SUMMARYThere are physiological variations in the levels of leucocytes. Among these, the circadian rhythm is very important in terms of the magnitude. Since newly identified lymphocyte subsets (i.e. extrathymic T cells) have recently been detected, a comprehensive study of the circadian rhythm was conducted. All leucocytes were found to vary in number or proportion with a circadian rhythm and were classified into two groups. One group-granulocytes, macrophages, natural killer (NK) cells, extrathymic T cells, gd T cells, and CD8 þ subset-showed an increase in the daytime (i.e. daytime rhythm). The other group-T cells, B cells, ab T cells, and CD4 þ subset-showed an increase at night. Humans are active and show sympathetic nerve dominance in the daytime. Interestingly, granulocytes and lymphocyte subsets with the daytime rhythm were found to carry a high density of adrenergic receptors. On the other hand, lymphocyte subsets with the night rhythm carried a high proportion of cholinergic receptors. Reflecting this situation, exercise prominently increased the number of cells with the daytime rhythm. These results suggest that the levels of leucocytes may be under the regulation of the autonomic nervous system.

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T cells expressing high levels of the T cell receptor (TCRhigh) differentiate in the major intrathymic pathway and then distribute to the peripheral immune organs, whereas T cells expressing intermediate levels of the TCR (TCRint) differentiate in both extrathymic pathways and an alternative intrathymic pathway and localize in unique sites, including the liver and thymic medulla. Since TCRint cells constitutively express interleukin-2 receptor beta-chain (IL-2R beta), two-color staining for CD3 (or TCR) and IL-2R beta clearly distinguished IL-2R beta+ CD3int (or TCRint) cells from IL-2R beta-, CD3high cells. CD3int cells may be considered to be primordial T cells based on their phenotype, morphology and other functional properties. In this study, using anti-V beta mAb in conjunction with the endogenous superantigen Mls, the distribution of self-reactive clones among T cells generated in all of the above pathways was investigated in mice. Self-reactive T cell clones were confined to IL-2R beta+, CD3int cells, in all of the organs tested. A significant proportion of self-reactive clones was never identified among CD3high cells in the thymus and peripheral immune organs in either young (8 week old) or old (50 week old) mice. Possibly reflecting their self-reactivity, CD3int cells, but neither NK cells nor CD3high cells had a potent cytotoxic effect against a syngeneic hepatoma in the presence of anti-CD3 mAb. These results raise the possibility that CD3int cells seen in the liver and thymus might belong to a similar primordial lineage of T cells, and that self-reactive clones are not generated through the major intrathymic pathway, but only through extrathymic pathways and an alternative intrathymic pathway.

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SUMMARYSelf-reactive clones, estimated by anti-Vb monoclonal antibodies (mAb) in conjunction with the Mls system, are confined to a population of intermediate (int) T-cell receptor ( TCR) (or CD3) cells (i.e. TCRint cells), but are not found among TCRhigh cells. The next questions to be answered are whether autologous killing is confined to TCRint cells and how such killing is mediated. In this study, 51Cr-labelled thymocytes of syngeneic or allogeneic origin were used as target cells (4-hr assay). When liver and splenic mononuclear cells (MNC ) obtained from B6 mice were used as eÂector cells, prominent autologous killing was seen in liver MNC, but not splenic MNC. Such killing was not seen when thymocytes from B6-lpr/lpr mice (i.e. Fas−) were used as target cells, nor when liver MNC from MRL-gld/gld mice (i.e. Fas ligand−) were used as eÂector cells (target thymocytes of MRL-+/+ mice). Cell separation experiments using a cell sorter revealed that autologous killing was mediated for the most part by CD3int cells, while allogeneic killing was mediated entirely by natural killer (NK ) cells, TCRint cells and TCRhigh cells. Among CD3int cells, the NK1.1+ subset (i.e. NK1.1+ T cells) manifested a higher level of autologous killing than did the NK1.1− subset. Consistent with the results of a functional assay, it was found by reverse-transcription-polymerase chain reaction ( RT-PCR) assay that CD3int cells among liver MNC showed the expression of Fas ligand mRNA, while thymocytes expressed Fas mRNA. When class I major histocompatibility complex (MHC )− thymocytes (from b 2 -microglobulindeficient mice) were used as target cells, NK cells, but not CD3int cells, showed potent cytotoxicity. These results suggest that autologous killing is a major function of TCRint cells with self-reactivity, and that such killing is mediated by means of Fas ligand/Fas molecules.

