Splenic Natural Killer Cell Research Articles

Modulation of splenic natural killer cell activity in rats mediated by the hypothalamo-autonomic system

Modulation of splenic natural killer cell activity in rats mediated by the hypothalamo-autonomic system

Islet cells but not thyrocytes are susceptible to lysis by NK cells

BB rats develop both pancreatic insulitis and lymphocytic thyroiditis, but whereas spontaneous autoimmune diabetes is common, hypothyroidism is rare. Splenic natural killer (NK) cells from acutely diabetic (AD) BB rats and from athymic nude rats are known to be cytotoxic to rat islet cells in vitro. To investigate possible differential tissue susceptibility to lysis by NK cells or their cytokines such as cytolysin (perforin) or NK cytotoxic factor (NKCF), we used an in vitro 51Cr-release assay to measure the cytotoxicity of splenocytes, cytolysin or NKCF against Wistar Furth (WF) and Fischer 344 (F-344) rat islet cells, and FRTL-5 F-344-derived and WRT Wistar-derived rat thyrocytes. The results demonstrated that spleen cells from AD-BB (RT1 u) rats and athymic F-344 nude (RT1 1) rats are cytotoxic to WF (RT1 u) islets and F-344 (RT1 1) islets, but not to FRTL-5 (RT1 1) or WRT (class IRT1 1) thyrocytes. WF and F-344 rat spleen cells were not cytotoxic to any of these cells. Thyrocytes are known to express class II molecules on their surface in chronic thyroiditis. We found that treatment of thyrocytes with interferon-gamma (IFN-γ) induced class II expression but did not increase the cytotoxicity of splenocytes against these cells. Cytolysin and NKCF were both cytotoxic to islets in a dose dependent manner, but FRTL-5 thyrocytes were resistant to killing by these cytokines. These findings suggest that islet cells and thyrocytes in vitro are differentially susceptible to lysis by NK cells.

Role of Natural Killer Cells in Resistance to Systemic Cryptococcosis

These studies demonstrate that Cryptococcus neoformans infection induced a dose-dependent augmentation of splenic natural killer (NK) cell activity by bg/+, but not bg/bg mice. To directly assess the role of NK cells in resistance to C. neoformans, bg/+ and bg/bg mice were treated with anti-NK-1.1 monoclonal antibody (mAb). Anti-NK-1.1-treatment abrogated the augmented NK cell activity observed during C. neoformans infection in bg/+ mice. Anti-NK-1.1-treated bg/+ mice had higher C. neoformans colony forming units (CFU) in their lungs on days 3 and 7 after intravenous (i.v.) challenge than control bg/+ mice. Moreover, the number of C. neoformans CFU in the lungs of anti-NK-1.1-treated bg/+ mice on days 3 and 7 were similar to those observed for infected bg/bg mice. By day 14, however, no differences in C. neoformans CFU were evident in the lungs of anti-NK-1.1-treated and control bg/+ mice. Anti-NK-1.1-treatment did not alter either the growth of C. neoformans in the spleens, livers, kidneys, or brain of bg/+ mice or the susceptibility of bg/bg mice to systemic cryptococcosis. These studies suggest that NK cells do not play a role in resistance to systemic cryptococcosis in the spleen, but do appear to play an early, but transient role in resistance to C. neoformans in the lungs. Overall, congenital defects in polymorphonuclear neutrophils (PMNs) and macrophages (M phi s), in addition to defects in NK cells, contribute to the enhanced susceptibility of bg/bg mice to systemic cryptococcosis.

Effects of splenectomy on pulmonary metastasis and growth of SC42 carcinoma transplanted into mouse liver

The carcinoma SC42 was transplanted into the liver of its syngeneic mice DS, and the immunological integrity of the spleen and the effects of splenectomy on the growth and pulmonary metastasis of the liver tumor were assessed. On day 7 after liver tumor transplantation, the natural killer (NK) activity of the splenocytes was significantly elevated; it subsequently decreased at a later stage of the tumor. The response of the splenocytes to PHA and Con-A decreased significantly from the early stage of the tumor. However, the mixed lymphocyte-tumor cell reaction increased significantly from day 14 to day 28. The survival rate of the mice, which had undergone simultaneous splenectomy and liver tumor transplantation, was significantly lower than that of sham-operated control mice. The number of pulmonary metastases in splenectomized mice was significantly greater than in the control mice. There was, however, no difference between the two groups in the weight of the liver tumor. By contrast, splenectomies performed 14 days before or 14 days after tumor transplantation had no significant influence on the survival of the mice. Splenectomies performed on day 0 and on day 3 after tumor transplantation significantly increased the number of pulmonary metastases. Furthermore, the intravenous injection of anti-asialo GM1 antisera on day 0 and day 3 significantly increased the number of pulmonary metastases, but injection of anti-Thy 1.2 antisera had no effect. These results suggest that splenic NK cells may play an important role in the suppression of pulmonary metastasis at early stages of the liver tumor.

