Yasuyuki Imai

Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators : diaminofluoresceins

The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green‐fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.

Initial steps in lymph node metastasis formation in an experimental system: possible involvement of recognition by macrophage C-type lectins.

Abstract We used histological observations and experiments with fluorescent cell tracers to investigate the roles of tissue macrophages in recognition through a galactose/N-acetylgalactosamine-specific C-type lectin (mMGL) in lymph node metastasis formation by mouse ovarian tumor OV2944-HM-1 (HM-1) cells. Lymph node metastasis from subcutaneous sites was shown to be initiated by the entry of tumor cells into the subcapsular sinus of lymph nodes where mMGL-positive cells were mainly located. To investigate whether mMGL-positive cells contributed to host resistance against lymph node metastasis, we repeatedly treated mice bearing transplanted tumors with an mMGL-blocking monoclonal antibody that was known to inhibit mMGL binding to its ligands. The number of HM-1 cells recovered from lymph nodes 2 weeks after subcutaneous injections was significantly greater when the mice were treated with the blocking anti-mMGL antibody. These results suggested that mMGL-positive macrophages contributed to the hosts defense against lymph node metastasis.

Enhancement in accessibility to macrophages by modification of mucin‐type carbohydrate chains on a tumor cell line: role of a C‐type lectin of macrophages

We investigated the role of mucin‐type (O‐linked) carbohydrate chains of tumor target cells in the recognition by macrophages through a Gal/GalNAc‐specific calcium‐dependent lectin. Binding of a soluble form of this lectin to P815 mastocytoma cells was increased by treatment with benzyl‐GalNAc, which presumably inhibited the extension of mucin‐type carbohydrate chains. The levels of cell surface expression of GalNAc residues were elevated after benzyl‐GalNAc treatment, as revealed by the binding of Vicia villosa agglutinin B4 and Dolichos biflorus agglutinin. Adhesion of treated P815 cells to this lectin immobilized on plastic surfaces also increased. Furthermore, the binding of P815 cells to macrophage‐like RAW 264.7 cells and to peritoneal macrophages also increased after the same treatment. We concluded that elevated levels of cell surface terminal GalNAc in mucin‐type carbohydrate chains increased accessibility of P815 cells to macrophages through Gal/GalNAc‐specific calcium‐dependent lectins. J. Leukoc. Biol. 57: 407–414; 1995.

Tumor site-selective localization of an adoptively transferred T cell line expressing a macrophage lectin.

CTLL‐2 cells were transfected with an expression vector that contained cDNA of a mouse macrophage galactose/N‐acetylgalactosamine‐specific calcium‐type lectin (MMGL) and a stable transfectant (CTL‐ML) was established. These cells and mock transfectant cells (CTL‐CEP) were labeled with a long‐term fluorescent cell tracer, DiI. The labeled cells were intravenously injected into mice that contained established lung metastases produced by OV2944‐HM‐1 ovarian tumor cells. Analyses with fluorescence microscopy of a series of frozen lung sections from the recipient mice revealed that CTL‐ML preferentially accumulated in the lung metastatic nodules, whereas CTL‐CEP did not Time course studies showed that the preferential accumulation was evident 3 days after adoptive transfer. We also found that OV2944‐HM‐1 cells bound peanut agglutinin and Vicia villosaagglutinin B4, whose sugar specificity overlaps with the specificity of MMGL. These results suggested that MMGL molecules expressed on CTLL‐2 cells contributed to their selective trafficking or retention in tumor foci possibly through recognition of tumor‐associated cell surface carbohydrate antigens. These results also suggested that MMGL could be used for the selective targeting of effector cells in adoptive immunotherapy. J. Leukoc. Biol. 62: 761–770; 1997.

Quantitative measurement of carbohydrate binding activity of mouse macrophage lectin

A simple ELISA assay measuring lectin activity of a mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectin (MMGL) was developed. The binding of galactosylated poly-lysine (termed a ligand) to the immobilized soluble form of MMGL (rML) was measured quantitatively. Consistent with the characteristics of MMGL, the binding was calcium dependent and inhibited by galactose and N-acetylgalactosamine. An antiserum against rML inhibited the ligand binding, demonstrating the usefulness of this method for the screening of blocking antibodies. Using this assay, we found a significant interaction between MMGL and carrageenans, a group of sulfated polygalactans. The inhibitory effect of carrageenans was not attributable to a nonspecific interaction because other types of sulfated polysaccharides, such as glycosaminoglycans and fucoidin, did not interfere with the ligand binding. The relevance of the present finding to the biological activities of carrageenans is discussed.

Separation of mouse T cell subsets by a fluorescent activated cell sorter using fluorescence-labeled peanut agglutinin.