SUMMARYThe existence of nicotinic acetylcholine receptors (nAChR) on lymphocytes remains controversial. We attempted to show the existence of nAChR on murine lymphocytes. The intraperitoneal injection of nicotine induced the lymphocytosis in the spleen on day 3. Although freshly isolated lymphocytes bound small quantities of fluorescein isothiocyanate ( FITC )-conjugated a-bungarotoxin (aBuTx), they began to bind aBuTx after incubation in medium. In contrast to granulocytes, various lymphocyte subsets obtained from various lymphoid organs were found to bind aBuTx. Affinity purification of aBuTx-binding protein revealed that lymphocytes expressed the same nAChR molecules as those of muscle. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that lymphocytes expressed the a-subunit mRNA of nAChR. These results suggest that lymphocytes carry nAChR on the surface and are stimulated directly via their nAChR by parasympathetic nerve stimuli. INTRODUCTIONunder specific pathogen-free conditions in the animal facility of Niigata University (Niigata, Japan). Mice were injected The existence of nicotinic acetylcholine receptor (nAChR) on intraperitoneally with 20 mg nicotine (Sigma, St Louis, lymphocytes remains controversial. In some reports, lympho-MO)/mouse. cytes have been shown by binding assays and cell-proliferation assays to bear nAChR.1-3 Whether thymic componentsCell preparation express nAChR, in connection with the search for the aetiology Splenic mononuclear cells (MNC ) were purified by Ficollof myasthenia gravis (MG), has also been examined.4,5 Some Isopaque gradient (1·090) centrifugation. Thymocytes and of these experiments showed that thymic or peripheral lympholymph node cells were obtained by teasing the thymus and cytes express nAChR but others showed that only thymic inguinal lymph nodes, respectively, and passing through a epithelial or myoid cells express nAChR.6-8 The results are stainless steel mesh. Liver lymphocytes were obtained by therefore controversial and a more definite identification Percoll gradient centrifugation ( 35% Percoll containing of nAChR on lymphocytes remains to be achieved. 100 U/ml heparin).9 Peripheral blood cells were obtained from In this study, we attempted to show the existence of nAChR the buffy coat ( 2% dextran sedimentation for 40 min). The on murine lymphocytes. We show that lymphocytes contaminating erythrocytes were lysed by ammonium chloride carried nAChR on the surface and it is suggested that they buffer (0·83% NH 4 Cl-Tris buffer, pH 7·6). Macrophages were are stimulated directly via their nAChR by parasympathetic purified from splenic MNC by the plastic adherence method.9 nerve stimuli.Immunofluorescence test Lymphocytes, granulocytes and macrophages were morpho-MATERIALS AND METHODS logically identified in cell smears after staining with haematoxylin and eosin. Granulocytes and macrophages were also Mice and nicotine administration identified by immunofluorescence tests using monoclonal anti-C3H/He mice (aged 6-8 weeks) were used. All m...

SUMMARYIt has been reported that human CD161 (NKR-P1A) + T cells are counterparts of murine natural T (NT) cells and predominantly accumulate in the liver. However, NT cells in the human intestine have not been well analysed. The aim of this study was to assess the existence of NT cells in human intestinal epithelium and determine their phenotypical characterization. Intra-epithelial lymphocytes (IEL) were isolated from surgical specimens (jejunum, ileum and colon). The surface phenotype of IEL was analysed using a FACScan and compared with that of mononuclear cells (MNC) from other organs. CD161 + T cells were abundant in human intestinal epithelium as well as the liver. The majority of CD161 + T cells in IEL were CD8 + cells. About 50% of CD161 + T cells in hepatic lymphocytes (HL) expressed CD56, whereas only 14% of CD161 + T cells in IEL expressed CD56. The jejunum showed the greatest abundance of CD161 + T cells among the intestinal regions investigated. These results suggest that CD161 + T (NT) cells predominantly exist in human intestinal epithelium and may play an important role in local immunity.