Effect of PTX on the rhC5a-induced PAI-1 production in HMC-1 cells.
(A) HMC-1 cells were incubated for 120 minutes at 37°C in the absence or presence of PTX at a concentration of 0.5 μg/mL. Thereafter, cells were washed and incubated for 24 hours in the presence or absence of rhC5a at a concentration of 0.5μ M as described in “Materials and methods.” (B) Effect of a monoclonal anti-C5aR/CD88 antibody on the rhC5a-induced PAI-1 production in HMC-1 cells. HMC-1 cells were incubated for 60 minutes at 4°C in the absence or presence of anti-C5aR/CD88 mAbs at a concentration of 20 μg/mL. As a control, HMC-1 cells were incubated with mAbs against CD123 at the same concentration under the same conditions. Thereafter cells were washed and incubated for 24 hours in the presence or absence of rhC5a at a concentration of 0.5 μM, as described in “Materials and methods.” (C) Effect of plasmin treatment on the ability of rhC5a to stimulate PAI-1 production in HMC-1 cells. rhC5a was incubated with plasmin immobilized to Sepharose 4B for 60 minutes at 37°C before its addition to HMC-1 cells. As a control, rhC5a was incubated with BSA coupled to Sepharose 4b under the same conditions. Thereafter, such treated rhC5a was added to HMC-1 cells at a concentration of 0.5 μM, and the cells were incubated for 24 hours as described in “Materials and methods.” Conditioned media of such treated cells were collected, and total PAI-1 antigen was determined. Values are given in ng/106 cells per 24 hours and represent mean values ± SD of 3 independent determinations. Experiments were performed 3 times, and a representative experiment is shown. Total PAI-1 antigen was significantly decreased in conditioned media obtained from cells pretreated with PTX before the addition of rhC5a (C5a + PTX) compared with cells not pretreated with PTX (C5a; P < .001). Total PAI-1 antigen was significantly increased in cells not pretreated with PTX before the addition of rhC5a compared with control cells treated with neither PTX nor rhC5a (control;P < .001) (A). Total PAI-1 antigen was significantly decreased in conditioned media obtained from cells pretreated with anti-C5aR/CD88 antibodies before the addition of rhC5a (C5aR/CD88 mAB + C5a) compared with cells pretreated with anti-CD123 antibodies (CD123 mAB + C5a; P < .001) and to cells not pretreated with antibodies before the addition of rhC5a (C5a;P < .001). Total PAI-1 antigen was significantly increased in cells pretreated with anti-CD123 antibodies and in cells not pretreated with antibodies before the addition of rhC5a compared with control cells treated with neither antibodies nor rhC5a (control;P < .001) (B). Plasmin-treated rhC5a lost its activity to induce PAI-1 production in HMC-1 cells (P < .001 when compared with BSA-treated or -untreated rhC5a) (C).
