Masahiro Minagawa

Circadian rhythm of leucocytes and lymphocyte 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, γδ T cells, and CD8+ subset—showed an increase in the daytime (i.e. daytime rhythm). The other group—T cells, B cells, αβ 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.

Numerical and functional characteristics of lymphocyte subsets in centenarians

The immune system in the aged is a very interesting subject for study. In this study, analysis was extended to extrathymic T cells as well as NK cells and “conventional” T cells (i.e., thymus-derived T cells) in terms of their constitution and function in both healthy and unhealthy centenarians. Middle-aged persons were used as controls. Healthy and unhealthy centenarians showed lower levels in the proportion and absolute number of lymphocytes. The major change in the constitution of lymphocyte subsets was increased levels in the proportion of NK cells (CD56+/CD57+) and extrathymic T cells (CD3+CD57+). Inversely, conventional T cells decreased in proportion and function (i.e., proliferative response to mitogen). Although NK cells increased in centenarians, NK activity by whole lymphocytes and the purified NK fraction decreased. The difference between healthy and unhealthy centenarians was small in all parameters, the only difference being a lower level of expression of CD56 antigens on CD57+ T cells in unhealthy centenarians. These results indicate that there is a major shift in lymphcyte population from conventional T cells to NK cells and extrathymic T cells with aging. Concerning the age-associated increases in CD56+ T and CD57+ T cells, these cells correspond to NK1+ T cells in mice.

The differential effect of stress on natural killer T (NKT) and NK cell function

When C57Bl/6 mice were exposed to restraint stress for 12 h or 24 h, lymphocytopenia was induced in the liver, spleen, and thymus. We examined which types of lymphocytes were sensitive or resistant to such stress by a immunofluorescence test. T cells of thymic origin were sensitive while NKT and NK cells were resistant. In contrast to the increase in the proportion of NK cells, NK activity of liver lymphocytes against YAC‐1 targets decreased at 24 h after stress. On the other hand, their NKT cytotoxicity against syngeneic thymocytes increased in parallel with an increase in their proportion. In perforin −/– B6 mice and B6‐gld/gld (Fas ligand−) mice, NK cells were found to mediate cytotoxicity through perforin while NKT cells mediated self‐reactive cytotoxicity through Fas ligand. These results suggest that stress increases the proportion of both NK and NKT cells, but that NK cytotoxicity is suppressed while self‐reactive NKT cytotoxicity is not, due to a diversity of their functional mechanisms.

P2X7 Receptor-Dependent and -Independent T Cell Death Is Induced by Nicotinamide Adenine Dinucleotide

Adding NAD to murine T lymphocytes inhibits their functions and induces annexin V binding. This report shows that NAD induces cell death in a subset of T cells within seconds whereas others do not die until many hours later. Low NAD concentrations (<10 μM) suffice to trigger rapid cell death, which is associated with annexin V binding and membrane pore formation, is not blocked by the caspase inhibitor Z-VADfmk, and requires functional P2X7 receptors. The slower induction of death requires higher NAD concentrations (>100 μM), is blocked by caspase inhibitor Z-VADfmk, is associated with DNA fragmentation, and does not require P2X7 receptors. T cells degrade NAD to ADP-ribose (ADPR), and adding ADPR to T cells leads to slow but not rapid cell death. NAD but not ADPR provides the substrate for ADP-ribosyltransferase (ART-2)-mediated attachment of ADP-ribosyl groups to cell surface proteins; expression of ART-2 is required for NAD to trigger rapid but not slow cell death. These results support the hypothesis that cell surface ART-2 uses NAD but not ADPR to attach ADP-ribosyl groups to the cell surface, and that these groups act as ligands for P2X7 receptors that then induce rapid cell death. Adding either NAD or ADPR also triggers a different set of mechanisms, not requiring ART-2 or P2X7 receptors that more slowly induce cell death.

Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor β-Chain-Deficient Mice

IL-2/IL-15Rβ-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rβ-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rβ-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rβ-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.

LOW LEVEL OF MIXING OF PARTNER CELLS SEEN IN EXTRATHYMIC T CELLS IN THE LIVER AND INTESTINE OF PARABIOTIC MICE : ITS BIOLOGICAL IMPLICATION

c‐kit+ stem cells have recently been found in the liver and intestine of adult mice. We examined whether such stem cells give rise to extrathymic T cells in these organs in situ. To this end, we used parabiotic B6.Ly5.1 and B6.Ly5.2 mice, i.e. mice sharing the circulation. The origin of lymphocytes was identified by anti‐Ly5.1 and anti‐Ly5.2 monoclonal antibodies in conjunction with immunofluorescence assays. Lymphocytes in the blood, spleen, lymphnodes and liver had become a half‐and‐half mixture of Ly5.1+ and Ly5.2+ cells in both individuals by day 14. However, this level of mixing decreased in extrathymic T cells in the liver ( i.e. NK T cells) and intestine by day 14 and thereafter. The same was observed in T cells of the thymus. The data from immunohistochemical staining supported the results of immunofluorescence assays for suspension cells. The present results raise the possibility that extrathymic T cells in the liver and intestine may arise from their own pre‐existing precursor cells, possibly from their own stem cells. Another important finding was that the composition pattern of lymphocyte subsets in one individual was quite similar to that in its partner at various sites. This result was interpreted to mean that only selected partner cells migrate to specific sites in the other partner individual.

