[Discovery of immature thymocyte proliferation factor].

An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of N-9F at 10(-12)-10(-9)M in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative, single positive and double positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF neutralized the increasing effect of NPF on immature thymocytes. All effects of NPF was inhibited by mAb QR6.6 to recognize a 100 kDa surface molecule of N-9F. The amino-terminal 20 amino acid sequence of NPF was identified and identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A Hitrap-mouse recombinant His-tag-saposin A antibody column bound NPF, pulled down the NPF activity in TCGF, and the antibody recognized a 16kDa molecule in western-blotting of TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes. The physiological significance of NPF in the growth and differentiation of immature thymocytes was discussed in view of the characteristic distributions of NPF and the molecule recognized by its mAb QR6.6 in fetal thymi.

Aminopeptidase N/CD13 regulates the fetal liver microenvironment of hematopoiesis.

Fetal liver (FL) hematopoiesis is thought to be important for expanding the cell number during ontogeny. In order to investigate the cellular interaction molecules among FL stromal and hematopoietic cells, we established a monoclonal antibody, Ndk-10, that reacts with FL stromal cells but not with dish non-adherent cells. When Ndk-10 was added to an FL stromal and hematopoietic cell-coculture, it inhibited the survival of c-kit+ cells. The inhibitory activity of Ndk-10 was also observed in the fetal liver organ culture. The Ndk-10 recognized a 150 kD molecule in the adherent cells of FL and kidney, and the N-terminal amino acid sequence was identical to that of mouse aminopeptidase N/CD13. The peptidase activity of CD13 was inhibited by Ndk-10, and addition of its specific inhibitor resulted in the same inhibitory activity as Ndk-10. We propose that aminopeptidase N/CD13 is a critical molecule that regulates the survival of c-kit+ cells in the FL microenvironment.

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody.

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.

Role of gamma delta T cells in the inflammatory response of experimental colitis mice.

We examined the severity of experimental colitis induced by dextran sulfate sodium (DSS) using immunologically manipulated mice. C57BL/6 mice showed more severe colitis than BALB/c mice, but mice of both strains recovered fully from the disease after the removal of DSS from their drinking water. The infiltrated cells at the lesions were mainly granulocytes in normal littermates. However, C.B-17 scid, IL-7Ralpha deficient, and TCR-Cbetadelta double-deficient mice showed severe colitis and did not recover from the disease even after the removal of DSS. It was found that the infiltrated cells at the lesions in the lethal strains were monocytes. Although both TCR-Cdelta(-/-) and TCR-Cbeta(-/-) mice showed severe colitis phenotypes, infiltration in the former is monocyte-dominant while that in the latter is granulocyte-dominant. Thus the type of cells that infiltrate at the lesions of DSS-induced experimental colitis may be controlled by functional T cell subsets. Immunohistological and RT-PCR analyses of the inflamed colon revealed that the murine homologue of human GROalpha released by some cells under the control of gammadeltaT cells is a possible candidate determining the severity of DSS-induced experimental colitis.

Isolation of proliferation factor of immature T-cell clone in concanavalin A-stimulated splenocyte culture supernatant.

An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of NPF at 10-11-10-8 g/ml in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative thymocytes and CD8 single positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF decreased double negative thymocytes. The amino-terminal amino acid sequence of NPF was identified to be Ser-Leu-Pro-Cys-Asp-Ile-Cys-Lys-Thr-Val-Val-Thr-Glu-Ala-Cys-Asn-Leu-Leu-Lys-Asp- and was identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A rabbit anti-mouse recombinant His-tag (mrH)-saposin A antibody recognized a 16000 MW molecule in TCGF. A Hitrap-saposin A antibody column bound NPF and pulled down the NPF activity in TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes.