Levels Of Natural Killer Activity Research Articles

Both CD4+CD25+Foxp3+ Regulatory T Cells and CD3-CD56+ NK Cells Affect the Response of Newly Diagnosed Chronic Myeloid Leukemia Patients to Treatment with a Tyrosine Kinase Inhibitor

Background: Regulatory T cells (Treg) play a role in the maintenance of chronic myeloid leukemia (CML). In contrast, natural killer (NK) cells have spontaneous cytolytic activity against various tumor cells, including leukemic cells, and play an important role in immune surveillance against tumor cells. In this study, we examined changes in the number of circulating Treg and NK cells and the NK activity of patients with CML before and after treatment with a tyrosine kinase inhibitor (TKI). Our goal was to evaluate the clinical significance of this treatment on these two different cell types.Patients and Methods: Treg and NK cells were characterized and quantified by flow cytometry in 13 newly diagnosed CML patients, 5 newly diagnosed patients with other myeloproliferative neoplasms (MPN), and 15 healthy controls. We analyzed all CML patients diagnosed with chronic phase (CP) disease and MPN patients from January 2015 and June 2016. Patients with CML, who were initially treated with TKI between January 2015 and June 2016 and were followed up for more than one year, were enrolled in the study. The mRNA level of the BCR-ABL fusion gene was analyzed by real-time PCR analysis at approximately 6-month intervals during the first year and at 12-month intervals thereafter. After obtaining informed consent, blood was drawn from all patients at initial diagnosis and 12 months after the start of treatment. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at effector: target (E: T) ratios of 20:1.Results: The median age for CML patients was 65 years (range: 47-84), and 9 of the patients were male. The median period of observation was 543 days. After 12 months, 8 CML patients (MMR-group) had a major molecular response (MMR), and 5 patients (NR-group) did have not a MMR. Before treatment, the absolute number of Treg cells was significantly lower in the MMR-group than that in the NR-group (27.6 ± 10 vs. 50.0 ± 24.0 /μL, p=0.037). Similarly, the absolute number of Treg cells after treatment was significantly lower in both the MMR-group (27.3±10.3 /μL) and the MPN-group (27.6±11.5 /μL) than that in the NR-group (40.0±9.9 /μL, p=0.05, p<0.01, respectively). Treg% values in peripheral lymphocytes before treatment in the NR (7.4±2.1%) were significantly higher than that in MMR group and in the healthy group (4.7±1.7%, 5.5±1.1%, p=0.024, p=0.016, respectively). The absolute number and percentages of CD3-CD56+ NK cells before treatment as well as after treatment did not differ between each group. However, NK activity before treatment in the NR-group (15±1.4%) was significantly lower than that in MMR-group (47.2± 11.6 vs 41.7±4.1%, p<0.01, p<0.01, respectively). With time, the number of CD8+ T cells in the NR group decreased significantly from 940±570 to 348± 167/μL (p=0.040), and the Treg% increased significantly from 0.18± 0.13 to 0.84± 0.29% (p<0.01). The relative percentage of CD3+CD4+ T cells (4.7±4.4%) in the 15 CML patients increased remarkably after treatment (12.8±4.3%, p<0.01) but the absolute number did not change.Conclusions: Treg cell numbers were significantly lower in the CML patients with a MMR compared to that in the patients without a MMR. Although the number of NK cells did not change in the MMR group compared to that in the NR group, the NK activity was significantly higher. While the mechanisms for these changes in cell populations are not completely understood, it is also important to note that we have not yet studied the cellular source and production of cytokines which enhance NK activity. The data suggest that some immunological mechanisms are involved in the response to TKI. Moreover, the increased levels of NK activity in patients with MMR suggest that not only Treg cell numbers but also NK activity may have clinical importance in evaluating the effects of TKI therapy. DisclosuresNo relevant conflicts of interest to declare.

Immunological mechanism of low-dose priming radiation resistance in walker-256 tumor model mice.

