Splenic natural cytotoxic activity is enhanced during growth of a murine fibrosarcoma

We have shown previously that the natural killer (NK) cell activity of DBA/2J mice bearing M-1 fibrosarcomas is consistently depressed at the later stages of tumor growth. The apparent mechanisms of inhibition are suppressor cell activation and prostaglandin E (PGE) production by tumor and lymphoid cells. In contrast, we show here that the natural cytotoxic (NC) activity of cells from the spleen, blood, and lymph nodes of mice bearing M-1 tumors is enhanced when compared to that of age- and sex-matched control mice. This enhanced NC activity does not appear to be due to increased cytolytic activity of macrophages but, rather, to enhanced cytolytic activity of multiple populations of non-adherent cells including B and T cells. Correlated with this is the finding that the NC activity of normal spleen cells is not inhibited in vitro by either PGE1 or PGE2 at levels which are inhibitory to NK cells. NC activity, although independent of PGE, is in fact enhanced by PGE1 in a dose-related fashion. These data indicate that NK and NC cells are regulated differently by PGE and during tumor growth. Utilizing a Winn assay, we also demonstrate that a cloned cell line with NC activity is capable of slowing tumor growth in vivo and that this action is improved if mice are treated with indomethacin concomitantly.     

In vivo acute depletion of CD8(+) T cells before murine cytomegalovirus infection upregulated innate antiviral activity of natural killer cells

In this study, we investigated the effect of depletion of CD8(+) T cells on the activity of natural killer (NK) cells at an early phase of murine cytomegalovirus (MCMV) infection. For CD8(+) T cell depletion, mice were intraperitoneally treated with anti-CD8 mAb, purified from 2.43 hybridoma, for 2 consecutive days before or after infection. Three days after infection, we found that an acute depletion of CD8(+) T cells before infection caused a significant decrease in the viral load in liver and spleen. This effect coincided with an increase in numbers of CD3(-) NK1.1(+) cells in spleen and their expression of the early activation molecule CD69. Although cytolytic activity of NK cells increased on day 3 of infection in CD8-depleted mice, the level of IFN-gamma decreased in serum and supernatant of cultured spleen cells. In contrast to the effect of acute depletion of CD8(+) T cells before infection, the depletion after infection had no effect on the viral load or number and cytolytic function of NK cells. Lack of effects of CD8(+) T cell depletion on the viral load and NK cytolytic activity is also observed in CD8(+) knockout mice. In conclusion, the results suggest that an acute depletion of CD8(+) T cells before MCMV infection effectively upregulated the antiviral activity of NK cells. This effect appears to be mediated through an increase in numbers, activation and cytolytic activity of NK cells.     

Altered Natural Killer and Natural Cytotoxic Cellular Activities in lpr Mice

Three strains of mice bearing the autosomal recessive lpr gene (MRL, C57BL/6, and C3H) that had spontaneously developed a lupus-like disease were studied sequentially for functional natural killer (NK) and natural cytotoxic (NC) cell activity. Natural killing was impaired in spleen and bone marrow cells from all the lpr strains, as well as from the congenic strain MRL--+/+, which develops a late onset lupus-like disease. The NK cell activity was found to be depleted as early as 2 months of age in all lpr strains, and decreased further with age. NK activity was augmentable by Poly I:C and interleukin 2 (IL-2), suggesting that the residual cells can respond to NK modulators. In contrast with NK cell activity, NC activity was not decreased in lpr mice but could be augmented by IL-3-rich supernatants. The spontaneous decrease in NK cell activity was associated with an increased autologous plaque-forming cell (APFC) response to bromelin-treated mouse red blood cells, which is produced primarily by B cells possessing the Ly-1 phenotype (Lyt-1+ B). When NK cell activity was increased by exogenous administration of Poly I:C, the APFC response diminished. Treatment of spleen cells with anti-asialo GM1 prior to Poly I:C treatment resulted in a decreased NK response but increased both APFC and Lyt-1+ B cells. The possible regulation of autoreactivity by NK cells is discussed.     

