Masaharu Ohbayashi

Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.

We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5 – 1.5 day newborns of lines 304 and 192 and on 7.0 – 7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma.

The ret oncogene can induce melanogenesis and melanocyte development in WvWv mice

We recently reported the establishment of transgenic mouse lines carrying the mouse metallothionein/ret fusion gene in which severe melanosis and melanocytic tumors developed. In the present study, we demonstrate that a significant number of pigmented hairs developed in Wv/Wv mice crossed to one of the transgenic mouse lines. The pigmented hair of Wv/Wv mice carrying the ret oncogene did not lose color during aging and reappeared after shaving, indicating that the melanocytes in the hair follicle function. The melanocytic tumors also developed in these mice, although the incidence was lower than that in the wild transgenic mice. Furthermore, the neutral tube culture of mouse embryos indicated that neural crest cells of the transgenic mice gave rise to a cell population that autonomously produced melanin even in the absence of melanocyte stimulating hormone. These results strongly suggested that the introduced ret oncogene could compensate for the defect of c-kit in Wv mice during both embryogenesis and postnatal life and induce a high level of melanin synthesis in the process of melanocyte development.

Immunohistochemical characteristics of chicken spleen ellipsoids using newly established monoclonal antibodies

Ellipsoids, the extra-vasculature sites surrounding penicilliary capillaries of the chicken spleen, play critical roles in the immune response and also in the clearance of pathogens or other particles. The meshwork of ellipsoids is formed by fibroblastic reticular cells. To characterize ellipsoidal reticular cells, a series of monoclonal antibodies against the chicken spleen have been developed. Of these antibodies, CSA-1 antibody reacts with fibroblastic reticular cells in ellipsoids and with endothelial cells. The reticular nature of positive cells in ellipsoids is indicated by immuno-electron microscopy, and by double-staining with anti-heat-shock protein 47 kDa (hsp47) antibody. The reaction of CSA-1 with reticular cells is limited in ellipsoids; CSA-1 does not react with reticular cells in other lymphoid organs. These findings indicate that ellipsoidal reticular cells share the antigen with endothelial cells. Ontogenic studies reveal that, on embryonic day 18, the development of ellipsoids is completed, penicilliary capillaries become fenestrated, and CSA-1 expression in ellipsoids begins. These findings suggest that CSA-1 is expressed on the cell surface of ellipsoidal reticular cells once they are exposed to blood flow.

Monoclonal antibodies against mouse splenic stromal cells.

A panel of rat monoclonal antibodies directed against mouse splenic stromal cells were isolated. These monoclonal antibodies were Immunohistochemically divided into four groups which reacted with non‐lymphoid cells of the murine spleen; (1) in the white pulp, (11) at the marginal zone, (111) in the red pulp, and (IV) on the endothelium of splenic blood vessels. These monoclonal antibodies were studied Immunohistochemically In lymphoid organs by means of light and electron microscopy. Monoclonal antibodies SS‐4 (group I) reacted with fibroblastic reticulum cells that were distributed only in the white pulp of the spleen and In the follicular areas of lymph nodes. The SS‐4 staining cell, In clustered splenic stromal cells, formed colonies which Included a small number of Thy‐1 positive lymphocytes. Therefore, we concluded that SS4 staining stromal cells comprise the lymphoid cornpartment. In contrast, monoclonal antibodies SS‐1, SS‐3 and SS‐5 (group II) reacted with dendritic shaped cells in the marginal zone of the spleen. Examination of splenic extra‐medullary hematopolesis in mice rescued by bone marrow transplantation after lethal irradiation revealed that SS‐3 and SS‐5 reacted with dendritic shaped stromal cells in clonal nodules of engrafted marrow in the red pulp. SS‐3 and SS‐5 staining cells could not be observed in physiologic hematopoiesis of non‐transplanted mice. It was suggested that SS3 and SS‐5 staining stromal cells are Involved in primitive hematopoiesls. Monoclonal antibodies SS2, SS‐6 and SS‐7 (group 111) mainly reacted with dendritic cells and macro‐phages in the red pulp. Monoclonal antibodies SS‐8 and SS‐9 (group IV) reacted with endothelial cells of blood vessels and sinuses. These findings of heterogeneity in mouse splenic stromal cells are further evidence that specific micro‐envlronments are composed by speclalired stromal cells.