The aim of the present study was to investigate whether low-dose priming radiation induces antitumor immunity that can be augmented by the modulation of natural killer (NK) cell and cytokine activity using a mouse tumor model. Walker-256 cells were injected into the right flank of male BALB/c mice. At 7 days after inoculation, mice were divided into three groups, including group 1,2,3. In group 1 the mice were without radiation, in group 2 the mice were received 2 Gy radiation only, and in group 3 the mice were radiated with a priming dose of 75 mGy followed by 2 Gy radiation after 24 h. On day 21 following the radiation, the tumors were removed and the tumor index (tumor weight as a percentage of body weight) was calculated. At 1, 7, 14 and 21 days following the 2 Gy radiation, mouse splenocytes were isolated to analyze the NK activity and measure the production of the cytokines interleukin-1β, interferon-γ and tumor necrosis factor-α by ELISA. Apoptosis was also measured by flow cytometry. The results demonstrated that priming radiation significantly delayed the tumor growth and prolonged the median survival time to 38 days compared with the 31-day survival in the 2 Gy radiation group. The percentage of apoptotic cells was significantly higher in the mice that received 75 mGy + 2 Gy radiation compared with that in the mice that received 2 Gy alone; by contrast, mice that were not irradiated exhibited a relatively low level of apoptosis. The primed mice had a higher level of NK activity as compared with the mice exposed to 2 Gy radiation only or mice that were not irradiated. Furthermore, cytokine expression remained at a higher level in mice receiving priming dose of radiation compared that in the mice receiving only 2 Gy radiation. In conclusion, the results indicated that low-dose priming X-ray radiation may enhance the NK activity and the levels of cytokines, and that the immune response serves an important role in anticancer therapy, including radiotherapy.

Job strain in different types of employment affects the immune response

The immune system, in cooperation with neuroendocrine functions, defends from cancer and infections mainly by the activity of blood natural killer (NK) cells. Blood NK activity may be influenced by the type of employment since work is the central part of life; moreover, job stress is a situation affecting both neuroendocrine and immune systems. This study examines anxiety (by STAI 1 and 2), job strain (by the Karasek's JCQ) and blood NK activity (by an in vitro radio-isotopic method) of 134 male workers. These men, over 38 years old with stable employment, were working in factories, in construction yards, in offices, as hospital attendants or as self-employed craftsmen. Workers in factories and in construction yards, with high job strain, showed lower NK activity, while office employees, with low job demand, and craftsmen with low anxiety and elevated decision latitude, showed higher NK activity; the level of NK activity of the hospital attendants was between the other groups. In conclusion, this study confirms that the type of employment, related to job stress, affects blood NK activity. Moreover, blood NK activity may be used in the bio-monitoring of workers at high risk.

Effect of forest bathing trips on human immune function

In Japan, a forest bathing trip, called "Shinrinyoku" in Japanese, is a short, leisurely visit to a forest; it is regarded as being similar to natural aromatherapy. This review focuses on the effects of forest bathing trips on human immune function. Beginning in 2005, adult Japanese individuals, both male and female, participated in a series of studies aimed at investigating the effect of forest bathing trips on human immune function. The subjects experienced a 3-day/2-night trip to forest areas, and blood and urine were sampled on days 2 (the first sampling during each trip) and 3 (the second sampling during each trip), and on days 7 and 30 after the trips. Natural killer (NK) activity, the numbers of NK, granulysin-, perforin-, and granzymes A/B-expressing lymphocytes in the blood, and the concentration of urinary adrenaline were measured. The same measurements were made before the trips on a normal working day as a control. The mean values of NK activity and the numbers of NK, granulysin-, perforin-, and granzymes A/B-expressing cells on forest bathing days were significantly higher than those on the control days, whereas the mean values of the concentration of urinary adrenaline on forest bathing days were significantly lower than that on the control days in both male and female subjects. The increased NK activity lasted for more than 30 days after the trip, suggesting that a forest bathing trip once a month would enable individuals to maintain a higher level of NK activity. In contrast, a visit to the city as a tourist did not increase NK activity, the numbers of NK cells, or the level of intracellular granulysin, perforin, and granzymes A/B. These findings indicate that forest bathing trips resulted in an increase in NK activity, which was mediated by increases in the number of NK cells and the levels of intracellular granulysin, perforin, and granzymes A/B.

