Serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog

After i.p. infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guerin, macrophages recovered from the peritoneal cavity display classical signs of immune activation. We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guerin infection. Serpin 2a (sp12a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products. The cytokine IFN-gamma also induces sp12a expression in macrophages, and this induction is synergistic with bacterial products. We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to sp12a in activated macrophages. The ISG15-sp12a conjugates were identified by tandem mass spectrometry and contained sp12a conjugated to either one or two molecules of ISG15. Whereas sp12a was induced by either bacterial products or IFN-gamma, ISG15 was induced only by bacterial products. Although many protein targets have been described for ubiquitin conjugation, sp12a is the first ISG15-modified protein to be reported. Macrophage activation is accompanied by the activation of a variety of proteases. It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.