Mikio Kuraya

L-Ficolin Specifically Binds to Lipoteichoic Acid, a Cell Wall Constituent of Gram-Positive Bacteria, and Activates the Lectin Pathway of Complement

The lectin pathway of complement is activated when a carbohydrate recognition complex and associated serine proteases binds to the surface of a pathogen. Three recognition subcomponents have been shown to form active initiation complexes: mannan-binding lectin (MBL), L-ficolin, and H-ficolin. The importance of MBL in antimicrobial host defense is well recognized, but the role of the ficolins remains largely undefined. This report shows that L-ficolin specifically binds to lipoteichoic acid (LTA), a cell wall component found in all Gram-positive bacteria. Immobilized LTA from Staphylococcus aureus binds L-ficolin complexes from sera, and these complexes initiate lectin pathway-dependent C4 turnover. C4 activation correlates with serum L-ficolin concentration, but not with serum MBL levels. L-ficolin binding and corresponding levels of C4 turnover were observed on LTA purified from other clinically important bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. None of the LTA preparations bound MBL, H-ficolin, or the classical pathway recognition molecule, C1q.

Activation of the Lectin Complement Pathway by H-Ficolin (Hakata Antigen)

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.

Analysis of C5a receptor by monoclonal antibody

We prepared a mouse monoclonal antibody (mAb), termed 4C8, to the human C5a receptor (C5aR, CD88) by fusing spleen cells from mice immunized with mouse Ltk- cells transfected with cDNA of human C5aR (Ltk-/C5aR) to the mouse myeloma cell line P3U1. This mAb belonging to the IgM kappa subclass, detected a 43 kDa band on cell lysates of Ltk-/C5aR by immunoblotting analysis. Flow cytometry revealed that 4C8 specifically bound to Ltk-/C5aR, suggesting that this antibody is specific for C5aR. Furthermore, 4C8 was found to partially block both intracellular Ca2+ increase in PMN stimulated by C5a and 125I-C5a binding to C5aR on PMN. When cross-linked by anti-mouse IgM, 4C8 completely inhibited the binding of C5a to C5aR on PMN and Ltk-/C5aR. Therefore, it seems likely that this mAb does not recognize the C5aR active site but sterically inhibits the binding of C5a to its receptor. Using this mAb, we detected a 50 kDa band of C5aR on cell lysates of PMN, monocytes and platelets by immunoblotting. C5aR was expressed on PMN and monocytes as determined by flow cytometry, whereas it was not demonstrated on the surface of platelets. Based on these results, this mAb should be useful for analysis of C5aR expression in various immunological conditions and inflammatory diseases.

Changes in mRNA expression patterns for cytokines in blood leukocytes of a rat tourniquet model

We examined changes in mRNA expression patterns for proinflammatory cytokines and growth factors in blood samples after application of a tourniquet to the rat hind limb. Slight upregulations of interferon (IFN)-gamma, macrophage colony-stimulating factor (M-CSF) and transforming growth factor (TGF)-beta1 mRNA began at 2h after tourniquet application and were short-lived. The levels of activating transcription factor (ATF)-3, a stress-inducible gene, had increased at 1h after tourniquet application. No significant expression of interleukin (IL)-6 mRNA was observed in most samples. There were no significant temporal changes in the levels of IL-1beta, cardiotrophin (CT)-1 mRNA compared to the control levels, but, downregulation of gp130, a receptor of the IL-6 family, began at 1h after tourniquet application. Nerve growth factor (NGF) mRNA gradually increased and reached a significantly high level at 4h after application of the tourniquet. Gene expression induction in blood leukocytes occurred soon after application of the tourniquet and was short-lived. The transient mRNA expressions probably trigger secondary events that may be beneficial to wound repair and regeneration.

Semen specific γ-glutamyltransferase carries ABH antigens: a sandwich ELISA for simultaneous semen detection and its ABO blood typing

Semen type of gamma-glutamyltransferase (gamma-GTP) is different from the membrane bound type of the enzyme in both biochemical and immunological properties, and consists of two subunits (150 and 95 kDa). We found that anti-ABH antibodies recognize a 150-kDa subunit of seminal gamma-GTP by Western blot and immunoprecipitation analyses. Using SG2, one of anti-semen specific gamma-GTP monoclonal antibodies which we had produced, and anti-ABH antibodies, we established a sandwich ELISA for identifying human seminal gamma-GTP and its ABO type simultaneously. This sandwich ELISA allows ABO typing of highly diluted semen. The dilutions for ABO typing were 10(5) times for A or O, and 10(4) times for B. Furthermore, ABO typing of semen was successfully performed by this ELISA, even in the mixed presence of vaginal fluid, saliva and blood. Thus, seminal gamma-GTP carries ABH antigens and the sandwich ELISA with SG2 and anti-ABH antibodies enables ABO typing of semen. The sandwich ELISA is extremely useful for ABO typing originated from semen in the mixture of biological fluids.

C3d and Epstein-Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV-I Line, MT-2

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT‐2 cells. First, we confirmed CR2 expression on MT‐2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS‐PAGE analysis. When MT‐2 lysates were incubated with anti‐CR2 mAb HB5 and thereafter with 32P‐labeled ATP, 52‐ and 74‐kDa proteins were phosphorylated, suggesting the activation of MT‐2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT‐2 cells when incubated with C3d or EBV. The cytotoxicity of the MT‐2 supernatant against L929 cells was elevated in a dose‐ and time‐dependent manner. Next, we confirmed EBNA expression on EBV‐infected MT‐2 cells and attempted to establish an EBV‐positive MT‐2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT‐2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT‐2 cells 3 days after EBV treatment gave a 3.0‐kb signal which comigrated with the EBV BamHI‐W probe. The 3.0‐kb signal of genomic EBV‐DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2‐positive MT‐2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT‐2 cells might be transient.