AbstractUrokinase-type plasminogen activator (uPA) signaling via its receptor uPAR inhibits late events in HIV-1 replication in acutely infected primary monocyte-derived macrophages (MDMs) and promonocytic U937 cells. Here we show that U937-derived, chronically infected U1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) express integrins, uPA, and soluble uPAR at levels similar to those of MDMs. uPA inhibited HIV expression in U1 cells incubated with either PMA or tumor necrosis factor-α (TNF-α), but not with other HIV-inductive cytokines or lipopolysaccharide. Of interest, only PMA and TNF-α, but not other HIV-inductive stimuli, induced surface expression of the αM chain CD11b in U1 cells constitutively expressing CD18, the β2 chain of the Mac-1 integrin. Like uPA, fibrinogen, a Mac-1 (CD11b/CD18) ligand, and M25, a peptide homologous to a portion of the β-propeller region of CD11b preventing its association with uPAR, inhibited HIV virion release in PMA-stimulated U1 cells. Both uPAR small-interference RNA (siRNA) and soluble anti-β1/-β2 monoclonal antibodies abolished the anti-HIV effects of uPA, whereas CD11b siRNA reversed the anti-HIV effect of M25, but not that induced by uPA. Thus, either uPA/uPAR interaction, Mac-1 activation, or prevention of its association with uPAR triggers a signaling pathway leading to the inefficient release of HIV from monocytic cells.