紫色杆菌LPS对小鼠脾细胞免疫活性的抑制作用

细菌内毒素或脂多糖(LPS)是机体内强烈的免疫调节剂。天然低毒性紫色杆菌LPS 体内处理小鼠,能促进脾细胞的分化、增殖,但降低脾细胞对 Con A 和同种细菌 LPS 的反应性,抑制混合淋巴细胞反应(MLR)和自然杀伤(NK)细胞的活性。LPS 处理供体小鼠还抑制其脾细胞在 F_1 小鼠内诱导的移植物抗宿主反应。应用混合培养方法,在 Con A和 LPS 诱导的淋巴细胞转化反应中分别检测到非特异性抑制细胞活性,但在MLR和NK 活性测定中未发现抑制细胞的存在。上述结果说明 LPS体内抑制T、B 淋巴细胞功能和 NK细胞活性,而这种抑制作用除由抑制细胞介导外,还存在其它尚未明瞭的机理。     

Chrysotile A affects YAC-1 cytolytic activity of spleen cells

Effect of short exposure of C57Bl/6 and F1/NZB x C57Bl/6/ mice to i.p. injected chrysotile A on YAC-1 cytolytic potential (NK cell function) of spleen cells was investigated. It was found that 3 days after injection, cytolytic activity of spleen cells was significantly abrogated and this was paralleled with an increase of alpha-naphtyl acetate esterase positive cells/macrophages in spleen cell population. In addition, effect of exposure of mice to i.p. injected chrysotile A coated with benzo(a)pyrene (BaP) was investigated. BaP did not modify the abrogative effect of chrysotile A on NK cell function. Asbestos fibres themselves were sufficient for depletion of NK cell function of spleen cells.     

Analysis of natural killer effector and suppressor activity by intraepithelial lymphocytes from mouse small intestine.

Intraepithelial lymphocytes (IEL) are morphologically similar to NK cells in other tissues and we have studied the NK activity of IEL isolated from mouse small intestine. In contrast to spleen NK cells, IEL showed little activity against YAC-1 over 4 h but had high levels of NK activity when the assay was extended to 18 h. IEL from nude mice did not show the enhanced NK activity found in other tissues. IEL were also found to suppress the NK activity of spleen cells and this suppressor function was not mediated by T lymphocytes or macrophages. The results indicate that the intestinal epithelium contains a population of potent NK cells which may represent a type of NK cell different to that found in other tissues. In addition, there are also cells capable of regulating NK cell function in the epithelial layer.     

NK cell activation and protection occur independently of natural killer T cells during Trypanosoma cruzi infection

Natural killer T (NKT) cells regulate aspects of pro-inflammatory and anti-inflammatory responses and contribute to the control of infections and chronic inflammatory diseases. During Trypanosoma cruzi infection both NKT cells and NK cells are critical to the protective response. How NKT cells interact and possibly regulate NK cells during infections remains uncertain. In vivo studies have demonstrated that specific activation of NKT cells with alpha-galactosylceramide (alpha-GalCer) leads to NK cell activation. These results suggest that during some infections activated NKT cells might regulate NK cell activation and functions. Therefore, using gene-deficient mice that lack NKT cells and antibody-treated mice that lack NK cells, we investigated the interactions of NKT cells and NK cells during experimental T. cruzi infection. We report here that during acute T. cruzi infection spleen and liver NK cell activation and cytolytic activity occur independently of NKT cells. Moreover, NK cell protection occurs independently of NKT cells. In contrast to these results that fail to demonstrate an interdependence, at day 4 of infection the number of liver NK cells is controlled by NKT cells. Thus, during T. cruzi infection, regulation of the number of liver NK cells requires NKT cells, but the activation of NK cells and protection by NK cells does not. The data presented here argue that during infections NK cell activation and protection occur independently of NKT cells.     

Immunological senescence. I. The role of suppressor cells.

The in vitro anti-SRBC response of several murine strains declined markedly with age in parallel with an increase in the activity of suppressor cells in the spleen and bone marrow which prevented early events during the induction of the immune response. These suppressor cells released soluble mediators and lacked the characteristics of mature T cells or macrophages. In addition the suppressor cell in the bone marrow could be removed on anti-Ig columns and fractions of old splenic suppressor cells sedimenting at 0.32 cm/h were greatly enriched in surface Ig bearing cells. Old immunodepressed mice did not lack potentially immunocompetent cells since the antibody response of old spleen cells could be restored by specifically activated T cells or lipopolysaccharide which act on B cells. These results suggest that a rise in the activity of non-T suppressor cells in the spleen and bone marrow may account, in part, for the depression in humoral immunity observed in aging mice.     