The role of natural killer cells in resistance to coccidiosis: investigations in a murine model.

Natural killer (NK) activity, detected by the lysis of Yac-1 target cells, was examined in splenic and mesenteric lymph node (MLN) cells throughout the course of infection with Eimeria vermiformis in BALB/c and C57B1/6 (B6) mice. These strains are, respectively, relatively resistant and susceptible to primary infections, which render them equally, and completely, resistant to challenge. Resting levels of NK activity were higher in B6 than in BALB/c, and B6 responded earlier in the course of infection than BALB/c, but splenic peak values were higher in BALB/c; the pattern of response in MLN cells was similar in both strains, but the peak was higher in BALB/c. At the time (7 days p.i.) of peak NK response in BALB/c mice there was, depending upon the choice of NK-resistant/lymphokine-activated killer (LAK)-sensitive target cells, either little (P388D1), or no (P815) splenic LAK activity. Challenge of immunized BALB/c mice did not evoke a detectable NK response. Although the higher NK activity in BALB/c mice correlated with greater control of primary infection, depletion of NK activity (demonstrated in splenic cells) in vivo by treatment with anti-asialo GM1 antibodies did not greatly affect the course of infection. Furthermore, this treatment did not augment the exacerbation of infection produced by treatment with anti-interferon-gamma (IFN-gamma) MoAb, indicating that, at least in this system, NK cells are not a fundamentally important source of this controlling cytokine of eimerian infections. The results suggest that NK cells may not greatly influence the outcome of coccidial infections.

Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis

Enhanced natural killer (NK) activity and normal lymphocyte antibody-dependent cellular cytotoxicity (ADCC) were observed in 16 patients with a diagnosis of progressive systemic sclerosis (PSS). Higher NK activity levels were observed against NK-sensitive K562 target cells, while the NK-resistant P815, Daudi and Raji cell lines were not lysed. Cytofluorimetric studies and morphological analysis of peripheral blood lymphocytes (PBL) showed an increased number of CD16 positive cells and large granular lymphocytes (LGL), indicating that the enhancement observed was probably attributable to an increase in the number of circulating NK cells.

Correlation between NK function and response to trastuzumab in metastatic breast cancer patients

BackgroundTrastuzumab is a monoclonal antibody selectively directed against Her2 and approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include mediation of antibody-dependent cellular cytotoxicity (ADCC) by triggering FcγRIII on natural killer (NK) cells. This study addresses the correlation between overall NK function and trastuzumab's clinical activity.Subjects and methodsClinical and immunological responses were assessed in 26 patients receiving trastuzumab monotherapy as maintenance management after chemotherapy (8 mg/kg load and then standard doses of 6 mg/kg every 3 weeks). Cytotoxic activity against the MHC class I-negative standard NK target K562 cell line and HER2-specific ADCC against a trastuzumab-coated Her2-positive SKBR3 cell line were assessed in peripheral blood mononuclear cells (PBMC) harvested after the first standard dose. After six months, seventeen patients were scored as responders and nine as non-responders according to the RECIST criteria, while Progression-Free Survival (PFS) was calculated during a 12 months follow-up.ResultsThe responders had significantly higher levels of both NK and ADCC activities (p < 0.05) that were not different from those of eleven normal controls. The NK activity of the non-responders was significantly (p < 0.05) lower than that of the normal controls. At twelve months, there was a marked correlation between PFS and NK activity only. PFS was significantly longer in patients with high levels of NK activity, whereas its pattern was unrelated to high or low ADCC activity.ConclusionOne of the mechanisms of action of trastuzumab is NK cell-mediated ADCC lysis of the Her2-positve target cell. We show here that its potency is correlated with the short-term response to treatment, whereas longer protection against tumor expansion seems to be mediated by pure NK activity.