Left Posterior Approach to the Superior Mesenteric Vascular Pedicle in Pancreaticoduodenectomy for Cancer of the Pancreatic Head

CONTEXT Dissection of the superior mesenteric artery is the most important part of a pancreaticoduodenectomy for pancreatic cancer. Since 2005, we have used the left posterior approach for superior mesenteric vascular pedicle dissection, in which the superior mesenteric artery and the superior mesenteric vein are dissected first in a clockwise fashion. OBJECTIVE This article presents the technique of a left posterior approach and the clinical outcome. PATIENTS Forty patients underwent a left posterior approach and were compared to 35 patients treated with a conventional dissection. MAIN OUTCOME MEASURES The differences in surgical technique between the left posterior approach and the conventional method were described, and the short- and long-term surgical results compared patients who underwent the left posterior approach to those who were treated with the conventional method. INTERVENTION The superior mesenteric vascular pedicle was first dissected from the left lateral border of the superior mesenteric artery. The superior mesenteric vein was also dissected from the left side. Then, the uncinate process and perivascular soft tissue were separated en bloc from the vasculature. RESULTS No life-threatening complications occurred after the pancreaticoduodenectomies using a left posterior approach. Diarrhea requiring the administration of antidiarrheal agents occurred in 65% of patients; however, planned adjuvant chemotherapy was completed in all patients who did not have an early tumor recurrence. Survival rate was 52.8% at 3 years after surgery. CONCLUSION After a pancreaticoduodenectomy with a left posterior approach, most patients had various degrees of diarrhea, but the adjuvant chemotherapy was able to be continued with close monitoring. The left posterior approach facilitates understanding of the topographic anatomy in the superior mesenteric vascular pedicle.

Portal Vein Resection in Surgery for Cancer of Biliary Tract and Pancreas: Special Reference to the Relationship Between the Surgical Outcome and Site of Primary Tumor

BackgroundEarly and late outcomes after superior mesenteric-portal vein resection (VR) combined with pancreaticoduodenectomy, major hepatectomy, or both for pancreaticobiliary carcinoma were retrospectively evaluated. VR is the most frequently used vascular procedure in this field, but an exact role of VR has not been compared according to the primary site of tumor.Materials and MethodsPostoperative outcomes were compared between surgery with and without VR in each of the three disease-based groups: hilar cholangiocarcinoma and intrahepatic cholangiocarcinoma with hilar extension (HIC, 56), middle and distal cholangiocarcinoma and gallbladder carcinoma (DGC, 118), and pancreatic head adenocarcinoma (PHC, 77).ResultsVR was performed in 19.6% of HIC, 8.5% of DGC, and 45.5% of PHC. In-hospital death was 7.1% (4 of 56) patients with VR (3 of DGC and 1 of PHC). Operations with VR in DGC showed a larger amount of blood loss and more increased ratio of R1operation than those with no VR. In HIC, DGC, and PHC, median survival time of patients with VR was 37, 6.8, and 20 months and that of patients without VR was 42.9, 28.6, and 20.3 months, respectively. VR did not affect survival either in HIC or in PHC; however, in DGC, VR was accompanied with dismal outcome compared with no VR (p = 0.001).ConclusionsAggressive surgery with VR can be justified both in HIC and in PHC but should not be recommended for DGC. Surgical outcomes of VR differed considerably, depending on the sites of the primary tumor.

The Majority of Lymphocytes in the Bone Marrow, Thymus and Extrathymic T Cells in the Liver Are Generated In Situ from Their Own Preexisting Precursors

Parabiotic pairs of B6.Ly5.1 and B6.Ly5.2 mice were used to investigate how lymphocytes in various organs and various lymphocyte subsets mixed with partner cells. The origin of partner cells was determined by using anti‐Ly5.1 mAb in conjunction with immunofluorescence tests. Parabiosis was also produced after the irradiation of B6.Ly5.2 mice at various doses to prepare an immunosuppressive partner. Irrespective of irradiation, lymphocytes and other hematopoietic cells in the bone marrow and lymphocytes in the thymus showed a low mixture of partner cells in comparison with those of all other organs tested. On the other hand, lymphocytes in the blood, spleen, and lymph nodes became a half‐and‐half mixture of their own cells and partner cells by 14 days after parabiosis. Among lymphocyte subsets, intermediate CD3 cells (i.e., CD3int cells) and NKT cells (i.e., NK1.1+ subset of CD3int cells) in the liver also showed a low mixture of partner cells. The present results raise the possibility that lymphocytes in the bone marrow and thymus, and extrathymic T cells in the liver might be in situ generated from their own preexisting precursor cells. Another observation was that, after irradiation, partner cells showed accelerated mixture even if they showed a low mixture under non‐irradiated conditions. However, only lymphocyte subsets with the same phenotype as those of preexisting cells entered the corresponding sites.

Quick recovery in the generation of self-reactive CD4low natural killer (NK) T cells by an alternative intrathymic pathway when restored from acute thymic atrophy.

The thymus comprises the mainstream of T cell differentiation which produces conventional T cells and an alternative pathway which produces primordial T cells with intermediate density of T cell receptor (TCR)–CD3 complex on the surface (i.e. intermediate TCR cells or TCRint cells). We induced acute thymic atrophy in mice by an administration of hydrocortisone (10 mg) or irradiation (6.5 Gy). It was demonstrated that CD3intCD4lowNK1.1+ T cells were immediately generated by an alternative intrathymic pathway without passing through the double‐positive CD4+8+ stage, when restored from thymic atrophy (days 3–14). These CD3intCD4lowNK1.1+ T cells mediated self‐reactivity and appeared even in the periphery. mRNA of an invariant chain of TCR Vα14Jα281 gene product was detected in these CD4low T cells, but not remaining CD4high T cells. The mainstream of T cell differentiation in the thymus was not restored up to day 14 and there was no leakage of self‐reactive clones into the population generated through the mainstream. These results reveal that an alternative intrathymic pathway is associated with the generation of self‐reactive T cells, in an early restoration phase after thymic atrophy.