Increase of natural killer (NK) activity of mouse lymphocytes following in vitro treatment with cytosine-arabinoside

The in vitro influence of cytosine-arabinoside (Ara-C) on mouse NK activity was studied treating effector cells, target cells or effector and target mixture with graded concentrations of the drug. Ara-C increased the NK efficiency of mouse splenocytes without enhancing the susceptibility of target cells or the cytolytic events when added to effector-target mixture. This phenomenon was confirmed with splenocytes collected from congenitally athymic (nude) or conventional donors of different ages, untreated or depressed or boosted for NK activity by various agents. In addition Ara-C increased the NK activity of spleen cells of nude mice deprived of nylon-adherent cells, and did not affect suppressor cells capable of inhibiting the lytic phase of NK process. The drug was able to significantly augment the binding ability of spleen cells to the NK-sensitive YAC-1 target. It was concluded that Ara-C would increase the efficiency of natural cytotoxicity presumably through a direct influence on effector lymphocytes.     

Characterization of Corynebacterium Parvum-Induced Suppressor Cells of Mouse NK and ADCC Activity

Treatment of mice with Corynebacterium parvum (Cp) resulted in a substantial decrease in natural killer activity in the spleen at 10 days. The decrease in cytotoxicity was associated with the presence of splenic nonadherent (NA) suppressor cells, capable of inhibiting natural as well as antibody-dependent cellular cytotoxicity (ADCC). The nonadherent suppressor cells appeared to be null cells, lacking detectable expression of Thy 1, L3T4 (CD4), Lyt 2 (CDE), or asialo-GM1 and could be physically separated from cells with NK activity by centrifugation on Percoll discontinuos density gradients. Our results indicate that Cp can negatively modulate cytolytic functions of NK cells by inhibiting the effector phase of cytotoxicity.     

Syngeneic B lymphoma cells provide a unique stimulus to natural killer (NK) cells in genetically low-NK SJL/J mice

SJL/J mice are a genetically low-NK strain, and their cytotoxic activity cannot be augmented with conventional NK inducers. In contrast, effector cells taken from the lymphoid tissues of SJL mice bearing a syngeneic B cell lymphoma (RCS) show variable, but significant levels of cytotoxic activity against NK-susceptible targets, such as YAC-1. Previous results suggested that the RCS cells themselves contributed to this cytotoxicity. However, results presented here indicate the most, if not all of the activity present within the lymphoid tissues of RCS-bearing mice is mediated by RCS-activated, host NK cells. These results were confirmed by in vitro studies, which demonstrate that both gamma irradiated (gamma-) RCS cells and gamma-allogeneic spleen cells induce cytotoxic activity in SJL spleen cells against YAC targets. However, the cytotoxicity induced by gamma-allogeneic cells is mediated largely by lymphokine-activated killer (LAK) cells, since these effectors also lyse NK-resistant target cells, such as L1210. In contrast, the cytotoxic effector cells that are induced by syngeneic gamma-RCS cells cause lysis of YAC targets, but not L1210 target cells. These data indicate that the syngeneic B cell lymphomas of SJL mice are a unique stimulus for host NK cells in this strain. Since activated NK cells produce a variety of lymphokines, RCS stimulation of host NK cells in SJL mice may provide some of the growth-promoting lymphokines that are known to be necessary for progressive growth of these lymphoma cells.     

Evidence of dual function of macrophage migration inhibitory factor relevant to tumor progression and regression

Macrophage migration inhibitory factor (MIF) is known to play an important role in broad-spectrum inflammation and immune responses. To evaluate the role of MIF in tumor growth, we established transgenic (Tg) mice (ICR strain) driven by cytomegalovirus (CMV) enhancer and beta-actin promoter. We inoculated Tg mice in the back with murine sarcoma cell line S-180 cells. The tumor growth rate was more enhanced in Tg mice than in littermate non-Tg mice up to day 9 after tumor inoculation. Surprisingly, most tumors embedded on the back of Tg mice regressed at day 10 after inoculation and eventually disappeared. Tumor volumes of non-Tg mice incessantly increased until death. We reinoculated the Tg mice with S-180 cells, which had been recovered from the first challenge, and found that the tumor cells were completely rejected in all cases. To identify the effector cells that eradicated the tumor cells, we prepared spleen cells from tumor-bearing Tg mice and carried out cell lysis assay. The magnitude of cytolytic activity of spleen cells obtained from Tg mice was significantly higher against S-180 cells, as well as natural killer cell-sensitive YAC-1 cells, than was the activity of cells from non-Tg mice. Furthermore, we observed that CTL activity of Tg mice against S-180 cells was significantly decreased by the deletion of CD8+ T cells or NK cells. On the other hand, the deletion of CD4+ cells minimally affected the cytolytic activity. Taken together, these results suggest that MIF has the potential to promote tumor growth and angiogenesis in the early phase and, by contrast, this protein could activate CD8+ cytotoxic T cells and NK cells, leading to tumor regression.     