Dissociation of inflammatory markers and natural killer cell activity in major depressive disorder

Major depressive disorder is associated with increases in infectious disease risk as well as the incidence of inflammatory disorders. Declines of natural killer (NK) cell activity are reliably found in depression, whereas other studies report evidence of inflammation in depressed patients. The potential association between NK activity and circulating markers of immune activation has not been previously examined in the context of major depression. In this study, we measured levels of NK activity, circulating levels of interleukin-6 (IL-6), soluble interleukin-2 receptor, and acute phase proteins in 25 male patients with current major depressive disorder and 25 age, gender, and body weight comparable controls. As compared to controls, patients with major depressive disorder showed lower NK activity ( p = .05) and higher circulating levels of IL-6 ( p < .05). Levels of NK activity were not correlated with IL-6 or with other markers of immune activation. The independent effect of depression on inflammatory markers and natural killer immune responses has implications for understanding individual differences in the adverse health effects of major depressive disorder.

Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on day 13 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the nuclear suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector : target system at a ratio of 50:1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 30% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 +/- 13.4 and 75.0 +/- 12.5 microm2, respectively) and pseudopregnant (97.5 +/- 4.9 and 69.2 +/- 3.5 microm2) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 +/- 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109.1 +/- 5.2 microm2. The data obtained confirm the known data on a low activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.

Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome

Macrophage activation syndrome (MAS) has been reported in association with many rheumatic diseases, most commonly in systemic juvenile rheumatoid arthritis (sJRA). Clinically, MAS is similar to hemophagocytic lymphohistiocytosis (HLH), a genetic disorder with absent or depressed natural killer (NK) function. We have previously reported that, as in HLH, patients with MAS have profoundly decreased NK activity, suggesting that this abnormality might be relevant to the pathogenesis of the syndrome. Here we examined the extent of NK dysfunction across the spectrum of diseases that comprise juvenile rheumatoid arthritis (JRA). Peripheral blood mononuclear cells (PBMC) were collected from patients with pauciarticular (n = 4), polyarticular (n = 16), and systemic (n = 20) forms of JRA. NK cytolytic activity was measured after co-incubation of PBMC with the NK-sensitive K562 cell line. NK cells (CD56+/T cell receptor [TCR]-αβ-), NK T cells (CD56+/TCR-αβ+), and CD8+ T cells were also assessed for perforin and granzyme B expression by flow cytometry. Overall, NK cytolytic activity was significantly lower in patients with sJRA than in other JRA patients and controls. In a subgroup of patients with predominantly sJRA, NK cell activity was profoundly decreased: in 10 of 20 patients with sJRA and in only 1 of 20 patients with other JRA, levels of NK activity were below two standard deviations of pediatric controls (P = 0.002). Some decrease in perforin expression in NK cells and cytotoxic T lymphocytes was seen in patients within each of the JRA groups with no statistically significant differences. There was a profound decrease in the proportion of circulating CD56bright NK cells in three sJRA patients, a pattern similar to that previously observed in MAS and HLH. In conclusion, a subgroup of patients with JRA who have not yet had an episode of MAS showed decreased NK function and an absence of circulating CD56bright population, similar to the abnormalities observed in patients with MAS and HLH. This phenomenon was particularly common in the systemic form of JRA, a clinical entity strongly associated with MAS.

Preferential apoptosis of CD56dim natural killer cell subset in patients with cancer.

Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.

