Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: Role of proteases

Granulocytes undergoing apoptosis are recognized and removed by phagocytes before their lysis. The release of their formidable arsenal of proteases an d other toxic intracellular contents into tissues can create significant da mage, prolonging the inflammatory response. Binding and/or uptake of apopto tic cells by macrophages inhibits release of proinflammatory cytokines by m echanisms that involve anti-inflammatory mediators, including TGF-beta. To model the direct effects of necrotic cells on macrophage cytokine productio n, we added lysed or apoptotic neutrophils and lymphocytes to mouse and hum an macrophages in the absence of serum to avoid complement activation. The results confirmed the ability of lysed neutrophils, but not lymphocytes, to significantly stimulate production of macrophage-inflammatory protein 2 or IL-8, TNF-alpha, and IL-10. Concomitantly, induction of TGF-beta1 by lysed neutrophils was significantly lower than that observed for apoptotic cells . The addition of selected serine protease inhibitors and anti-human elasta se Ab markedly reduced the proinflammatory effects, the lysed neutrophils t hen behaving as an antiinflammatory stimulus similar to intact apoptotic ce lls. Separation of lysed neutrophils into membrane and soluble fractions sh owed that the neutrophil membranes behaved like apoptotic cells. Thus, the cytokine response seen when macrophages were exposed to lysed neutrophils w as largely due to liberated proteases. Therefore, we suggest that anti-infl ammatory signals can be given by PtdSer- containing cell membranes, whether from early apoptotic, late apoptotic, or lysed cells, but can be overcome by proteases liberated during Iysis. Therefore, the outcome of an inflammat ory reaction and the potential immunogenicity of Ags within the damaged cel l will be determined by which signals predominate.