Monoclonal antibody 4H12 recognizes subsets of adherent-lymphokine activated killer cells and splenic natural killer cells from pregnant and neonatal mice.

Using a new mAb, 4H12, that recognizes a plasma membrane-associated Ag on granulated metrial gland cells, we identified subsets of murine NK cells in spleen cell-derived adherent lymphokine activated killer cells and in the spleens of neonatal and pregnant mice. In spleen cell adherent-lymphokine-activated killer cultures, 4H12 Ag was detected on a small subset of cells after 7 days culture and expression increased with time to 70% of the cells after 21 days culture. 4H12+ cells were large (up to 70 microns) and granular. The Ag was also detected in the cytoplasmic granules of some, but not all 4H12 surface positive cells. Coexpression studies indicated 4H12+ cells were predominantly positive for the NK1.1 and ASGM1 Ag, negative for the MAC-1 and F4/80 Ag, and +/- for the LGL-1 and CD3 Ag. Subsets of 4H12+/-, LGL-1+/- exhibited morphologic characteristics restricted to specific phenotypic subsets. The 4H12-/LGL-1+ subset was shown to contain the smallest, least granular cells, whereas the 4H12+/LGL-1+/- subsets contained the largest and most granular cells. Although 4H12 expression was negligible in the spleens of normal adult mice, spleen cells of neonatal and pregnant mice contained subsets of NK1.1+ cells that coexpressed 4H12. The 4H12+/NK1.1+ and 4H12-/NK1.1+ subsets displayed differential levels of NK1.1 expression. 4H12+ NK cells were NK1.1 low to high, whereas 4H12- NK cells were NK1.1 high only. The functional significance of subsets of NK cells in IL-2 culture and in the spleens of neonatal and pregnant mice remains to be elucidated.

Effects of vaccinia virus-expressed interleukin 2 on the immune system of sublethally irradiated mice

Vaccinia virus that expressed interleukin 2 (IL 2) was cleared from immunodeficient irradiated mice more efficiently than virus that did not express interleukin 2. These results extend the previously observed protection from nude mice to another model of immunodeficiency. No antibody or cytotoxic T lymphocyte response could be detected in sublethally irradiated mice that had been inoculated with IL 2-expressing vaccinia virus, but levels of splenic natural killer cell activity were elevated. Sublethally irradiated mice that had recovered from IL 2-plus hemagglutinin-expressing vaccinia virus were partially protected against both influenza virus and vaccinia virus. These results indicate that vaccinia virus-expressed IL 2 mediates clearance of primary viral infection via a mechanism that does not involve antibody or cytotoxic T lymphocytes. They also indicate that inclusion of lymphokine genes in live recombinant viral vaccine vectors may increase vaccine safety.

Central administration of interferon-α enhances rat sympathetic nerve activity to the spleen

Intracerebroventricular injection of recombinant human interferon-α (IFN-α) in a dose of 1 × 10 4 U, but not 3 × 10 3 U, produced a long-lasting (at least for 60 min) increase (2.5–5 times the preinjection level) in the electrical activity of splenic sympathetic nerve filaments in urethane-α chloralose-anesthetized rats. Intravenous injection of IFN-α (3 × 10 4 U) also produced a similar excitation of splenic sympathetic nerves. There were no changes in arterial blood pressure and body temperature. The result, together with the previous findings, provides further evidence that the central IFN-α-induced suppression of cytotoxic activity of splenic natural killer cells may be mediated by the sympathetic innervation.

Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat

Recent evidence suggesting that immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders. The mammalian osteopetroses are a pathogenetically heterogeneous group of skeletal disorders characterized by skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption. Using a 51Cr-release microcytotoxicity assay we demonstrated that splenic natural killer (NK) cell activity was significantly reduced in two distinctly different osteopetrotic mutations in the rat, osteopetrosis (op) and toothless (tl). To determine whether this reduction in NK cell-mediated cytotoxicity is caused by decreased cell number and/or function in these osteopetrotic mutants, we quantitated NK cells by analyzing mononuclear cell suspensions labeled for two-color fluorescence with OX8 and OX19 monoclonal antibodies in a fluorescence-activated cell sorter. Flow cytometry of these double-labeled cells revealed that the percentage of NK cells (OX8+/OX19- subset) in op and tl spleens was not significantly different from that of normal spleens. These results suggest that NK cells in these osteopetrotic mutants are functionally defective. Thus aberrations in osteoclast and NK cell function coexist in these mutations, and their developmental relationships deserve further study.