The Effects of Sex, Menstrual Cycle, and Oral Contraceptives on the Number and Activity of Natural Killer Cells

Objectives. The aim of this study was to study the impact of sex, the menstrual cycle, and the use of oral contraceptives (OC) on the number and activity of natural killer (NK) cells.Methods. Both the number and the activity of NK cells were assessed per milliliter of blood, and NK activity (NKA) per NK cell and per lymphocyte was calculated. NKA was measured in each subject using a whole blood assay, which preserves the plasma and all blood cells, and using a washed blood assay, in which plasma is replaced with an artificial medium. The subjects were young (20–29 years old) women with a regular menstrual cycle (n = 39; 26 tested on both the follicular and the luteal phases), age-matched women who use OC (n = 26), and age-matched men (n = 20).Results. Men showed markedly and significantly higher NKA than women with regular menstrual cycles or women using OC, who had the lowest levels of NKA. No significant differences in blood concentration of NK cell were found. Differences in NKA were of similar magnitude in the whole and washed blood assays per milliliter of blood, per NK cell, or per lymphocyte. The menstrual cycle had no significant effect on activity levels of NK cells, but during the periovulatory phase, the number of NK cells per milliliter of blood increased significantly.Conclusions. The observed differences are independent of the presence of serum factors during the in vitro assessment of NKA, but may be related to chronic exposure to sex steroids and to fluctuation in the NK cell expression of β-adrenoceptors.

Natural Killer Cell Activity and Resistance to Tumor Metastasis in Prepubescent Rats: Deficient Baselines, but Invulnerability to Stress and β-Adrenergic Stimulation

Although young children and animals exhibit high rates of tumor development, little is known about natural killer (NK) cell activity in the very young. We recently provided direct evidence that reduced levels of NK activity in prepubescent rats underlie higher levels of susceptibility to metastasis. The aim of the current study was to further characterize NK activity and tumor resistance in prepubescent rats, specifically with respect to the effects of stress and sex, as these factors have been shown to modulate tumor development in adult populations. Two NK outcomes were assessed: levels of whole blood NK cytotoxicity and lung tumor retention of NK-sensitive MADB106 tumor cells which metastasize only to the lungs. The corticosterone (CS) response to surgery was also assessed. In the first set of experiments, prepubescent males and females (36 days of age) and mature males (98 days) were subjected to abdominal surgery and 5 h later were either tested for plasma CS levels or challenged with MADB106 tumor cells. The findings indicated that whereas surgery increased CS levels in the young rats to similar levels observed in mature animals, surgical stress did not increase lung tumor retention in the young animals, despite exerting marked and significant effects in the mature rats. These findings persisted when lower tumor loads were used in the young rats to compensate for the markedly reduced resistance to metastasis in this population. Because surgery involves activation of the sympathetic nervous system (SNS) which is known to regulate NK activity, we assessed the impact of the β-adrenergic agonist, metaproterenol, on NK activity and on lung tumor retention. Metaproterenol (0.8 mg/kg, 1 h before testing) resulted in a large suppression of NK activity and resistance against MADB106 metastasis in mature males and females, but not in prepubescent animals. In mature, but not in young animals, males exhibited higher baseline levels of NK activity. Taken together, these findings indicate that NK cells of prepubescent rats are resistant to β-adrenergic stimulation, and suggest that prepubescent rats are markedly less responsive to SNS-induced suppression of NK activity, which may underlie their invulnerability to the effects of surgery on MADB106 metastasis.

Decreased Natural Killer Cell Responses and Altered Interleukin‐6 and Interleukin‐10 Production in Alcoholism: An Interaction Between Alcohol Dependence and African‐American Ethnicity

