Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression

Hyperthermia, which is used as an adjunctive therapy for cancer, is known to modulate the activity of natural killer (NK) cells in vitro, but its effect in vivo is unclear. In the present study, we used a whole body hyperthermia (WBH) device heated by infrared rays to evaluate the effect of WBH on mice models. We demonstrate here that wild type C57BL/6J mice exposed to 42 degrees C for 60min had reduced NK cell cytolytic activity against YAC-1 target cells as determined by cytolytic assay. This result was confirmed using Rag-2 knockout mice, which possess functional NK but not cytolytic T or NK-T cells. Moreover, WBH decreased the mRNA expression of perforin and granzyme B in spleens of mice. But the expression of TNF cytokines (Fas ligand and TRAIL) was unchanged. These data suggest that the suppression of NK cell activity induced by WBH could be mediated through the perforin/granzyme pathway.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.     

Killing of target cells by redirected granzyme B in the absence of perforin

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.     

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. JayneBrahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl.Physiol. 83(3): 845-850, 1997.This study wasdesigned to investigate the effects of moderate-intensity endurancetraining on basal natural killer (NK) cell cytolytic activity in murinesplenocytes that were enriched for1)NK1.1⁺ cells or2) macrophages andNK1.1⁺ cells. Mice were assignedto sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn)groups. Splenocyte number, the percentages ofNK1.1⁺, large granular lymphocytes(NK1.1⁺, LGL-1⁺),and other subpopulations did not change in Trn mice. Approximately 70%of cells enriched for NK1.1⁺expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantlylower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remainedin the in vitro assay, LU perLGL-1⁺ cell did not differ acrossgroups. The results indicate that highly enrichedNK1.1⁺ cells from Trn mice hadlower NK cell activity compared with Sed mice. No differences in NKcell activity were observed when cells were enriched forNK1.1⁺ cells and macrophages.These findings support the hypothesis that macrophage modulation of NKcells may be one mechanism contributing to augmented basal NK cellactivity in endurance-trained individuals.

     

Modulation of NK cell activity by moderate intensity endurance training and chronic ethanol consumption.

Chronic ethanol consumption can suppress natural killer (NK) cell activity. Exercise after ethanol administration may enhance blood ethanol clearance, which may benefit the immune response. This study examined the effects of moderate intensity endurance training and chronic ethanol consumption (20% wt/vol) on splenic NK cell activity. Mice were assigned to one of four groups: sedentary, water drinking (SED-H2O); sedentary, ethanol consuming (SED-EtOH); trained, water drinking (TR-H2O), and trained, ethanol consuming (TR-EtOH). TR groups ran 60 min/day, 5 days/wk, at 12 m/min for 10 wk. Mice were killed 48 h after exercise. Baseline NK cell activity was suppressed 30% in TR and EtOH groups compared with SED-H2O controls. Activation with recombinant human interleukin-2 increased cytolytic activity in all groups four- to fivefold. These results indicate that training did not abrogate the effects of chronic ethanol consumption on NK cell activity. Furthermore, moderate endurance training may contribute to suppressed nylon wool-enriched NK cell activity in murine splenocytes for as long as 48 h after exercise.     

Impaired NK cell development in an IFN-gamma transgenic mouse: aberrantly expressed IFN-gamma enhances hematopoietic stem cell apoptosis and affects NK cell differentiation

Aberrant expression of IFN-gamma has been demonstrated to cause a wide variety of alterations in cell function and development. Previously we reported that constitutive expression of IFN-gamma in bone marrow (BM) and thymus results in a total absence of B cells and a substantial decrease in the number of hematopoietic progenitor cells. In this study, we demonstrate a severe deficiency of NK1.1(+)CD3(-) cells in this transgenic mouse model. Compared with normal control littermates, we found a pronounced reduction of NK cells in IFN-gamma transgenic mouse spleen and liver despite maintenance of normal function. In addition, we observed a reduced number of BM cells in the IFN-gamma transgenic mouse despite normal expression of hematopoietic growth factors in the BM. Interestingly, these cells were less responsive to stem cell factor (SCF) despite c-kit expression on hematopoietic stem cells (HSCs). We observed that addition of exogenous IFN-gamma inhibited proliferation of HSCs and differentiation of NK precursors from HSCs in normal mice in response to SCF, IL-7, fms-like tyrosine kinase 3 ligand, and IL-15. Furthermore, we found that HSCs express the IFN-gammaRalpha subunit and undergo apoptosis in response to exogenous IFN-gamma. Thus, we have demonstrated the occurrence of a severe deficiency of NK cells and lower numbers of BM cells in an IFN-gamma transgenic mouse model. Furthermore, because exogenous IFN-gamma affects the responsiveness to hematopoietic growth factors such as SCF in vitro, our results indicate that chronic expression of IFN-gamma in vivo leads to widespread immune system defects, including alterations in NK cell differentiation.     

