Michiyo Hatanaka

Regulation of complement-mediated swine endothelial cell lysis by a surface-bound form of human C4b binding protein.

BACKGROUND Human C4b-binding protein (C4bp) functions as a cofactor for factor I in the degradation of C4b and C3b and, in addition, accelerates the rate of decay of the C4b2a complex. METHODS In this study, we constructed a surface-bound form of human C4b-binding protein (C4bp-PI) consisting of a short consensus repeat 1-8 of the alpha-chain of C4bp and a glycosyl phosphatidylinositol (GPI) of the decay-accelerating factor (CD55) and established stable swine endothelial cell (SEC) lines expressing C4bp-PI by transfection of cDNA. Amelioration of complement-mediated lysis by the transfectant molecules was tested as an in vitro hyperacute rejection model of swine to human discordant xenograft, using the lactate dehydrogenase assay. RESULTS Flow cytometric profiles of the stable SEC lines with C4bp-PI showed a high level of expression of this molecule. The cell lysate of the SEC line with C4bp-PI showed strong cofactor activity in not only C4b but also C3b, whereas the activity of plasma C4bp to bind to C3 was very weak. Approximately 150 x 10(4) molecules of C4bp-PI per SEC blocked human complement-mediated cell lysis by approximately 75%. CONCLUSIONS The results suggest that the surface-bound form of C4bp will be very useful in clinical xenotransplantation.

High Expression of Membrane Cofactor Protein of Complement (CD46) in Human Leukaemia Cell Lines: Implication of an Alternatively Spliced Form Containing the STA Domain in CD46 Up‐Regulation

Human membrane cofactor protein (MCP, CD46) is a receptor for the measles virus and serves as a complement regulator which protects host cells from autologous complement attack. MCP is highly polymorphic due to a variety of mRNA splice products. The levels of MCP expression on T and myeloid cell lines are usually two‐eightfold higher than those on their normal counterparts, whereas Burkitts lymphoma B cell lines express less MCP than B cell lineages carrying no EB virus. The molecule has a Ser/Thr‐rich (ST) domain adjacent to the functional domain, namely short consensus repeats (SCR). The ST domain and a cytoplasmic tail (CYT) contribute to the MCP polymorphism. The ST domain is encoded by three exons (A, B and C) and major ST iscforms are STABC, STBC and STC. The authors investigated the relationship between the expression levels and isoform usage of MCP by flow cytometry using specific antibodies against STA and STC, by reverse transcriptase‐polymerase chain reaction (RT‐PCR) with size markers for each splice variant, and by RT‐PCR/Southern blotting using a specific probe for STA. The results were (1) the profiles of mean shifts of myeloid and T cell lines were STC < STA on flow cytometry while those of B cell lines and normal blood cells were STa < STC; (2) all cell lines tested by RT‐PCR expressed the messages for the isoforms STBC/CYTl, STC/CYTl, STBC/CYT2, and STC/CYT2. The band for STABC/CYT2 overlapped that for STC/CYTl, and the band for STABC/CYTl was marginal in all cell lines examined; (3) semi‐quantitative analysis of the STABC isoforms by Southern blotting indicated the presence of high levels of the STABC messages in myeloid and T‐cell lines in comparison with B lymphoid cells and normal leucocytes. Thus, the quantity of MCP expressed parallels the STABC message level, which is up‐regulated in T and myeloid leukaemia cell lines.

Distribution of C3-Step Regulatory Proteins of the Complement System, CD35 (CRl), CD46 (MCP), and CD55 (DAF), in Hematological Malignancies

The distribution and levels of three membrane proteins, CD35, CD46, and CD55, which serve as complement regulators, were examined in normal peripheral blood and hematologically malignant cells. CD35 was negative in most leukemia cells regardless of the type of leukemia, although granulocytes, monocytes, and some populations of lymphocytes were CD35+. CD46 was present in all blood cells except erythrocytes, and levels were 2-8 times higher in most leukemia cells than in their mature counterparts, particularly in CML and CLL cells, except for those of B cell lineage. CD55, a widely-distributed phosphatidyl inositol-anchored protein, was more frequently lost in NHL cells than in other types of hematological malignancies. In this review, we discuss the roles, mechanisms, and clinical applications of cell-associated complement regulatory proteins in hematological malignancies.