A monoclonal antibody to gamma interferon blocks augmentation of natural killer cell activity induced during systemic cryptococcosis

These studies demonstrate that the cytotoxic activity of splenic natural killer (NK) cells is augmented in both nu/nu and nu/+ mice during systemic cryptococcosis. Both the kinetics and the regulation of NK cell activity differed in Cryptococcus neoformans-infected nu/nu and nu/+ mice. Greater augmentation was observed following challenge with 10(5) cells than with smaller inocula, and augmented NK cell activity was not always associated with enhanced control of systemic cryptococcosis. Infection with a nonencapsulated strain of C. neoformans induced an early but transient increase in splenic NK cell activity in nu/nu and nu/+ mice. Injection of capsular polysaccharide induced a transient augmentation of splenic NK cell activity in nu/+ mice but caused a persistent increase in splenic NK cell activity in nu/nu mice. In vivo treatment with monoclonal antibody to gamma interferon abrogated the augmentation of splenic NK cell activity induced during cryptococcal infections in both nu/nu and nu/+ mice and enhanced the susceptibility of nu/+ mice to C. neoformans to a greater extent than it did that of nu/nu mice. These results suggest that gamma interferon is an important mediator of resistance to C. neoformans.

Suppression of Natural Killer Cell Activity by Ethanol Consumption and Food Restriction

Effects of 4-week food restriction and ethanol consumption on natural killer (NK) cell activity and carcass composition were evaluated. Female, C57BL/6 mice given water (H2O) or ethanol (20% w/v, ETOH) ad libitum were placed in one of three dietary groups: unrestricted (UNR), moderately restricted (MR, 2.2 g/day), or severely restricted (SR, 1.8 g/day). Food restriction alone (MR, SR) significantly reduced body, spleen, and thymus weights; carcass lipid content (SR only); spleen cell number; and baseline and interleukin-2 (rIL-2) stimulated NK cell activities. Ethanol consumption was unaffected by food restriction and in restricted mice it did not suppress food intake. Thus, average calories derived from ethanol increased from 30% (UNR) to 40% (SR) with the degree of food restriction in these groups. Mice given ethanol and restricted food intake had at least as heavy or heavier body, spleen, and thymus weights than water-drinking (H2O) counterparts. Spleen cell number was reduced in ethanol-consuming (ETOH), food restricted groups compared with UNR H2O control. Baseline NK cell activity was suppressed 50% to 90% in all ETOH and food-restricted groups. rIL-2 stimulated NK cell activity was suppressed 18% to 76% in food restricted mice independent of ethanol intake. These results indicate that supplementary ethanol calories did not enhance NK cell activity in UNR ETOH mice, nor did they protect splenic NK cell activity from the suppressant effects of food restriction. Ethanol consumption significantly increased carcass lipid content in all groups compared with their H2O counterparts. This increase was largely responsible for the preservation of body weight in ETOH mice especially during food restriction.

Cyclosporine promotes the induction of thymic lymphomas in C57BL/6 mice initiated by a single dose of γ-radiation

We previously demonstrated that a single dose of gamma-radiation (350 rads) was able to induce thymic lymphomas in C57BL mice when followed by promoting treatment with oral cyclosporine (CsA), a non-genotoxic immunosuppressant. We have now tested the efficacy of various doses of gamma-radiation as an initiator of CsA promotion of the induction of thymic lymphomas in male C57BL mice. The effects of oral CsA on the splenic natural killer (NK) cell activity of non-irradiated and irradiated (400 rads, 1x) mice were tested by the standard 51Cr release assays against YAC-1 cells. The cumulative incidence of thymic lymphomas induced by a single dose of gamma-radiation at 100, 200, 400 and 600 rads were 10, 25, 63 and 75% respectively, after 42 weeks of CsA promotion. The splenic NK cell activity in non-irradiated mice given CsA for 4 weeks was twice as high as that in the control mice. CsA inhibited poly I:C-induced augmentation of the splenic NK cell activity. In mice given a single dose (400 rads) of gamma-radiation and CsA for 4 weeks, a similar but reduced enhancement of the splenic NK cell activity as seen in non-irradiated mice was observed. These results indicate that the efficacy of CsA promotion in the induction of thymic lymphomas is dependent on the initiating doses of gamma-radiation, and that CsA enhances host splenic NK cell activity during the early stage of tumor promotion.