Circadian rhythms of oviposition and feeding activity in Japanese quail: effects of cyclic administration of melatonin

The aim of these experiments was to test the effect of a cyclic administration of melatonin, by mimicking the daily rhythm of hormone levels, on the circadian organization of two distinct functions in quail: oviposition and feeding activity. Laying and feeding rhythms under photoperiodic conditions and constant darkness (DD) were investigated. Under DD, where the two rhythms were free running, a daily rhythm of melatonin was administered. In LD 14h:10h, two different individual profiles of laying were established, with stable females laying at the same time each day and delayed females laying progressively later each day. For feeding activity, all birds were clearly synchronized to the photoperiodic cycle. In DD, the laying birds showed a free-running rhythm of oviposition with a period longer than 24 h for both profiles but the delayed profile females had a longer period than stable profile females. In comparison, the free-running period of feeding rhythm of the same birds was shorter than 24 h. A cyclic administration of melatonin had no effect on laying rhythm, which continued to free-run in DD, whereas feeding activity was synchronized as soon as the first cycle of melatonin was administered. From these results, it seems that two different circadian systems drive each of the two types of behavior separately. Melatonin could be the main synchronizer for the temporal control of feeding behavior, but it does not play a part in the control of oviposition in Japanese quail.     

Circadian rhythms of demand-feeding and locomotor activity in rainbow trout

Under free-running conditions, most rainbow trout displayed circadian feeding rhythms, although the expression of circadian rhythmicity depended on the experimental condition: 16·7% of fish under constant dim light (LL dim), 66·1% under a 45 :45 min light-dark cycle (LD pulses), and 83·8% under constant light (LL). Under LD pulses, the period length of the free-running rhythms for feeding was significantly shorter (21·9 ± 0·7 h, n =8) than under LL (26·2 ± 0·3 h, n =10). Period length for locomotor activity under LL was 25·8 ± 0·6 h ( n =4). Under LD conditions, the daily demand-feeding profile was always confined to the light phase and chiefly composed of two main episodes, directly after lights on (light elicited) and in anticipation to lights off (endogenous). Contrasting to feeding, the diel locomotor activity profile varied remarkably: a diurnal activity pattern at the bottom, while a clearly nocturnal pattern at the surface. These results contribute to a better understanding of feeding and locomotor rhythms of rainbow trout, providing evidence for the existence of a biological clock involved in their circadian control. This finding contrasts with the previously recorded lack of an endogenous oscillator in the pineal organ driving the rhythmic secretion of melatonin, which suggests different locations from the pineal for the circadian pacemakers in this species.     

Induction of natural killer cell activity and perforin and granzyme B gene expression following continuous culture of short pulse with interleukin-12 in young and old mice

Anticancer immunotherapy with cytokines is often limited by the occurrence of severe toxicity, particularly in older age groups, which are characterized by a reduced tolerance to antineoplastic therapies. We, and others, have recently demonstrated the efficacy of pulsing procedures with IL-2 as a new therapeutic strategy to induce antitumor cytotoxic cells. The aim of this paper was to evaluate the effect of IL-12 on NK cell activity in young and old mice and to investigate the possibility of inducing NK cytotoxicity and perforin and granzyme B gene expression through a brief exposure of spleen lymphocytes from young and old mice to IL-12. Pulsed lymphocytes were compared with non-pulsed cells cultured continuously in IL-12. IL-12 was able to boost both endogenous and IL-2-induced NK cell activity in young and old mice; the levels of cytotoxicity were lower in old than in young animals although the relative increase of IL-12 plus IL-2 versus IL-2 alone was greater for old mice. Comparable levels of NK cell activity were obtained in pulsed (5 min-1 hour) and non-pulsed lymphocytes from both young and old mice after one or three days of culture. The efficacy of the pulsing procedure was evident in both endogenous and IL-2-induced NK cytotoxicity. The mRNA encoding perforin and granzyme B were markedly and similarly enhanced in both IL-12-pulsed and non-pulsed lymphocytes in comparison with control cells. The results demonstrate the effectiveness of IL-12 pulsing in inducing antitumor cytotoxic cells, suggesting the possibility of using IL-12 pulsing, alone or in combination with IL-2, in the immunotherapy of both young and old subjects.     

Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland

Conclusion The circadian rhythm of melatonin synthesis in the pineal glands of various species has been summarized. The night-time elevation of melatonin content is in most if not all cases regulated by the change of N-acetyltransferase activity. In mammals, the N-acetyltransferase rhythm is controlled by the central nervous system, presumably by suprachiasmatic nuclei in hypothalamus through the superior cervical ganglion. In birds, the circadian oscillator that regulates the N-acetyltransferase rhythm is located in the pineal glands. The avian pineal gland may play a biological clock function to control the circadian rhythms in physiological, endocrinological and biochemical processes via pineal hormone melatonin.     