Involvement of GTP-regulatory protein in brain prostaglandin E2 receptor and separation of the two components

The specific binding protein for prostaglandin (PG) E2 solubilized from porcine brain was sensitive to guanine nucleotides. GTP inhibited the association and enhanced the dissociation of the specific [3H]PGE2 binding. Scatchard analyses showed that GTP (10 microM) decreased the binding affinity more than 3-fold without major change in the number of binding site. Gel filtration separated the binding site from GTP-regulatory component (N). The separated binding protein had a reduced affinity to PGE2 and lost its sensitivity to GTP. The addition of the separated N restored its responsiveness to GTP, and also increased the binding affinity to the original level. These results provide direct evidence for the molecular interaction between the PGE2 binding protein and N in the brain.

A monoclonal antibody against human decay-accelerating factor (DAF, CD55), D17, which lacks reactivity with semen-DAF☆

Human decay-accelerating factor (DAF, CD55) is a phosphatidyl inositol-anchored glycoprotein consisting, from the N-terminus, of 4 short consensus repeats (SCR), a Ser/Thr (ST)-rich region providing O-glycosylation sites, and the membrane-anchoring unit. A mAb, named D17, was raised against purified erythrocyte-DAF. This mAb recognized DAF on blood cells and most cell lines as determined by flow cytometry and immunoblotting. Its reactivity was similar to but weaker than that of two other well-characterized mAbs to DAF, IA10 (seeing an epitope within SCR1) and 1C6 (seeing an epitope within SCR3). The reactivity of D17 with erythrocyte DAF became increased by treatment with sialidase/O-glycanase, suggesting that its epitope is located close to the O-glycosylation sites, probably within the ST-rich region or SCR4. D17 barely blocked the decay-accelerating activity of DAF. Using the three mAbs, tissue-associated and soluble forms of DAF were identified by SDS-PAGE/immunoblotting and immunohistochemical staining. IA10 and 1C6 recognized a 50 kDa protein in spermatozoa lysate and two proteins of Mr 70 and 55 kDa, respectively, in seminal fluid. These represented membrane-associated and soluble forms of DAF, which were neither recognized by mAb against membrane cofactor protein (MCP, CD46) and C3b/C4b receptor (CR1, CD35) nor by non-immune IgG. In contrast to IA10 and 1C6, D17 did not recognize either spermatozoa-DAF or seminal plasma-DAF, or the deglycosylated or untreated forms of them. Immunohistochemical analysis showed that testis was stained with IA10 but not with D17.(ABSTRACT TRUNCATED AT 250 WORDS)

A monomeric human C4b-binding protein (C4bp) more efficiently inactivates C3b than natural C4bp : participation of C-terminal domains in factor I-cofactor activity

We designed a cDNA construct encoding an artificial membrane molecule consisting of all 8 short consensus repeats (SCRs) of human monomeric C4b-binding protein (C4bp) followed by DAFs GPI anchor, named mC4bp, and expressed the protein on swine endothelial cells (SEC). At the same level of expression, mC4bp protected host cells as effectively as DAF, the most potent complement (C) regulator on the membrane. This result was unexpected from the reported functional properties of natural multimeric C4bp. Here, we investigated the mechanism whereby mC4bp has potent cell-protective activity. Our results were as follows: (1) mC4bp serves more efficiently as a methylamine-treated C3 (C3ma)-inactivating factor I-cofactor than natural C4bp and as efficiently as MCP as a methylamine-treated (C4ma)-inactivating cofactor by fluid-phase cofactor assay: (2) the potency of C3ma inactivation by mC4bp and factor I is quite high compared to those of other cofactors: (3)blocking studies using mAbs against C4bp suggested that both the 48 kDa N-terminal fragment and the C-terminal domain near the portion responsible for bundle formation participate in the high C3ma-inactivating capacity of mC4bp. Thus, acquiring high C3ma-inactivating capacity secondary to monomeric alteration leads to high C regulatory activity of mC4bp. These results infer that mC4bp differs from C4bp in its potent factor I-cofactor activity and is a good candidate as a safeguard against hyperacute rejection of xenografts.

