No abstract

Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm with dismal prognosis, and no optimal therapy has been developed. We tested the defucosylated chimeric anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, KM2760, to develop a novel immunotherapy for this refractory tumor. In the presence of peripheral blood mononuclear cells (PBMCs) from healthy adult donors, KM2760 induced CCR4-specific antibody-dependent cellular cytotoxicity (ADCC) against CCR4-positive ATLL cell lines and primary tumor cells obtained from ATLL patients. We next examined the KM2760-induced ADCC against primary ATLL cells in an autologous setting. Antibody-dependent cellular cytotoxicity mediated by autologous effector cells was generally lower than that mediated by allogeneic control effector cells. However, a robust ADCC activity was induced in some cases, which was comparable with that mediated by allogeneic effector cells. It suggests that the ATLL patients' PBMCs retain substantial ADCC-effector function, although the optimal conditions for maximal effect have not yet been determined. In addition, we also found a high expression of FoxP3 mRNA and protein, a hallmark of regulatory T cells, in ATLL cells, indicating the possibility that ATLL cells originated from regulatory T cells. KM2760 reduced FoxP3 mRNA expression in normal PBMCs along with CCR4 mRNA by lysis of CCR4 ؉ T cells in vitro. Our data suggest not only that the CCR4 molecule could be a suitable target for the novel antibody-based therapy for patients with ATLL but also that KM2760 may induce effective tumor immunity by reducing the number of regulatory T cells.

SUMMARYDecay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembraneanchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf 2). In this report, we describe a Daf1 gene knock-out mouse arising as the ®rst product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf 2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide¯uores-cence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identi®ed both GPI and transmembrane forms of Daf 2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.

Neuraminidase-treated human erythrocytes become sensitive to haemolysis by heterologous serum via activation of the alternative complement pathway (ACP), while remaining insensitive to homologous serum because of the presence of inhibitors on the cell membrane. We obtained a monoclonal antibody which renders the neuraminidase-treated erythrocytes sensitive to haemolysis by homologous human serum via the ACP. This antibody reacts with a 20 KDa membrane glycoprotein which interferes with the terminal stage of complement action on cell membranes. The 20 KDa protein is anchored to the membrane via phosphatidylinositol.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b‐9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b‐9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59− human B‐cells, which formed non‐selective ion channels with a total conductance of 360 ± 24 pS as measured at the beginning of the steady‐state phase of the inward currents. C5b‐8 and small‐size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59+ human B‐cells in spite of small‐size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59− cells inhibited the serum‐evoked inward current. The ion channels formed by the small‐size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b‐8 as well as that of small‐size C5b‐9. These results offer an explanation as to why CD59‐expressing cells are not leaky in spite of a buildup of homologous C5b‐8 and small‐size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3.4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.

The complement (C) regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), which control C3 convertases, together with CD59, an inhibitor of the membrane attack complex (MAC), were found to be present in the developing human placenta from at least 6 weeks of gestation until term. Immunostaining revealed differences in the distribution of these proteins on the fetally derived trophoblast epithelium, especially in early placentae which contain trophoblast populations of diverse proliferative potential and differentiation status. Expression of all three proteins occurred on the terminally differentiated syncytiotrophoblast epithelium covering chorionic villi and which is in direct contact with maternal blood. CD59 was also expressed on the underlying villous cytotrophoblast cells and on their extra-villous derivatives. These two populations showed differential expression of the C3 convertase regulators. Villous cytotrophoblast cells expressed MCP but were largely devoid of DAF. Proliferation of this population to generate extra-villous cytotrophoblast cell columns was associated with both an increase in DAF expression and a decrease in MCP expression. Throughout placental development, expression of DAF appeared to be lower than that of MCP and CD59 as assessed by solid-phase binding assays on isolated trophoblast membranes. Early placentae were also found to contain both DAF+ and DAF- chorionic villi. Conversely, expression of CD59 appeared comparatively high and transcripts for CD59 were found to be much more abundant than those for DAF in purified trophoblast cells. C regulatory proteins appear to play an important role throughout gestation in protecting the fetally derived human conceptus from maternal C. The differential expression patterns of the proteins on trophoblast may reflect differences in requirement for specific functional activities at different locations within the placenta.