Itsuro Yoshida

Identification and Characterization of a Novel Human Collectin CL‐K1

Collectins are a family of C‐type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host‐defense. Here we report the cloning and characterization of a novel collectin CL‐K1. RT‐PCR analyses showed CL‐K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL‐K1 cDNA expressing CHO cells revealed that CL‐K1 is expressed as a secreted protein. CL‐K1 is found in blood by immunoblotting and partial amino acid analyses. CL‐K1 showed Ca2+‐dependent sugar binding activity of fucose and weakly mannose but not N‐acetyl‐galactosamine, N‐acetyl‐glucosamine, or maltose, though mannose‐binding lectin (MBL) containing similar amino acid motif. CL‐K1 can recognize specially several bacterial saccharides due to specific sugar‐binding character. Elucidation of the role of two ancestor collectins of CL‐K1 and CL‐L1 could lead to see the biological function of collectin family.

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Role of the UL25 gene product in packaging DNA into the herpes simplex virus capsid: location of UL25 product in the capsid and demonstration that it binds DNA.

ABSTRACT Recent studies have suggested that the herpes simplex type 1 (HSV-1) UL25 gene product, a minor capsid protein, is required for encapsidation but not cleavage of replicated viral DNA. This study set out to investigate the potential interactions of UL25 protein with other virus proteins and determine what properties it has for playing a role in DNA encapsidation. The UL25 protein is found in 42 ± 17 copies per B capsid and is present in both pentons and hexons. We introduced green fluorescent protein (GFP) as a fluorescent tag into the N terminus of UL25 protein to identify its location in HSV-1-infected cells and demonstrated the relocation of UL25 protein from the cytoplasm into the nucleus at the late stage of HSV-1 infection. To clarify the cause of this relocation, we analyzed the interactions of UL25 protein with other virus proteins. The UL25 protein associates with VP5 and VP19C of virus capsids, especially of the penton structures, and the association with VP19C causes its relocation into the nucleus. Gel mobility shift analysis shows that UL25 protein has the potential to bind DNA. Moreover, the amino-terminal one-third of the UL25 protein is particularly important in DNA binding and forms a homo-oligomer. In conclusion, the UL25 gene product forms a tight connection with the capsid being linked with VP5 and VP19C, and it may play a role in anchoring the genomic DNA.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells

Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Comparison of human blood concentrations of collectin kidney 1 and mannan-binding lectin.

Mannan-binding lectin (MBL) was first discovered as a collectin in animal blood, and was shown to have such unique characteristics as a collage-like domain and a carbohydrate recognition domain. We recently identified human collectin kidney 1 (CL-K1, COLEC11) from a human kidney cDNA library. To quantitate the CL-K1 concentration in blood, we developed several polyclonal and monoclonal antibodies using recombinant human CL-K1 in CHO cells and the CL-K1 fragment in Escherichia coli. Using these antibodies, we established a sandwich enzyme-linked immunosorbent assay (ELISA) system. The concentration of CL-K1 in human plasma was 0.34 ± 0.13 µg/ml and that in MBL was 1.72 ± 1.51 µg/ml. Concentrations of MBL are often low due to its single nucleotide polymorphisms (SNPs) which seem to be related to an opsonic defect. However, no low concentrations of CL-K1 were observed on testing over two hundred blood samples. We also found that the blood concentration of CL-K1 was not dependent on gender or age and did not correlate completely with that of MBL. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of CL-K1 in humans.

Immunolocalization of a Novel Collectin CL-K1 in Murine Tissues

We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central veins in liver, which suggests that murine CL-K1 may be mainly produced in the liver and secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase‐negative herpes simplex virus type 1 (HSV‐1; VRTK− strain) and type 2 (HSV‐2; UWTK− strain) was studied in comparison with that of their parental strains (VR‐3 and UW‐268, respectively) in an encephalitis model of adult (4‐week‐old) and newborn (3‐day‐old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV‐1, because the 50% lethal dose (LD50) of VRTK− was 60 times higher than that of VR‐3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK− strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK− strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV‐1 and HSV‐2 was confirmed with the one‐step growth of virus strains in L‐M and L‐M(TK−) cells.

Participation of Type I Interferon in the Decreased Virulence of the UL13 Gene-Deleted Mutant of Herpes Simplex Virus Type 1

We isolated a UL13 gene-deleted mutant of the herpes simplex virus type 1 (HSV-1) strain VR3 (VRDelta13) and its revertant virus (VRDelta13R). This deletion mutant still had virus host shutoff (vhs) activity, although a previous report had suggested the possibility of a functional relation between the UL13 product, that is protein kinase (PK), and vhs activity. We compared the in vivo growth of these viruses in BALB/c mice. VRDelta13 was cleared in the early period of intraperitoneal infection. VRDelta13 had a higher sensitivity to the mouse type I interferon (IFN) and showed a higher level of IFN induction in the study period of infection than did VR3 and VRDelta13R. These results suggest that a nonspecific antiviral response (i.e., the IFN system) may contribute to this rapid inhibition of viral replication in vivo.

Molecular cloning and functional analysis of scavenger receptor zebrafish CL-P1

BACKGROUND Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis. METHODS We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO). RESULTS Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6hours post-fertilization (hpf), reached its highest level at 24hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. CONCLUSIONS In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor. GENERAL SIGNIFICANCE These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.

Mechanisms of augmented resistance of cyclosporin A-treated mice to influenza virus infection by trehalose-6,6'-dimycolate

Cyclosporin A (CsA), which is an immunosuppressive drug of helper T lymphocytes, diminished a resistance of mice to influenza virus infection. Mice inoculated intravenously with trehalose‐6,6′‐dimycolate (TDM, a glycolipid component of the cell wall of Mycobacterium) in an oil‐in‐water emulsion (TDM emulsion) recovered the resistance to influenza virus infection impaired by CsA. Number of antibody‐producing cells was markedly reduced in CsA‐ and/or TDM‐treated mice. Interferon production in lung of TDM‐treated mice was augmented; however, it was extremely reduced not only in CsA‐treated mice, but also in CsA‐ and TDM‐treated mice. Activities of natural killer cells of CsA‐ and/or TDM‐treated mice were not different from that of control mice. Numbers of lymphocytes in lung of TDM‐treated mice and CsA‐ and TDM‐treated mice were more predominantly increased than that of control mice. Analysis of lung lymphocytes by flow cytometry revealed no difference between the populations of L3T4+ T lymphocytes and Lyt‐2.2+ T lymphocytes in CsA‐ and/or TDM‐treated mice and the populations in control mice. However, the population of γδ T cell receptor positive (γδ TCR+) lymphocytes increased markedly in lung of TDM‐treated mice and also CsA‐ and TDM‐treated mice. In vitro experiments showed that macrophage cultures treated with TDM emulsion released a mediator(s) which activates T lymphocytes, but not B lymphocytes. These and our earlier results suggest that the recovered anti‐influenza virus resistance of CsA‐treated mice by treatment with TDM emulsion was caused by elicitation of macrophages with TDM, then activation of T lymphocytes, especially γδ TCR+ lymphocytes.