Epidemiological data indicate that among alcoholics African-American men have higher rates of infections compared with white alcoholics. Alcohol is thought to reduce natural killer (NK) cell responses and to alter cellular immunity by changing the relative balance of Th1 versus Th2 cytokine response profiles. However, translation of these basic immunological observations into the clinical setting is limited, and what clinical data are available are contradictory, in part, because of heterogeneity within the alcoholic patient population. Whether ethnic characteristics contribute to variation in the immunological effects of alcohol is not known. This study compared NK activity, interleukin (IL)-2-stimulated NK activity, and concanavalin A-stimulated peripheral blood mononuclear cell production of Th1 (IL-12 and IL-2), Th2 (IL-10), and proinflammatory (IL-6) cytokines in 31 hospitalized chronic alcoholic patients and 31 age-matched controls who were stratified on the basis of ethnicity. NK-cell responses were significantly different across the four groups, and African-American alcoholics showed the lowest levels of NK activity (F = 9.5;p < 0.001) and IL-2-stimulated NK activity (F = 2.9; p < 0.05). Compared with the other three groups, African-American alcoholics also showed lower levels of IL-6 (F = 7.2;p < 0.01) and higher levels of IL-10 (F = 4.9;p < 0.05). Stimulated production of IL-2 and IL-12 were similar in the four groups. Regression analyses showed that alcohol dependence and ethnicity predicted NK activity, whereas the interaction between alcohol dependence and ethnicity predicted levels of IL-6 and IL-10. In summary, the immunological effects of alcohol dependence are modified by ethnicity status, consistent with the increased health risks found in African-American alcoholics.

Decreased Natural Killer Cell Responses and Altered Interleukin-6 and Interleukin-10 Production in Alcoholism: An Interaction Between Alcohol Dependence and African-American Ethnicity

Background: Epidemiological data indicate that among alcoholics African-American men have higher rates of infections compared with white alcoholics. Alcohol is thought to reduce natural killer (NK) cell responses and to alter cellular immunity by changing the relative balance of Th1 versus Th2 cytokine response profiles. However, translation of these basic immunological observations into the clinical setting is limited, and what clinical data are available are contradictory, in part, because of heterogeneity within the alcoholic patient population. Whether ethnic characteristics contribute to variation in the immunological effects of alcohol is not known. Methods: This study compared NK activity, interleukin (IL)-2–stimulated NK activity, and concanavalin A–stimulated peripheral blood mononuclear cell production of Th1 (IL-12 and IL-2), Th2 (IL-10), and proinflammatory (IL-6) cytokines in 31 hospitalized chronic alcoholic patients and 31 age-matched controls who were stratified on the basis of ethnicity. Results: NK-cell responses were significantly different across the four groups, and African-American alcoholics showed the lowest levels of NK activity (F= 9.5;p < 0.001) and IL-2–stimulated NK activity (F= 2.9;p < 0.05). Compared with the other three groups, African-American alcoholics also showed lower levels of IL-6 (F= 7.2;p < 0.01) and higher levels of IL-10 (F= 4.9;p < 0.05). Stimulated production of IL-2 and IL-12 were similar in the four groups. Regression analyses showed that alcohol dependence and ethnicity predicted NK activity, whereas the interaction between alcohol dependence and ethnicity predicted levels of IL-6 and IL-10. Conclusions: In summary, the immunological effects of alcohol dependence are modified by ethnicity status, consistent with the increased health risks found in African-American alcoholics.

Differences in NK Cell Function in Mice Bred for High and Low Aggression: Genetic Linkage between Complex Behavioral and Immunological Traits?

In previous studies, we found differences in cellular immune responsiveness in Institute for Cancer Research (ICR) mice selectively bred for high and low levels of aggression. Compared to the high aggressive line, the low aggressive line had low levels of natural killer (NK) and T cell activity and increased susceptibility to tumor development. To dissect further this novel association, experiments were designed to test two competing hypotheses. The first hypothesis was that the phenotypic expression of the line differences in NK cell activity are dependent on and regulated by the expression of high and low levels aggressive behavior in the lines. The alternative hypothesis was that the differences in immune status are independent of the expression of aggression by the lines, suggesting linkage between a subset of genes involved in determining these complex behavioral and immunological traits or pleiotropic gene effects on both traits. In Experiment 1, three conditions of postweaning social experience (mice singly housed, group housed within line, or group housed between lines) were tested in males to determine whether experiential conditions which modify the expression of aggression would in turn modify the line differences in NK cell activity. This experiment revealed that the difference in NK cell activity between high aggressive and low aggressive male mice was attributable to line only. The different postweaning social conditions examined had no effect on modifying the differences in NK activity, and social dominance hierarchy did not correlate with levels of NK cell activity. Whereas males of the high and low lines exhibit differences in aggressive behaviors across most contexts, females do not exhibit such differences except in response to an intruder during the postpartum period. Therefore, in Experiment 2 we compared the NK cell activity of nulliparous females of the high and low aggressive lines. Under these conditions, females of the low aggressive line had low levels of NK activity compared to high aggressive females (differences comparable to those seen between males of the high and low lines). Taken together, these experiments lend support to the hypothesis that this association may be due to a genetic linkage between subsets of genes involved in determining these complex behavioral and immunological traits, or may possibly represent a fortuitous association which occurred during the selective breeding.