マウスのナチュラルキラー細胞とマクロファージの活性におよぼす<I>Mycoplasma pulmonis</I>感染および二酸化チッ素暴露の影響

A marked enhancement was seen in activities of mouse splenic natural killer (NK) cells and macrophages one week after infection with Mycoplasma pulmonis (Mp) or exposure to nitrogen dioxide (NO2). Macrophage activity of the Mp-infected mouse showed further augmentation by exposure to NO2 while NK activity of Mp-infected mice was not affected by NO2-exposure.

Imexon and biological response modifiers in murine models of aids

The Rauscher murine leukemia retrovirus system provides an in vivo model of the human acquired immune deficiency syndrome for testing the ability of antiviral agents and biological response modifiers (BRM) to suppress viremia and retroviral disease. In the present report we examined three agents in the Rauscher retrovirus model: imexon, Ampligen and poly[I,C]-LC. Imexon reduced splenomegaly, viremia, and serum reverse transcriptase levels even when treatment was not initiated until 7 days after virus infection. Imexon also significantly prolonged the survival of infected mice. Thus it proved to be an effective antiviral agent in this system, although imexon did not completely eliminate retroviral infection in treated mice. Poly[I,C]-LC and Ampligen had immunomodulatory effects. Both of these BRM augmented the cytolytic activity of splenic natural killer (NK) cells in infected animals when treatment was initiated 24 h after infection. Poly[I,C]-LC had antiretroviral activity when administered on this schedule. In order to examine the role of NK cell augmentation in the antiviral activity of poly[I,C]-LC, we attempted to deplete NK activity by treatment with rabbit antibody to asialo GM 1, a ganglioside on the surface of murine NK cells. Combined treatment of infected mice with poly[I,C]-LC and anti-asialo GM 1 decreased the antiviral activity of poly[I,C]-LC. This finding suggests that NK cells may be involved in the antiviral effect of this BRM.

Effect of imexon treatment on friend virus complex infection using genetically defined mice as a model for HIV-1 infection

Imexon (4-imino-1,4-diazobicyclo-3.1.0-hexan-2-one) was moderately effective in the treatment of a retroviral infection in a genetically defined murine model. The animal model consisted of a Friend virus complex (FV) infection in a hybrid mouse strain, (B10.A × A/WySn)F 1, which has similarities with acquired immune deficiency syndrome (AIDS). Intraperitoneal imexon initiated 1 or 3 days after FV inoculation and continued through 13 days after inoculation significantly reduced splenomegaly, splenic cell-free virus titers and viral RNA. Viral infectious centers/10 6 splenocytes and FV titers in the plasma were reduced, though not to a statistically significant level. The effect of imexon on survival was not statistically significant which suggested that the antiviral effects were only transiently effective. Phytohemagglutinin-induced blastogenesis and percent of total T cells, T helper cells and T suppressor/cytotoxic cells in the spleens were increased, and the percentage of B cells decreased by imexon treatment of both FV-infected and uninfected mice. The splenic natural killer cell activity and interleukin-1 production were not markedly affected. Virus specific neutralizing antibody developed in both imexon- and placebo-treated FV-infected mice, although titers were lower in the imexon-treated animals.

Exercise Stress and Murine Natural Killer Cell Function

Male C3He mice were trained to run on a treadmill (final speed, slope, and duration of 30 m/min, 8 degrees, 30 min/day, 5 days/week, respectively) for 10 weeks or they remained sedentary. At the end of the training program, half of the mice were sacrificed and half were given a single bout of exercise to exhaustion (50% stepwise increases in final running speed for 2-min intervals). Splenic catecholamine concentrations, splenic natural killer cell cytolytic activity against YAC-1 tumor targets, and frequency of asialo GM1 (a murine natural killer cell surface glycolipid)-positive splenocytes were assessed. Exhaustive exercise in both trained and untrained mice reduced the in vitro killing of tumor targets by splenic natural killer cells relative to killing by splenocytes from mice which did not undergo the acute exercise bout (P less than 0.05). The frequency of asialo GM1-positive splenocytes was also reduced in the exhaustively exercised animals (P less than 0.05). Training alone, without the additional stress of exhaustive exercise, reduced the frequency of asialo GM1-positive splenocytes relative to a sedentary condition (P less than 0.05), but did not compromise natural killer cell cytolytic activity against the tumor targets. Splenic epinephrine concentrations in the exhaustively exercised animals were elevated 3- to 5-fold above the concentrations observed in trained and sedentary mice. These results suggest that a single, acute exercise bout reduces the capacity of splenic natural killer cells to kill tumor targets in vitro and that training enhances splenic natural killer cell cytolytic activity, on a per cell basis, against tumor targets.

