Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). We have shown that neutrophil recruitment to the lung during P. aeruginosa pneumonia is impaired in uPAR-deficient (uPAR-/-) mice but is normal in uPA-/- mice. However, both uPA-/- mice and uPAR-/- mice have impaired lung clearance of P. aeruginosa compared with wild-type (WT) mice. To determine the role of uPA and uPAR in antibacterial host defense, we compared neutrophil bacterial-phagocytosis, respiratory burst, and degranulation among uPA-/-, uPAR-/-, and WT mice. Neutrophil phagocytosis was significantly diminished comparing uPA-/- and uPAR-/- mice with WT mice at all time points. The generation of superoxide by both uPA-/- and uPAR-/- neutrophils was about half of that seen in WT neutrophils. Degranulation of azurophilic granules was significantly diminished in uPA-/- neutrophils compared with either uPAR-/- or WT neutrophils. By contrast, agonist-stimulated release of specific granules was not diminished in either uPA-/- or uPAR-/- mice compared with WT. We conclude that the uPA/uPAR system modulates several of the crucial steps in neutrophil activation that result in bacterial killing and effective innate host defense.

Signaling motifs located within the cytoplasmic domain of certain receptors contribute to lysosome fusion. Most studies have described lysosome fusion with respect to endocytic receptors. Phagolysosome fusion has not been extensively studied. To test the hypothesis that the tail of Fc␥RIIA participates in phagolysosomal fusion, a "reverse" genetic complementation system was used. It was previously shown that complement receptor type 3 (CR3) can rescue the phagocytic activity of a mutant Fc␥RIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fc␥ receptor-dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of Fc␥RIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type Fc␥RIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of Fc␥RIIA containing a full-length cytoplasmic tail with the 2 ITAM tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2) CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that observed with the wild-type receptor. In contrast, in the presence of CR3 and the tail-minus form of Fc␥RIIA, internalized particles did not colocalize with dextran. Electron microscopy revealed that the lysosomal enzyme acid phosphatase colocalized with immunoglobulin G-coated targets internalized by wild-type Fc␥RIIA but not by tail-minus Fc␥RIIA and CR3. Thus, the tail of Fc␥RIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence. IntroductionPhagocytosis is a crucial step toward the eventual destruction of foreign particles by the immune system. After separation from the plasma membrane, a phagosome must traffic to and fuse with lysosomes. Lysosomes contain a battery of hydrolytic enzymes within a low pH environment. 1 Endosome-to-lysosome recognition is mediated by signaling motifs located within the cytoplasmic domains of certain receptors. 2,3 Some investigators have suggested that the tyrosine-containing ITAM motif found within the cytoplasmic domain of receptors such as the ␥ chain of Fc␥ receptor I (Fc␥RI) and Fc␥RIII and the cytoplasmic domain of Fc␥RIIA is responsible for phagocytic signaling in antibodydependent phagocytosis. 4,5 It is possible that signal sequences located in the cytoplasmic tails of these receptors are responsible for mediating trafficking to lysosomes and the eventual fusion of the 2 organelles. However, when the cytoplasmic domain of Fc␥RIIA or its crucial tyrosine residues are removed, phagocytosis is abolished. 4,5 After the phagocytic activity of the receptor is lost, there is no mechanism to study downstream properties of the receptors such as lysosome fusion.Fc␥Rs are known to cooperate with complement receptors during immunoglobulin G (IgG)-dependent phagocytosis and oxidant production. 6-9 One mechanism of Fc␥-to-complement receptor cooperation involves physical associ...