Kinetics of monocytoid B lymphocytes in localized inflammatory lesions induced by lipopolysaccharide in mice

Immunohistochemistry was used to study the kinetics of B lymphocytes (B‐lys) In the early stages of the localized inflammatory response Induced in SMA mice by the subcutaneous injection of lipopolysaccharide (LPS). At the injection sites, medium‐sized B‐lys formed early inflammatory lesions with neutrophils and activated macrophages on days 1 and 2. The B‐lys were morphologically similar to monocytes, but were not stained with Mac1 antibody. Remarkably the B‐lys showed the phenotypes of B220+, lgM+, IgD (slight to negative), Ly‐1‐ and CD23‐ by double immunohistochemi‐cal staining. The B‐lys were also positive for alkaline phosphatase. Consequently the B‐lys could be identified as monocytoid B‐lys or marginal zone B‐lys. Plasmacytic B‐lys and plasma cells were first observed on days 3 and 4, but no lymphoid follicles were found at the injection sites. In the inguinal lymph nodes, the same B‐lys responses were mainly induced in the paracortical lesions (T cell areas) preceding the formation of activated germinal centers (GC). These findings suggested that the B‐lys, induced by injections of LPS, matured into plasma cells in the localized inflammatory lesions independent of GC, and that they were different from follicular B‐lys.

Immunohistochemical and functional studies on rat high endothelial venules using monoclonal antibodies

SummaryThe distribution of high endothelial venules (HEVs) in various rat organs has been investigated immunohistochemically using two monoclonal antibodies (MoAbs), REC16-11 and REC4-1. The staining patterns observed with REC16-11 and REC4-1 were compared with those obtained with the MoAb anti-rat ICAM-1, which is a cell adhesion molecule in HEVs. REC16-11 reacted not only with HEVs but also with all vascular endothelial cells (ECs) in the tissues examined. Electronmicroscopy showed that REC16-11 and REC4-1 reacted with the cell surface antigens of ECs and imniunoblot analysis of rat splenic stromal preparations showed that REC4-1 stained 42 kd and 60 kd bands. REC4-1 inhibited the binding of lymphocyte to HEVs in the mucosaassociated lymphoid tissues (MALT), but had no effects on lymphocyte binding to HEVs in peripheral (non-mucosal) lymph nodes. These findings suggested that the MoAb REC4-1 recognized the associated antigen of the lymphocyte-HEV recognition system in MALT.

Mucosa-associated Lymphoid Tissue Lymphoma(MALToma).

The distinctive low-grade B-cell mucosa-associated lymphoid tissue lymphoma (MALToma) of the extranodal lymphoid tissue has been well characterized in recent years. One hundred cases of gastrectomized primary gastric lymphomas, 30 cases of surgically resected primary intestinal lymphomas and five cases of autopsied pulmonary lymphomas were examined clinicopathologically. Fortyone cases of gastric lymphomas, nine cases of intestinal lymphomas and four cases of pulmonary lymphomas were classified as MALToma by using Isaacsons classification. Morphological feature of MALToma was lymphoepithelial lesion. All of gastric MALTomas were found out in advanced stage. Pulmonary lymphoma cases were not diagnosed correctly during life. Metastasis in gastrointestinal tract and serous membrane were seen in four cases of pulmonary MALToma. Tumor cells of MALTomas share an identical phenotype with splenic marginal zone B-cells. We examined the experimental gastric ulcer of rat by using immunohistochemistry. Centrocyte-like cells foci were observed in the ulcer bed, which express the phenotypical characters as same as splenic marginal zone B-cells. We discussed the cytopathological features of MALToma from the viewpoint of B-cell lineages.

Generation of monoclonal antibodies against murine bone marrow stromal cells.

We reported new monoclonal antibodies (moAb), MBS1-3, that raised against long term cultured mouse bone marrow stromal cells (MBSC). They were obtained from a hybridoma prepared by fusion of NS-1 mouse myeloma cells with splenic cells of rat immunized with MBSC. MoAb MBS1 (Ig G2a) recognized the cellsurface determinant mMBS1 (M.W. 10kD) on so-called blanket cells in MBSC. Immunohistochemical analysis showed that moAb MBS1 reacted with dendritic cells in red pulp cord and marginal inner zone of spleen and in perisinusoidal space of liver. We examined bone marrow, spleen and liver which were obtained from syngenic bone marrow transplanted mice (BMT mouse) after lethal irradiation and phenylhydrazine induced hemolytic anemia mice. Dendritic cells which beared MBS1 distributed in the erythroid developing colony in red pulp of spleen, bone marrow and liver of BMT mice and anemic mice. It was suggested that the dendritic cells were highly concerned with erythrocytes proliferation. We also examined the MBSC under some kinds of cytokines stimulation. The number of moAb MBS1 positive cells were increased under IL-1, IL-3 or GM-CSF stimulation. Other two moAbs, moAb MBS2 (Ig G1) reacted with fibrobalstic reticulum cells, moAb MBS3 (Ig G1) reacted with endoth-elial cells and macrophages in MBSC. Stromal cells which beared these two moAbs were little related with hematopoiesis by the examination of BMT or anemic mice.