Transient appearance of hepatic natural killer cells with high cytotoxicity and unique phenotype in very young mice.

There were few natural killer (NK) cells in the liver in very young mice at the age of 1-2 weeks. This was because the cell yield from the liver of young mice was low. The percentage of NK cells in the liver of young mice, however, was almost comparable with that in the liver of adult mice. Lymphocytes were isolated from the liver and spleen of C57BL/6 (B6) mice, and NK cytotoxicity and phenotype were herein examined in this study. NK cytotoxicity was extremely high in the liver of very young mice. This phenomenon was seen in the liver of various normal mouse strains. In contrast, the appearance of high cytotoxicity was not seen in NK cells of the spleen, irrespective of mouse strains. The quality of NK cells in the liver of young mice was different from that in adult mice. NK cells in the liver of young mice were mainly CD69(+)Mac-1(-) Fas ligand(+), whereas those in the liver of adult mice were CD69(-)Mac-1(+) Fas ligand(-). These results revealed that the quality of hepatic NK cells changes in the process of ageing. Namely, liver NK cells in very young mice temporarily show the highest NK cytotoxicity and a unique activated phenotype. Physiological meaning of the present phenomenon was discussed.

CD161+ T (NT) cells exist predominantly in human intestinal epithelium as well as in liver.

It 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.

An allogeneic microenvironment influences the phenotype of intermediate T-cell receptor cells expanding in MRL-lpr/lpr mice.

MRL-lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double-negative (DN) CD4- CD8- alpha beta T cells expressing a low density of interleukin-2 receptor beta-chain (IL-2R beta). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T-cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL-2R beta. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL-2R beta. A cell-sorting experiment for purified fractions demonstrated that only CD8- cells reconstituted TCRint cells in scid mice. Namely, DN CD4- CD8- cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.

Participation of NK1.1+ T cells in the rejection of lpr alphabetaT cells when bone marrow cells of lpr mice are transplanted into B6 mice.

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr-->B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3 IL-2Rbeta+ NK cells and IL-2Rbeta+ CD3int cells (i.e., CD3int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1+ and NK1.1-, among the IL-2Rbeta+ CD3int cells. However, the NK1.1+ subset (i.e., NK1.1- T cells) was much more radioresistant, and the majority of CD3int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1 mAb or anti-asialoGM1 antibody to eliminate NK cells alone or both NK cells and NK1.1+ T cells. When irradiated B6 mice were pretreated with anti-NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1+ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4 8 cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.

Calcitonin-producing pancreatic somatostatinoma: report of a case.

A 59-year-old woman was hospitalized due to a 1-year history of diarrhea and weight loss. Echography and computed tomography of the abdomen revealed a 10 x 7 cm solid mass in the tail of the pancreas and gallstones, while selective celiac angiography revealed the presence of a hypervascular mass. High levels of somatostatin and calcitonin were detected in the plasma, 70 pg/ml (normal range <28 pg/ml) and 5550 pg/ml (normal range 37 +/- 8 pg/ml), respectively. This tumor was thus removed by means of a distal pancreatectomy and a splenectomy. After the pancreatic tumor was removed, the elevated levels of plasma somatostatin and calcitonin returned to the normal ranges, and the persistent diarrhea also dramatically disappeared. A postoperative immunohistochemical study showed the tumor cells to be diffusely positive for somatostatin and calcitonin. These results clearly indicate this patient to be a case of calcitonin-producing pancreatic somatostatinoma.

Neonatal granulocytosis is a postpartum event which is seen in the liver as well as in the blood.