Impact of changes in housing condition on mouse natural killer cell activity

Various psychosocial stressors, such as housing condition and rotation, have been reported to influence tumour growth. This study assessed the influence of housing condition and change in housing condition on natural killer (NK) cell activity, an important component of natural immune defense against cancer. Mice which were individually housed for four weeks did not differ from group-housed mice in NK cell cytolytic activity against tumour targets in vitro or in the frequency of NK cells in the spleen. Switching of housing condition (group to individual, individual to group) for one week did not change the splenic NK cytolytic capacity relative to mice which were not switched. The two groups of mice which experienced a change in housing condition were, however, significantly different from each other. These data suggest that an acute change in housing condition, rather than the housing condition per se, has differential effects on the capacity to kill tumours by NK cells.     

Concomitant Immunity against Tumor Development is Enhanced by the Oral Administration of a Kampo Medicine, Hochu-Ekki-To (TJ-41: BU-ZHONG-YI-QI-TANG)

The oral administration of a kampo herbal medicine, Hochu-ekki-to (TJ-41: Bu-Zhong-Yi-Qi-Tang) using a water-supplying bottle resulted in a slight but significant inhibition of Meth A growth. The oral administration of TJ-41 with gastric gavage significantly enhanced the specific antitumor activity against Meth A at rechallenge on day 9. In a tumor-neutralizing assay, the tumor draining LN cells of the TJ-41 administered mice showed an antitumor activity against Meth A. In a cytolytic assay, the anti-Meth A specific cytolytic T lymphocyte activity was not detected in the spleen cells of the Meth A bearing and TJ-41 administered mice. The oral administration of TJ-41 enhanced the natural killer (NK) activity of the spleen cells in naive mice but could not improve the decreased NK activity of spleen cells from the tumor bearing mice. In a cytostatic assay, the peritoneal exudate cells from the Meth A bearing and TJ-41 administered mice showed a significantly higher amount of cytostatic activity against Meth A than that from either Meth A bearing or TJ-41 administered mice. These results indicate that the oral administration of TJ-41 into the tumor bearing mice may thus be able to enhance concomitant antitumor immunity through the augmentation of the cytostatic activity.     

Inhibition of natural killer activity by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Balb/c mice and nude mice was studied. CGRP dose-dependently (10(-9) to 10(-7) M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10(-7) M CGRP was about 60%. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Balb/c spleen cells. Furthermore, when cells were treated with 10(-9) to 10(-7) M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM1 antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM1 completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM1 injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Kinetic analysis of the inhibition of natural killer (NK) activity by multiple populations of tumor-activated suppressor cells

Previous studies have shown that the progressive growth of the M-1 fibrosarcoma in DBA/2J mice is associated with the activation of suppressor cells which inhibit both mitogen-induced proliferative responses and antibody synthesis. In this study, we have analyzed the effect of tumor growth on NK cell activity. Mice in the advanced stages of tumor growth did have a significant depression in NK activity, and this depression could not be overcome by the injection of the interferon inducer, polyinosinic-polycytidylic acid (Poly I:C). The decline in NK activity was associated with the presence in the spleens of suppressor cells capable of inhibiting the NK activity of spleen cells from Poly I:C-treated syngeneic mice. In order to characterize the suppressor cells, we used a combination of negative selection procedures and kinetic analysis. These studies demonstrated that the spleens of tumor-bearing mice contained two distinct populations of suppressor cells which were not evident in normal mice. One population was non-adherent to nylon wool, Thy-1-, non-phagocytic, did not bind target cells, and had a non-competitive mechanism of suppression. The second population was adherent, Thy-1-, phagocytic, and had a competitive mechanism of suppression. In addition, the spleens of both normal and tumor-bearing mice contained an adherent, non-competitive suppressor cell population which was enriched following negative selection procedures removing T cells or phagocytic cells.     

