Immunocytochemical localization of latent transforming growth factor- β1 activation by stimulated macrophages

Transforming growth factor-β1 (TGF-β) is secreted in a latent form consisting of mature TGF-β noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-β from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-β action. We have identified two events associated with latent TGF-β (LTGF-β) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-β concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-γ and lipopolysaccharide reportedly activate LTGF-β via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-β activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-β epitopes. The induction of TGF-β immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-β activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-β and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-β activation provides an important tool for studies of its regulation.