α2-macroglobulin functions as a cytokine carrier to induce nitric oxide synthesis and cause nitric oxide-dependent cytotoxicity in the RAW 264.7 macrophage cell line

Abstract

Nitric oxide (NO) is an important mediator of macrophage activities. We studied the regulation of macrophage NO synthesis by α2-macroglobulin (α2M), a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor-β (TGF-β). Native α2M and the α2M receptor-recognized derivative, α2M-methylamine (α2M-MA), increased nitrite generation by the RAW 264.7 murine macrophage cell line. The level of nitrite accumulation, which is an index of NO synthesis, was comparable to that observed with interferon-γ. Native α2M and α2M-MA also increased inducible nitric oxide synthase (iNOS) mRNA levels and substantially reduced the number of viable cells, as determined by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium/succinyl dehydrogenase assay or trypan blue exclusion. At slightly higher α2M concentrations, [3H]thymidine incorporation was inhibited. All of these activities were counteracted nearly completely when the iNOS competitive inhibitor N(G)-monomethyl-L-arginine was included. By in situ nick translation, native α2M and α2M-MA increased the percentage of cells with detectable single strand chromatin nicks from 4 to 12 and 17%, respectively. This change suggested apoptosis; however, electron microscopy studies demonstrated variability in the morphology of injured cells. To determine the mechanism by which α2M increases macrophage NO synthesis, we studied proteolytic α2M derivatives that retain partial activity. A 600-kDa derivative that retains growth factor binding activity increased RAW 264.7 cell NO synthesis and iNOS mRNA levels comparable to native α2M and α2M-MA. The purified 18-kDa α2M receptor-binding fragment had no effect on NO synthesis or iNOS expression. Thus, the growth factor-carrier activity of α2M and not its receptor-binding activity is essential for NO synthesis regulation. A TGF-β-neutralizing antibody mimicked the activity of α2M, increasing RAW 264.7 cell NO synthesis and decreasing cellular viability. These studies demonstrate that α2M can regulate macrophage NO synthesis and profoundly affect cellular function without gaining entry into the cell and without binding specific plasma membrane receptors.