indicate that cellular cholesterol content and inflammatory mediators are effective stimulants of the production and secretion of elastase-type enzymes by human monocyte-macrophages. Among these factors, cellular cholesterol content, OZ, PAF, and LPS may represent factors of relevance to the inflammatory role of the macrophage in atherogenesis and more specifically to the alteration of elastin structure in the extracellular matrix of the vessel wall.