Alterations in splenic natural killer cell activity induced by the Shionogi mouse mammary tumor

We have previously demonstrated that differential housing conditions alter the growth rate of the Shionogi mouse mammary carcinoma (SC115). The present study was undertaken to determine if natural killer (NK) cells are involved in mediating the effects of differential housing on SC115 tumour growth rate. Splenic NK cell activity was assayed at 24 h, 3 days and 1 week post-injection in both tumour- and vehicle-injected animals. Significant stimulation of splenic NK cell activity occurred 3 days post-injection of SC115 cells. However, no correlation was observed between the level of splenic NK cell activity and tumour growth rate induced by housing condition. We conclude that either splenic NK cell activity does not accurately reflect NK cell activity at the tumour site or that NK cells are not a significant regulator of the differential tumour growth rates seen in this model.

Vitamin A Deficiency Decreases Natural Killer Cell Activity and Interferon Production in Rats

We examined the effect of vitamin A deficiency on natural killer (NK) cell activity and interferon (IFN) production. Rats were weaned at 16 or 21 d of age onto semisynthetic diets containing either 0 or 4 micrograms retinol/g diet. At the time of study, retinol-depleted rats had serum vitamin A concentrations less than 7% of those of pair-fed controls. In two studies, rats exhibited no external signs of retinol deficiency, but with further depletion some symptoms were observed. Splenic NK cell activity against chromium-51-labeled YAC-1 cells was significantly decreased in vitamin A-depleted rats (22-80% of values for control rats, depending on the degree of retinol deficiency), regardless of the ratio of effector to target cells used. When vitamin A-depleted rats were repleted orally with retinol, NK cell activity was consistently normalized. To understand the possible mechanisms involved in decreasing NK cell activity, we investigated IFN production by concanavalin A-stimulated spleen cells from vitamin A-depleted, from repleted and from control animals. IFN titers were significantly decreased (22-33% of values for control rats) in supernatants of spleen cell cultures of the vitamin A-depleted rats. Repletion with vitamin A resulted in IFN activities ranging from 80 to 130% of controls. Adding alpha/beta IFN in vitro to the spleen cells of vitamin A-depleted animals increased their NK cell activity. The number of spleen cells reacting with a monoclonal antibody specific for rat NK cells was slightly lower in retinol-depleted rats, but not enough to account for the differences in NK cell and IFN activities. These data suggest that vitamin A deficiency affect the nonspecific arm of the immune system, possibly by altering the functional capacity of cells to produce lymphokines needed for the generation of an appropriate cytolytic response.

Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.

Aldicarb immunotoxicity: Functional analysis of cell-mediated immunity and quantitation of lymphocyte subpopulations

Adult female B6C3F1 mice received distilled water only or water containing 1.0, 10, or 100 ppb of aldicarb daily for 34 days. The target concentration of aldicarb present in the 100 ppb dosing solution was analytically verified. To further develop an immune profile of this compound, following aldicarb exposure, the ability of splenic natural killer cells and specifically sensitized cytotoxic T-lymphocytes to lyse YAC-1 lymphoma and P815 tumor cells, respectively, was evaluated. To supplement the functional assays, the impact of aldicarb exposure on the percentages and absolute numbers of total T-cells, T-suppressor, T-helper, and B-cells was evaluated. The absence of statistically significant effects on any of these parameters supports earlier reports that aldicarb does not result in adverse effects on the immune system of mice.

Characterization of immune alterations induced by a conditioned aversive stimulus

In this study, we investigated the immune alterations induced in rats by an aversive conditioned stimulus that had been developed through pairings with electric shock. The results showed that the conditioned stimulus induced a pronounced suppression of the mitogenic responsiveness of splenic and blood lymphocytes and a reduction in splenic natural killer cell activity. In contrast, the conditioned stimulus did not induce any alteration in the mitogenic responsiveness of lymphocytes from the mesenteric lymph nodes. The reduction in the mitogenic responsiveness of splenic lymphocytes was not related to a reduction in the level of interleukin-2 (IL-2) production, as splenic lymphocytes from subjects exposed to the conditioned stimulus showed normal levels of IL-2. Plasma corticosterone measurements showed that glucocorticoid secretion was related to the alteration of the mitogenic responsiveness of blood lymphocytes. However, plasma corticosterone levels were not related to any of the other immune measures. These findings establish that a signal for an aversive event can have a pronounced effect on immune function, but that such an effect is dependent on the particular compartment of the immune system studied. These results support the claim that glucocorticoids can induce immune alterations, but they suggest that additional pathways must be involved in the immune alterations induced by a conditioned aversive stimulus.