Endogenous PTX3 is released by PMNs during conidia phagocytosis and contributes to internalization through the molecular mechanisms used by recombinant PTX3. (A) Kinetics of PTX3 release from human PMNs in the presence of 10% NHS and conidia. PTX3 levels were measured in the supernatants of 8 × 10⁶/mL PMNs at different time points of incubation (0, 30, 60, and 120 minutes) with conidia and 10% NHS or NHS alone. (B) PTX3 levels were measured in PMN supernatants upon 2 hours of incubation with or without conidia and different concentrations of NHS (0%, 3%, 10%). (C) FACS analysis of FITC-conidia phagocytosis by wild-type (Ptx3+/+) and Ptx3-deficient (−/−) bone marrow PMNs (CD45⁺, Ly6G^high, CD11b^high) in the presence of 10% NHS or HHS. When indicated, conidia were pre-opsonized with recombinant PTX3 (50 or 100 μg/mL). Experiments were performed in duplicate with PMNs collected from 4-10 Ptx3^+/+ and Ptx3^−/− mice. Data were normalized and expressed as percentage of the mean in wild-type PMNs. *P ≤ .05; **P < .01; ***P < .0001; Student unpaired t test. (C) Confocal analysis of total CD11b location in wild-type, Ptx3^−/−, and FcRγ-deficient (FcRγ^−/−) PMNs during phagocytosis of conidia eventually opsonised with recombinant PTX3. (Top) CD11b (red) and nucleus (DAPI). (Middle) CD11b (red) and Normarski. (Bottom) CD11b in pseudocolor scale. Two or 3 internalized conidia per cell are indicated with asterisks. Note that location of CD11b in the phagocytic cup is impaired in Ptx3^−/− and FcRγ^−/− PMNs and is rescued by recombinant PTX3 in Ptx3^−/− PMNs but not in FcRγ^−/− PMNs.