Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo

Abstract

Plasma procarboxypeptidase B (proCPB) is activated by the endothelial thrombin-thrombomodulin [corrected] complex. Activated proCPB [corrected] (CPB) functions as a fibrinolysis inhibitor, but it may play a broader role by inactivating inflammatory mediators. To test this hypothesis, C5a-induced alveolitis was studied in wild-type (WT) and proCPB-deficient mice (proCPB-/-). C5a-induced alveolitis, as measured by cell counts and total protein contents in bronchoalveolar lavage fluids, was markedly enhanced in the proCPB-/- mice. E229K thrombin, a thrombin mutant with minimal clotting activity but retaining its ability to activate protein C and proCPB, attenuated C5a-induced alveolitis in WT but not in proCPB-/- mice, indicating that its beneficial effect is mediated primarily by its activation of proCPB. Lung tissue histology confirmed these cellular inflammatory responses. Delayed administration of E229K thrombin after the C5a instillation was ineffective in reducing alveolitis in WT mice, suggesting that the beneficial effect of E229K thrombin is due to the direct inhibition of C5a by CPB. Our studies show that thrombin-activatable proCPB, in addition to its role in fibrinolysis, has intrinsic anti-inflammatory functions. Its activation, along with protein C, by the endothelial thrombin-TM complex represents a homeostatic response to counteract the inflammatory mediators generated at the site of vascular injury.

Figure 1

C5a-induced alveolitis in WT and proCPB^−/− mice. C5a at 2 concentrations or saline control (vehicle) were instilled endotracheally in WT (□) or proCPB^−/− (■) mice. Six hours later, BAL was performed, and total WBC counts (A) and total protein contents (B) in the BAL fluids were determined. (C) The wet-to-dry weight ratio of the right upper lobe was determined as described in “Materials and Methods” (n = 6 in each group; ** P < .05). Data are presented as mean ± SD.

Figure 2

E229K thrombin attenuated C5a-induced alveolitis in WT but not in proCPB^−/− mice. C5a (0.1 mg/mL) or saline control was instilled endotracheally in WT (□) or proCPB^−/− (■) mice. E229K thrombin or saline control was administered intravenously into the right jugular vein 5 minutes before the C5a instillation. Six hours later, BAL was performed and total WBC counts (A) and protein contents (B) in the BAL fluids were determined. (n = 6 in each group). Data are presented as mean ± SD. *P < .05; **P < .01.

Figure 3

C5a-induced alveolitis in WT and proCPB^−/− mice. Representative lung tissue samples of C5a-induced alveolitis from WT mice in the absence (A) or presence (B) of pretreatment with E229K thrombin and from proCPB^−/− mice in the absence (C) or presence (D) of pretreatment with E229K thrombin. Black arrows show macrophages. Images at 200× total magnification were visualized using a Zeiss Axiovert 40 CFL microscope equipped with an A-plan 20×/0.30 numerical aperture ph1 objective lens (Zeiss, Dublin, CA). Images were captured and rendered using a Zeiss AxioCam camera and AxioVision AC software.

Figure 4

Chemokine and cytokine levels in BAL fluids. Cytokine levels for IL-5, IL-6, TNFα, and MCP-1 were determined from BAL fluids clarified by centrifugation by flow cytometry using the BD mouse Th1/Th2 and BD mouse inflammation cytometric bead array (CBA) kits using the methods outlined by the manufacturers. The levels of mouse KC and MIP-3α were determined by sandwich ELISA according to the manufacturer's instructions. Data are presented as mean ± SD.

Figure 5

Delayed administration of E229K thrombin did not rescue C5a-induced alveolitis in WT mice. E229K thrombin or saline control was administered intravenously 2 hours after the initiation of C5a (0.10 mg/mL) induced alveolitis in this study (n = 6 in each group). Data are presented as mean ± SD.