Test for ability of decay-accelerating factor (DAF, CD55) and CD59 to alleviate complement-mediated damage of xeno-erythrocytes.

We investigated the susceptibility to human complement (C) of xeno‐erythrocytes into which phosphatidylinositol (PI)‐anchored human C regulatory protein, decay‐accelerating factor (DAF) or CD59 had been incorporated. Erythrocytes of sheep (Esh), swine (Esw), dog (Edg), and guinea pig (Egp), unsensitized with human natural antibody (Ab), were used as xeno‐target. C‐mediated lysis of erythrocytes (E) was induced in both classical and alternative pathways in parallel with the density of the sensitized Ab, except for Egp. The efficacy of DAF/CD59‐mediated protection of the xeno E from human C, however, differed among these E species. In both classical and alternative pathways, Esh or Esw, which are non‐activator surfaces, were protected by the incorporated DAF or CD59, DAF being more effective than CD59. On the other hand, CD59 was more effective than DAF in both pathways in protection of Egp, which is an alternative pathway activator.

Acute promyelocytic leukemia with CD59 deficiency

CD59 is a phosphatidyl inositol-anchored protein (which is lost in paroxysmal nocturnal hemoglobinuria (PNH) cells) with the capacity to block the formation of membrane attack complex, and protects host cells from autologous complement-mediated cytolysis. We found a patient with acute promyelocytic leukemia (APL) accompanied by disseminated intravascular coagulation (DIC), the cells of which were CD59-negative. Although the CD59 deficiency in the malignant cells was not related to PNH, we offered the possibility that DIC was induced by APL lysis secondary to the deficiency of CD59.

C5b-8 Step Lysis of Swine Endothelial Cells by Human Complement and Functional Feature of Transfected CD59

The authors established several swine endothelial cell (SEC) lines expressing human CD59 by transfection of cDNA, and assessed the function of the transfectant molecules in comparison with those of membrane cofactor protein (MCP) and decay‐accelerating factor (DAF) in an in vitro hyperacute rejection model of swine to human discordant xenograft. At the usual expression rate, DAF and MCP protected SEC from human complement mediated cell lysis, but CD59 did not block human complement attack on SEC. However, CD59 protects SEC from cell lysis when sufficiently expressed as in human umbilical vein (HUVEC). The authors examined why CD59 needed so many molecules to protect human complement‐mediated SEC lysis and found that SEC underwent lysis by human C5b‐8. The degree of C5b‐8 step lysis of SEC was approximately 70% of the total activity (C5b‐9). Additionally, CD59 protected human complement activation less efficiently at the C5b‐8 step than at the C9‐step. Therefore, to overcome human complement mediated SEC lysis, C8 activity must be inhibited by dense expression of CD59.

Rapid purification of human complement receptor type 1 (CD35, CR1)

We established a rapid purification procedure for complement receptor type 1 (CR1, CD35). Human erythrocyte stromata were solubilized, and the extract was directly applied to a Red-Sepharose column. The eluate was diluted 2-fold, then subjected to an immunoaffinity column, anti-CR1 (named 31R)-conjugated to Sepharose. More than 50% of CR1 was recovered with purity of > 90%. The CR1 preparation showed sufficient cofactor activity for factor I as compared to that prepared by the conventional method. Since 31R recognizes a single epitope in CR1 including its rare variants and soluble forms, this method will allow us to recover with high efficiency these forms of CR1 which have been detected especially in some disease states.