In a recent series of studies, we demonstrated that stress in humans and animals, with resultant sympathetic nerve strain, induces severe granulocytosis, because granulocytes carry adrenergic receptors on the surface. Because activated granulocytes produce free radicals and superoxides, they sometimes induce tissue damage if the stress is too strong or continuous. Human neonates are also known to show high levels of granulocytes in the peripheral blood. In this study, we investigated whether such neonatal granulocytosis are a stress-associated response at birth. Both human and mouse materials, before and after birth, were used. The number of leukocytes in the blood, as well as some other factors in the serum, were measured. Although levels of granulocytes were found to be low in fetal humans and mice, they increased sharply after birth. In parallel with this postpartal granulocytosis, transaminases in sera increased transiently. In reference to results of a transient elevation in the levels of catecholamines at birth in mice, all these phenomena resemble stress-associated responses. Indeed, fatty liver and hematopoietic destruction in the liver were also observed in mice and humans. At this time, the production of inducible nitric oxide synthase (iNOS) by granulocytes in the liver was evident. These results suggest that neonatal granulocytosis is a postpartum event which results from various stresses (e.g., oxygen stress) at birth. This event may be responsible for such well-known neonatal phenomena as the termination of fetal hematopoiesis in the liver and as neonatal jaundice.

Circadian rhythm of leucocytes and lymphocytes subsets and its possible correlation with the function of the autonomic nervous system.

There 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, gammadelta T cells, and CD8+ subset--showed an increase in the daytime (i.e. daytime rhythm). The other group--T cells, B cells, alphabeta T cells, and CD4+ 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.

[Adult bochdalek hernia after playing at a tug of war].

A 38-year-old female was admitted to Shonai Hospital with severe abdominal pain and nausea after playing at a tug of war in the athletic meeting. The X-ray film showed air above the left diaphragm, and CT scan and barium enema revealed the incarcerated transverse colon to the left thoracic cavity. Operation was performed through a thoracotomy. Because of no evidence of trauma, the case was diagnosed as adult Bochdalek hernia. Repair could be done by direct suture and her postoperative course was uneventful.

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice.

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.

Mouse liver T cells: their change with aging and in comparison with peripheral T cells.

Mouse liver contains both IL-2Rbeta- (or low positive) high T-cell receptor (TCR(hi)) cells and IL-2Rbeta+ intermediate TCR (TCR(int)) cells. TCR(int) cells consist of natural killer 1.1 (NK1)+ and NK1- subsets. NK1- TCR(int) cells increase constantly with age whereas TCR(hi) cells decrease. NK1+ TCR(int) cell proportions in the liver increase until middle age and decrease thereafter. Although NK1+ TCR(int) cells in other organs are few regardless of age, NK1- TCR(int) cells gradually appear in other lymphoid organs with aging. Skewed usage of Vbeta7 and Vbeta8 TCR was observed in NK1+ TCR(int) cells in the liver but the predominance was less obvious in NK1- TCR(int) and TCR(hi) cells in the liver and other organs. TCR V alpha14 messenger RNA (mRNA) was detected in NK1+ TCR(int) cells but not in the other two populations. In contrast, although NK1+ TCR(int) cells contain virtually no V alpha11+ T cells, NK1- TCR(int) cells contain a much higher proportion (approximately 12%) of V alpha11+ T cells, whereas approximately 4% of TCR(hi) cells are V alpha11+. NK activities of liver mononuclear cells (MNC) and splenocytes decrease with aging, although the former is always greater than the latter. NK activity of liver MNC is a function of NK cells, partly NK1+ TCR(int) cells but not NK1- TCR(int) cells or TCR(hi) cells. These results suggest that lymphocytes of liver and other organs at old age are no longer occupied solely by conventional thymus-derived T cells, and the increase of extrathymic IL-2Rbeta+ NK1- TCR(int) cells in liver and periphery could be closely related to immunological changes with aging.

Autologous killing by a population of intermediate T-cell receptor cells and its NK1.1+ and NK1.1- subsets, using Fas ligand/Fas molecules.

Self-reactive clones, estimated by anti-V beta 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 effector 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 effector 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 beta 2-microglobulin-deficient 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.

Self-reactive forbidden clones are confined to pathways of intermediate T-cell receptor cell differentiation even under immunosuppressive conditions.

