ALPHA(2)-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

Nitric oxide (NO) is an important mediator of macrophage activities. W e studied the regulation of macrophage NO synthesis by alpha(2)-macrog lobulin (alpha(2)M), a proteinase inhibitor and carrier of certain gro wth factors, including transforming growth factor-beta (TGF-beta). Nat ive alpha(2)M and the alpha(2)M receptor-recognized derivative, alpha( 2)M-methylamine (alpha(2)M-MA), increased nitrite generation by the RA W 264.7 murine macrophage cell line. The level of nitrite accumulation , which is an index of NO synthesis, was comparable to that observed w ith interferon-gamma. Native alpha(2)M and alpha(2)M-MA also increased inducible nitric oxide synthase (iNOS) mRNA levels and substantially reduced the number of viable cells, as determined by thylthiazol-2-yl) -2,5-diphenyltetrazolium/succinyl dehydrogenase assay or trypan blue e xclusion. At slightly higher alpha(2)M concentrations, [H-3]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 alpha(2)M and alpha(2)M-MA increased the percentage of cells with detectable si ngle strand chromatin nicks from 4 to 12 and 17%, respectively. This c hange suggested apoptosis; however, electron microscopy studies demons trated variability in the morphology of injured cells. To determine th e mechanism by which alpha(2)M increases macrophage NO synthesis, we s tudied proteolytic alpha(2)M derivatives that retain partial activity. A 600-kDa derivative that retains growth factor binding activity incr eased RAW 264.7 cell NO synthesis and iNOS mRNA levels comparable to n ative alpha(2)M and alpha(2)M-MA. The purified 18-kDa alpha(2)M recept or-binding fragment had no effect on NO synthesis or iNOS expression. Thus, the growth factor-carrier activity of alpha(2)M and not its rece ptor-binding activity is essential for NO synthesis regulation. A TGF- beta-neutralizing antibody mimicked the activity of alpha(2)M, increas ing RAW 264.7 cell NO synthesis and decreasing cellular viability. The se studies demonstrate that alpha(2)M can regulate macrophage NO synth esis and profoundly affect cellular function without gaining entry int o the cell and without binding specific plasma membrane receptors.