The Regulation and Biological Activity of Interleukin 12

Interleukin 12 (IL-12) is a pleiotropic cytokine and mediates several biological activities on human T and natural killer (NK) cells, including induction of IFN-γ production, enhancement of cell-mediated cytotoxicity and comitogenic effects on resting T-cells. The major cellular sources producing IL-12 are antigen-stimulated monocytes, macrophages, and B-cells isolated from peripheral blood mononuclear cells (PBMC). Our laboratory has investigated the regulation of IL-12 gene expression in both cord blood and adult PBMC, and the effects of IL-12 on induction of IFN-γ production, NK, and lymphokine-activated killer (LAK) cytotoxicity. IL-12 mRNA expression and protein production in LPS-stimulated cord blood MNC were 3–4 fold decreased when compared with adult PBMC. There were no differences between cord blood and adult PBMC in both basal levels of transcription or the degree of transcriptional activation of the IL-12 gene. Additionally, the half-life of IL-12 p40 mRNA was 3-fold lower in activated cord blood compared to adult PBMC. Exogenous IL-12 induced a significant increase of IFN-γ from both cord and adult PBMC. Cord MNC has significantly reduced levels of NK activity, and IL-12 significantly enhanced cord blood NK cytotoxicity upto similar levels in adult PBMC. IL-12 also significantly enhanced cord blood NK and LAK activities against a broad range of neuroblastoma, leukemia, and lymphoma cell lines. Lower doses of IL-12 and IL-15 concomitantly generated either synergistic or additive effects on cord blood NK and LAK cytotoxicities. In light of the important biological functions of IL-12, reduced expression and production of IL-12 from activated cord blood may contribute to the immaturity of cord blood cellular immunity and contribute, in part, to decreased severe graft vs. host disease following unrelated cord blood stem cell transplantation. IL-12 enhancement of IFN-γ, NK, and LAK activity in activated cord blood MNC upto comparable levels in adult PBMC suggests that exogenous IL-12 stimulation can compensate for the immaturity in cord blood cellular immunity. These characteristics of IL-12 biological activity strongly suggest its potential usefulness in future cancer immunotherapy.

In vitro effects of IL-12 and IL-2 on NK cells, cytokine release and clonogenic activity in myelodysplastic syndromes (MDS).

We evaluated the in vitro effects of IL-12, alone and in association with IL-2 on MDS bone marrow and peripheral blood cells. Thirty-six patients and 14 healthy subjects were studied. Natural killer-activity (NK-a) levels and lymphocyte immunophenotypes were determined in fresh bone marrow (BMMNC) and peripheral blood mononuclear cells (PBMNC), which then were resuspended in medium containing IL-2, IL-12 or IL-2 + IL-12 for 7 days. Re-evaluation of NK-a levels, lymphocyte immunophenotypes, clonogenic activity and cytokine release showed that, unlike IL-2, IL-12 did not significantly increase NK-a or CD3-/56+ cell levels in either bone marrow or peripheral blood; IL-2 + 12 led to a significant increase that fell between the values reached by each cytokine alone. IL-2 + 12 and, although to a lesser extent, also IL-12 alone induced the release of large amounts of gamma-IFN and alpha-TNF. In addition, the number of clusters particularly decreased in the samples treated with IL-2 + 12 and IL-12 alone. Clonogenic activity was not modified after stimulation with any of the treatment. These data suggest that IL-12 induces the release of inhibitory cytokines in normal as well as MDS cells and that it could be used in patients with elevated bone marrow blastosis.