It is believed that self-reactive forbidden T-cell clones are generated by 'failure' of the pathway of T-cell differentiation in the thymus, if it is disturbed. We examined how such forbidden clones are generated under immunosuppressive conditions. Mice were treated with an injection of deoxyspergualin, FK506, or cycloporin A. From day 3, the number of cells yielded by various organs decreased. Because of the resistance of intermediate (int) T-cell receptor (TCR) cells (i.e. TCRint cells), they became more prominent in proportion than TCRhigh cells. TCRhigh cells are conventional T cells generated through the mainstream in the thymus, whereas TCRint cells are primordial T cells generated by the extrathymic pathway or an alternative intrathymic pathway. Similar to untreated mice, forbidden V beta 3+ and V beta 11+ clones in C3H/He (Mls-1b2a) mice were confined to TCRint cells after treatment; there was no leakage of forbidden clones into TCRhigh cells in the thymus and periphery. In parallel with the increase in the proportion of TCRint cells, the proportion of forbidden clones also increased under immunosuppressive states, especially in the liver. Liver mononuclear cells isolated from treated mice still had the potential to mediate autologous killing. The present results suggest that the generation of self-reactive clones is highly restricted to the pathways of TCRint cell differentiation even under immunosuppressive conditions.

Groove pancreatitis with recurrent duodenal obstruction. Report of a case successfully treated with pylorus-preserving pancreaticoduodenectomy.

Groove pancreatitis is a rare subtype of chronic pancreatitis that is difficult to distinguish from pancreatic carcinoma. Most reported patients have undergone a Whipple procedure because pancreatic cancer was not ruled out. We report a case of groove pancreatitis in a patient who presented with recurrent duodenal obstruction without biliary stricture. The diagnosis of groove pancreatitis was based on characteristic episodes of repeated duodenal obstruction and the absence of radiographic evidence of cancer. Subsequently, our patient underwent a successful pylorus-preserving pancreaticoduodenectomy (PPPD). PPPD is a favorable alternative to the Whipple operation for duodenal obstruction resulting from this disease.

Absolute dependence of T cell receptor(hi) cell generation and relative dependence of T cell receptor(int) cell generation on the thymus.

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCRhi). In contrast, primordial T cells (or NK1.1+ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCRint). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte-depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCRhi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor beta chain+ TCRint cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin alpha induced a slight expansion of CD3int cells in nude mice. To examine a possible interaction of TCRint cells with TCRhi cells in the periphery, B6 nu/nu mice (Ly5.2+) were injected with TCRhi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCRint (Ly5.2+) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCRhi cells is absolutely dependent on the thymus and that TCRint cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.

Identification of nicotinic acetylcholine receptors on lymphocytes in the periphery as well as thymus in mice.

The 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 alpha-bungarotoxin (alpha BuTx), they began to bind alpha BuTx after incubation in medium. In contrast to granulocytes, various lymphocyte subsets obtained from various lymphoid organs were found to bind alpha BuTx. Affinity purification of alpha BuTx-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 alpha-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.

Restricted appearance of self-reactive clones into T cell receptor intermediate cells in neonatally thymectomized mice with autoimmune disease.

Neonatally thymectomized (NTx) mice fall victim to such autoimmune diseases as gastritis and pancreatitis with aging. Self-reactive T cell clones are known to be consistently generated through TCR intermediate (i.e. TCRint) cell differentiation in normal mice (i.e. via the extrathymic pathways and an alternative intrathymic pathway). It was investigated whether the generation of such clones in NTx mice follows this rule or whether they are generated by default via mainstream T cell differentiation in the thymus. The majority of T cells generated in NTx mice were TCRint cells in all organs tested. In contrast to athymic mice, which carry only TCRint cells with aging, a leaky appearance of high TCR (i.e. TCRhi) cells emerged in the lymph nodes and other organs of NTx mice. Self-reactive clones estimated by anti-Vbeta monoclonal antibodies in conjunction with the Mls system were confined to TCRint cells in all tested organs, including a target organ, the stomach, in NTx mice. A leaky population of TCRhi cells did not contain a significant number of self-reactive clones. Moreover, such self-reactive clones among TCRint cells in NTx mice with autoimmune disease were shown to be nonanergic in the proliferation assay. The present results suggest that the generation of self-reactive clones is totally due to TCRint cell differentiation, although it is still undetermined whether the expanding TCRint cell population is generated via the extrathymic pathway or an alternative intrathymic pathway. It is shown here not to be due to a failure of the TCRhi cell-differentiation pathway in NTx mice.

Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen.

Morphological and phenotypic characterization in previous studies has indicated that intermediate (int) T-cell receptor (TCR) cells or T natural killer (TNK) cells may stand at an intermediate position between NK cells and high TCR cells of thymic origin in phylogenetic development. In this study, a functional study on cytotoxic activity against various tumour targets was performed in each purified subset. When a negative selection method entailing in vivo injection of anti-asialo GM, antibody or anti-interleukin (IL)-2R beta monoclonal antibody (mAb) was applied, IL-2R beta 1 CD3 NK cells were found to have the highest NK activity while IL-2R beta 1 int CD3 (or TCR) cells had a lower level of the NK activity. High CD3 cells (freshly isolated) did not have any such activity. Sorting experiments further revealed that the NK function mediated by int CD3 cells was augmented when they were exposed to anti-CD3 mAb. anti-TCR alpha beta, or anti-TCR-delta mAb. This phenomenon was not observed in NK cells and high CD3 cells. More importantly, when anti-CD3 mAb (or anti-TCR mAb) was added to the assay culture, int CD3 cells became cytotoxic against even NK-resistant tumour (Fc gamma R-. Fas+) targets. Liver mononuclear cells or int CD3 cells exposed to anti-CD3 mAb for 6 hr showed an elevated level of perforin in their cytoplasms. The present results suggest that int CD3 cells are usually non-cytotoxic against various tumours but become functional after being stimulated via the TCR CD3 complex.

Existence of a small population of IL-2R beta hi TCRint cells in SCG and MRL-lpr/lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene.

Mice carrying the lpr gene, SCG and MRL-lpr/lpr mice, were used to characterize the phenotype and lpr gene of abnormally proliferating T cells in these mice. A major population which expanded in these mice were T cells expressing intermediate (int) levels of T cell receptor (TCR) (and CD3) and the phenotype of interleukin-2 receptor (IL-2R) beta lo alpha- (possibly abnormal TCRint cells). The levels of TCRhi cells of thymic origin (generated through the mainstream of T cell differentiation in the thymus) profoundly decreased after the onset of disease. However, a small population of normal TCRint cells (i.e. IL-2R beta hi alpha-) were also found to exist in all tested organs. For example, the majority of abnormal IL-2R beta lo TCRint cells were CD4-8- CD2-, while normal IL-2R beta hi TCRint cells were a mixture of single-positive cells (mainly CD8+), CD4-8- cells and CD2+ cells. Moreover, normal TCRint cells preferentially produced normal Fas mRNA and Fas molecules from the lpr gene. This phenomenon explains the leaky appearance of normal Fas mRNA and Fas molecules in mice carrying the lpr gene. It is suggested that a small population of IL-2R beta hi TCRint cells are resistant to the lpr genetic abnormality.

Origin of CD57+ T cells which increase at tumour sites in patients with colorectal cancer.

Human T cells carrying natural killer (NK) markers, CD57 or CD56 antigens, appear to be distinguishable from other T cell subsets in terms of their granular lymphocyte morphology and their numerical increase in patients with AIDS and in recipients of bone marrow transplantation. At the beginning of this study, we observed that CD57+ T cells as well as CD56+ T cells were abundant at tumour sites in many patients with colorectal cancer. Since all these findings for CD57+ T cells are quite similar to those of extrathymic T cells seen in mice, we investigated how CD57+ T cells are distributed to various immune organs in humans. They were found to be present mainly in the bone marrow and liver, but to be completely absent in the thymus. Similar to the case of extrathymic T cells in mice, they were observed to consist of double-negative CD4-8- subsets as well as single-positive subsets (preponderance of CD8+ cells), and to contain a considerable proportion of gamma delta T cells. These features are striking when compared with those of CD57- T cells, which are characterized by an abundance of CD4+ subsets and alpha beta T cells. Not only at tumour sites but also in the peripheral blood, some patients with colorectal cancer displayed elevated levels of CD57+ cells. These results suggest that CD57+ T cells may be of extrathymic origin, possibly originating in the bone marrow and liver, and may be associated with tumour immunity, similar to another extrathymic population of CD56+ T cells in humans.