Circadian rhythms of locomotor activity in cockroach nymphs: free running and entrainment

The temporal organization of locomotor activity was investigated in nymphs of the cockroach Leucophaea maderae. Approximately 40% of the animals examined between 1 and 50 days of age exhibited a circadian activity rhythm in constant darkness (n = 172) with an average free-running period of 23.7 +/- 0.68 hr. Twelve of 17 animals in which activity was recorded for most or all of the final instar also exhibited periods of rhythmic activity. The rhythms of the nymphs could be entrained by light-dark (LD) cycles with periods of 22, 24, or 26 hr. In contrast, neither maternal influences during embryogenesis nor hatching from the egg was effective in synchronizing the rhythms. Although adult cockroaches can be readily entrained by temperature cycles, in nymphs temperature appeared at best to be a weak zeitgeber. Embryonic exposure to an LD cycle until 6 days prior to egg hatch was effective in synchronizing the activity rhythms of the nymphs, indicating that differentiation of an entrainable pacemaking system occurs prior to hatching.     

Circadian rhythms in the activity of a plant protein kinase.

Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5- to 10-fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2----3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2-free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.     

Murine anti-CD3 monoclonal antibody induces potent cytolytic activity in both T and NK cell populations

Antibodies specific for the CD3 complex have the capacity to both stimulate and inhibit a variety of T cell functions. We show here that a monoclonal antibody to the epsilon chain of CD3 can induce efficient non-MHC-restricted cytolytic activity in murine lymphocytes with peak activity occurring after 48 hr of incubation. In a panel of targets, the anti-CD3-activated effectors lysed tumor cells but not normal lymphoblasts. Cytolysis was not dependent on the presence of the antibody in the cytolytic assay. Moderate to high cytolytic activity was elicited from lymph nodes, spleen, and thymus by anti-CD3 treatment in vitro, whereas only low activity was apparent in bone marrow. The precursors of anti-CD3-activated cells consisted largely of mature T cells, although a smaller component of immature T cells was also involved. Thus, separation of thymocytes based on adhesion to peanut agglutinin revealed that both positive (immature) and negative (mature) fractions could be activated, while cytotoxic pretreatment of spleen cells with an antibody (J11d) to immature T cells before anti-CD3 activation significantly decreased the resulting cytotoxicity. The majority of precursors in spleen were Thy 1+ and CD8+ and/or AGM1+. Antibody depletion studies showed that the effector cells have both a T and a NK component consisting of Thy 1+, CD5+, CD8+, CD4-, and AGM1- cells and Thy 1-, CD5-, CD8-, CD4-, and AGM1+ cells, respectively. The production of significant amounts of IL-2 and TNF in culture following anti-CD3 treatment, along with the synergistic effect of exogenously added IL-2, suggests that one or both of the effector cell types could be induced by lymphokines. The intraperitoneal administration of the anti-CD3 antibody induces cytolytic activity in vivo. Therefore, the direct activation of cytolysis by anti-CD3 antibody and the additional effects, both direct and synergistic, of lymphokines produced by the activated lymphocytes could conceivably provide a potent anti-tumor therapy.     

Circadian rhythms of testosterone-dependent behaviors,crowing and locomotor activity,in male Japanese quail

Summary An apparatus was devised to record crowing (mate calling by males) together with locomotor activity and recorded data was analyzed by several methods for rhythm analysis. Crowing and locomotor activity of Japanese quail held on long days were recorded during sexual development as estimated from circulating gonadotropins and testosterone. Both behaviors were testosterone-dependent but commencement of crowing preceded the increase in locomotor activity. When the two behaviors attained their maximum levels, crowing showed consistent daily rhythms in which two peaks were apparent, a major one at the onset of light and a broader one 8 hours later. Locomotor activity also showed a clear daily rhythm with a peak between the two peaks of crowing rhythm suggesting a fixed phase relationship between the two rhythms.Both rhythms free-ran under constant dim light with periods shorter than 24 h. They persisted in birds which had been castrated and then supplied with exogenous testosterone via implanted Silastic capsules. The durations of both rhythms were quite comparable to each other and they maintained a fixed phase relationship similar to that found under LD cycles.The results indicate that testosterone is essential for the induction of crowing and for the enhancement of locomotor activity but the formation of the rhythms in behavior was strictly dependent on a circadian oscillatory mechanism.Abbreviations LH luteinizing hormone - FHS follicle-stimulating hormone - LD light-dark - LDim light-dim light     

All-trans retinoic acid (ATRA) induces miR-23a expression,decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity

NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model.