Enrichment of murine splenic natural killer (NK) cells by the sequential elimination of non-NK cells

A simple and reliable three-step procedure to enrich for murine endogenous splenic NK cells is described. The method is based on the sequential elimination of non-NK cell subsets by standard and inexpensive techniques executed in a specific order. First, macrophages and other adherent cells are eliminated by incubation on plastic surface. Secondly, the T cells are excluded from the multicellular aggregates formed by agglutination of the remaining cells with wheat germ lectin. Thirdly, after dissociation of the aggregates with N-acetyl-D-glucosamine and osmotic lysis of erythrocytes, NK cells are separated from other nucleated cells by nylon wool filtration. C57BL/6 spleen cells were used to establish the enrichment procedure. Usually their NK cell activity is intermediate but occasionally either low or high NK cell activity was observed in input cell suspensions. The NK cell activity recovery and the degree of enrichment varied inversely with the initial NK cell activity level of the input cell suspension. When initial NK cell activity was intermediate, it was enriched 10-30-fold. Experiments were done to establish if suppressor cells, and nylon wool-adherent, naturally activated NK cells, putatively present in input cells, could have been responsible for the abnormal initial NK cell activity detected in some C57BL/6 spleen cell suspensions and for the variations in the degree of enrichment achieved by the method here described. Either no or negligeable suppressor cell activity was noted in the cell fractions normally discarded at each step of the procedure. On the other hand, nylon wool-adherent NK cells were eliminated during the fractionation of spleen cells with higher than average initial NK cell activity and would account for the lower NK cell enrichment obtained in these conditions.     

Generation of cellular immunity to lymphocytic choriomeningitis virus is independent of CD1d1 expression.

CD1 molecules are cell surface glycoproteins, structurally similar to major histocompatibility complex (MHC) class I molecules. The murine CD1d1 molecule has been shown to be essential for the positive selection of a unique subpopulation of T cells [the natural killer (NK) T cells], as CD1d1-deficient mice lack NK T cells. These cells have recently been suggested to play an important role in the induction of innate immunity (i.e. NK cells) and the regulation of immune homeostasis. As such, it was asked whether NK T cells were necessary for the generation of cellular immunity to an acute virus infection. In these studies, the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), a classic inducer of NK cells, and its pathogenic variant clone 13 were used. When NK-cell activity was assessed on day 3 post-LCMV infection, surprisingly, it was found that CD1d1-deficient mice could generate NK-cell activity at wild-type levels. Likewise, LCMV-specific cytotoxic T-lymphocyte (CTL) activity in CD1d1-deficient mice was indistinguishable from that generated in wild-type mice. Additionally, viral titres in the spleen (LCMV Armstrong) and blood (LCMV clone 13) of infected CD1d1-deficient mice were at comparable levels to those found in wild-type mice, as were virus infection-induced increases in cell surface H-2Kb in the spleen. Therefore, these results suggest that the LCMV-induced generation of NK-cell and virus-specific CTL activity, as well as viral clearance, are independent of CD1d1 expression.     

Analysis of low natural killer cell activity in 89Sr-treated mice

Treatment of mice with the long-lived bone-seeking radioisotope 89Sr results in the selective irradiation and destruction of the bone marrow. This is accompanied by a marked reduction in natural killer cell activity against YAC-1 lymphoma [NK(YAC-1)]. To test for the presence of cellular suppressors of NK(YAC-1) in 89Sr-treated mice, in vitro and in vivo cell mixture protocols were used. In vitro, we did not observe any specific inhibitory effect of spleen cells from 89Sr-treated mice on NK(YAC-1) activity of normal spleen cells. The NK(YAC-1) activity of 89Sr-treated mice, measured in vivo by their ability to clear radiolabeled YAC-1 cells from the lungs, was impaired. However, spleen cells from 89Sr-treated mice, when adoptively transferred with normal spleen cells, failed to inhibit the NK(YAC-1) activity of the latter in the lung clearance assay. Further, when normal spleen cells were injected into 89Sr-treated mice, the ability of the transferred cells to mediate in vivo activity was not suppressed in the 89Sr-treated host. These experiments support the suggestion that the low NK(YAC-1) activity in 89Sr-treated mice is not mediated by suppressor cells, but may be due to the destruction of the marrow microenvironment which is essential for the generation of functional NK(YAC-1) cells.     

Suppression of murine natural killer cell activity by adherent cells from aging mice

Natural killer (NK) cell activity declines with age in mice. The purpose of this study was to investigate the effect of peritoneal and splenic adherent cells from young and old mice on NK activity to determine whether adherent cell suppressor function might contribute to this decline. Peritoneal adherent cells from old mice suppressed NK activity of young splenic non-adherent indicator cells more than peritoneal cells from young mice. Splenic adherent cells from old but not from young mice also suppressed this activity. That (1) the suppressive activity of the adherent cell populations was not affected by treatment with anti-Thy-1 plus complement, and that (2) the adherent cell population contained 77-92% cells positive for alpha-naphthyl acetate esterase activity, suggests that the active adherent suppressor cell may be a macrophage. Therefore, the age-related decline in NK activity in mice can be explained, in part, by an increase in adherent cell suppressor function.