Nylon non-adherent spleen T cells obtained from concanavalin A-injected mice were labeled with fluorescein-labeled peanut agglutinin and separated into peanut agglutinin-positive (PNA+) cells and peanut agglutinin-negative (PNA-) cells by a fluorescent activated cell sorter. PNA+ cells were found to exert marked suppressive effect on primary anti-sheep red blood cells antibody response, but PNA- cells did not affect the antibody response. From these results and from the sugar-binding specificity of PNA, suppressor T cells are supposed to possess abundant galactosyl residues exposed on the cell surface, which are not masked by sialyl residues.

Fractionation of mouse cytotoxic T cells by use of lectins

Mouse cytotoxic T lymphocytes (CTLs), induced in vivo and in vitro in mixed-lymphocyte cultures, were fractionated into agglutinated and unagglutinated cells by use of various lectins. Cytolytic activity was enriched in the cell fraction agglutinated by various 2-acetamido-2-deoxy-alpha-D-galactopyranose-specific lectins, namely, Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), and Phaseolus limensis agglutinin (LBA). Only a little cytolytic activity remained in the unagglutinated cell fraction. Furthermore, the relationship between the binding of lectins and the cytolytic activity in various CTL cell lines was investigated with a fluorescence-activated cell sorter, and high-rank correlation was found between the cytolytic activity of these CTL cell-lines and the binding of DBA or HPA to them, but weaker rank correlation in the cases of LBA and peanut agglutinin (PNA). DBA was found to bind to almost all cells of interleukin-2-dependent CTL cell-lines tested. Although the DBA-positive CTL cell-lines have strong cytolytic activity, the DBA-negative cells, which consist of a small proportion of the CTLs, also exhibited cytolytic activity. Thus, the major part of CTLs has binding sites for alpha-D-GalNAc-specific lectins, particularly for DBA, and this lectin is useful for the enrichment of CTLs. However, the binding sites for DBA cannot be regarded as an exclusive differential marker for CTLs.

NEUROTROPIN INHIBITS EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE) IN LEWIS RATS

The effects of Neurotropin, a substance extracted from the inflammatory dermis of rabbits inoculated with Vaccinia virus, for experimental allergic encephalomyelitis (EAE) in Lewis rats, a model for human multiple sclerosis (MS), was studied. The peptide defined by residues 68-84 (MB 68-84) which corresponds to the encephalitogenic portion of the guinea pig myelin basic protein (MBP) in complete adjuvant H37Ra (CFA) was injected into the hind foot pad of each rat. Neurotropin significantly suppressed the clinical and histological expression of actively induced EAE when administered i.p. daily from day 0 to day 6 after immunization. In addition, passive EAE induced by precultured spleen cells from rats immunized with MB 68-84 in CFA was also suppressed by daily administration of Neurotropin after cell transfer. Neurotropin treatment significantly suppressed the delayed-type hypersensitivity (DTH) response to MB 68-84. Furthermore, the ability of spleen cells from Neurotropin-treated rats to transfer EAE was significantly lower than that of saline-treated rats. It seemed that the suppression may be due to the inhibition of the activation by MB 68-84 of sensitized spleen cells, as demonstrated by proliferative response to MB 68-84. However, no difference was observed in Con A-induced proliferative response of the spleen cells between Neurotropin- and saline-treated rats. These findings indicate that Neurotropin inhibits EAE by suppressing the immune responses to encephalitogenic MBP with little non-specific suppression.

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F1 (B/W F1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F1 mice as compared with young B/W F1 mice. Neurotropin, when administered i.p. to aged B/W F1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F1 mice. Neurotropin, administered to the aged B/W F1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory functions of aged B/W F1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F1 mice by the recovery of IL-2 production.

Immunohistochemical study on a macrophage calcium-type lectin in mouse embryos: transient expression in chondroblasts during endochondral ossification

We investigated expression of mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin (MMGL) in mouse embryos using a rat monoclonal antibody (mAb) LOM-14 that we previously developed. Immunoblot analysis revealed that a significant expression of MMGL was first detected in detergent extracts of whole embryos of 11 days post coitus (dpc) and the level of its expression increased during further fetal development (examined up to 18-dpc embryos). Tissue sections of 12, 14, 16, and 18-dpc embryos, newborn and adult mice were investigated by immunohistochemical staining. In embryos of 12-dpc and later stages, mesenchymal cells (typically distributed in the embryonic skin) exhibited positive signals for MMGL. Interestingly, a conspicuous staining was observed during endochondral ossification in temporary cartilage tissue, in which chondroblasts were transiently positive for MMGL. The staining intensity for the chondroblasts peaked in 14-dpc embryos and then gradually decreased. The staining was diminished while hypertrophy and maturation of chondrocytes proceeded, and was eliminated in areas with calcification. Immunoelectron microscopic study demonstrated the presence of MMGL in rough endoplasmic reticulum in the chondroblasts in the temporary cartilage tissue in 14-dpc embryos. These results provide first evidence showing the expression of MMGL in cells other than macrophages.