Procarboxypeptidase R deficiency causes increased lethality in concanavalin A-induced hepatitis in female mice.

Carboxypeptidase R (CPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is an enzyme generated by proteolytic cleavage of its zymogen (proCPR). CPR removes the C-terminal arginine from inflammatory peptides such as C3a and C5a, bradykinin, enkephalin, and the thrombin-cleaved N-terminal fragment osteopontin (cleaved N-OPN). In the mouse model of concanavalin A (Con A)-induced immune-mediated fulminating hepatitis, cleaved N-OPN is one of the important peptides that induce the production of chemokines or cytokines. In the current study using proCPR deficient mice, we showed that injection of Con A into the mouse tail vein can induce a significantly higher lethality in proCPR-deficient female but not in male mice. Furthermore, a lack of CPR activity increased serum macrophage inflammatory protein-2 (MIP-2) and high-mobility group box 1 (HMGB1) levels after Con A injection. These in vivo findings suggest that CPR helps to protect against Con A-induced hepatitis.

Diagnostic surface expression of SWAP-70 on HIV-1 infected T cells.

Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.

Human IgM monoclonal antibodies reactive with HIV-1-infected cells generated using a trans-chromosome mouse.

The trans-chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock-out of its endogeneous genes coding for mu-and kappa-chains of immunoglobulin. One of these TC mice was immunized with HIV-1-infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV-1-infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 mug/ml of 9F11 was able to induce complement-mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of mu-and kappa-chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species-specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV-1-infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV-1-infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.

Hepatitis induced by an IgM monoclonal antibody against procarboxypeptidase R.

Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.

Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in lipopolysaccharide-primed mice.

Carboxypeptidase R (CPR) is a heat-labile enzyme found in serum in addition to stable carboxypeptidase N. CPR cleaves the C-terminal basic amino acids, arginine and lysine, from inflammatory peptides such as complement C3a and C5a, bradykinin, and enkephalin. This enzyme is generated from procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor, following cleavage by proteolytic enzymes such as thrombin, plasmin, and trypsin. We generated proCPR-deficient mice by knocking out exons 4 and 5 of the proCPR gene, which are regarded as essential for CPR function. At LPS challenge, there was virtually no difference in lethality among proCPR(+/+), proCPR(+/-), and proCPR(-/-) mice. However, challenge with cobra venom factor, which can activate and deplete almost all complement in vivo, induced a lethal effect on proCPR(-/-) mice following LPS sensitization which up-regulates C5a receptor expression. In contrast, proCPR(+/+) and proCPR(+/-) mice were able to tolerate the cobra venom factor challenge with the limited dose (30 U). Although carboxypeptidase N plays a role in inactivation of inflammatory peptides in vivo, CPR may also be important in the regulation of hyperinflammation.