Increased surgery-induced metastasis and suppressed natural killer cell activity during proestrus/estrus in rats.

We have previously reported sex- and estrous-related differences in host resistance to the metastatic development of a mammary adenocarcinoma cell line, MADB106, in the Fischer 344 (F344) rat. In other studies, we found that surgery suppressed natural killer (NK) cell activity and increased the NK-sensitive metastatic development of MADB106 tumor cells. The current study was designed to explore whether sex or estrous phase at the time of surgery impacts the degree of such deleterious effects of surgery. Such estrous effects could be related to an ongoing clinical debate regarding the importance of the timing of breast cancer surgery with the menstrual cycle in premenopausal women. Mature F344 males and cycling females underwent either experimental laparotomy with halothane anesthesia, halothane anesthesia alone, or were untreated. Five hours after surgery, animals either were injected with radiolabeled MADB106 tumor cells and assessed for lung tumor cell retention 12 hours later, or underwent blood withdrawal for in vitro assessment of NK cell activity. MADB106 tumor cells metastasize only to the lungs, and lung tumor cell retention is: a) an early indicator of the number of metastases that would develop weeks later, and b) highly sensitive to in vivo levels of NK activity. This mammary adenocarcinoma cell line is syngeneic to the inbred F344 strain of rats used in our studies, thus constituting a model for breast cancer metastasis. The results indicated that sex, estrous phase, and surgery interacted in their effects on NK cell activity and tumor metastasis. MADB106 lung tumor cell retention was increased by surgery in both sexes (2- to 3-fold) compared to the anesthesia only and control groups. This increase, however, was significantly greater in proestrus/estrus (P/E) females than in metestrus/diestrus (M/D) females. Among the control animals, females in P/E exhibited significantly less NK cytotoxic activity compared to the males, and the NK activity exhibited by females in M/D was between these two groups. Surgery suppressed NK cytotoxic activity to a similar level in all groups. Possible implications of these findings for the surgical care of women with breast cancer are discussed.

Alzheimer caregiver stress: basal natural killer cell activity, pituitary-adrenal cortical function, and sympathetic tone.

The association between Alzheimer caregiving and natural killer (NK) cell activity and basal plasma levels of adrenocorticotropic hormone (ACTH), cortisol, beta-endorphin, prolactin, epinephrine, norepinephrine, and neuropeptide Y was determined in 100 spousal Alzheimer caregivers and 33 age- and gender-comparable control volunteers upon intake into a study of the psychological and physiologic impact of caregiving. The relationship between these physiologic measures and individual characteristics such as age, gender, medical status, severity of stress, severity of depressive symptoms, and caregiver burden was tested. In addition, the association between NK activity and alterations of the neuroendocrine measures was investigated. As compared to controls, the Alzheimer caregivers had similar levels of NK activity and of basal plasma neuroendocrine hormones and sympathetic measures. While older age and male gender status were associated with increased levels of ACTH, neither medical caseness, severity of life stress, nor severity of depressive symptoms was associated with alterations in any of the multiple physiologic domains. Classification of Alzheimer caregiver burden identified caregivers who were mismatched in terms of the amount of care they were required to provide and the amount of respite time received. The mismatched caregivers had significantly higher basal plasma ACTH but no change in other physiological measures, as compared to non-mismatched caregivers. NK activity was negatively correlated with plasma levels of neuropeptide Y but not with any of the other neuroendocrine measures. Based on this cross-sectional evaluation of NK activity and neuroendocrine and sympathetic measures, we conclude that most Alzheimer caregivers do not show evidence of altered basal physiology.