Depressed natural killer cell activity in acute myocardial infarction.

Natural killer (NK) cell activity against K562 target cells was measured in patients within 24 h of acute myocardial infarction (AMI) and regularly thereafter for 6 weeks. NK cell activity was suppressed on days 1, 3, and 7 (P less than 0.01), day 14 (P less than 0.05) and at 6 weeks (P = 0.05) when compared to controls. Interferon, interleukin 2 and indomethacin enhanced NK cell activity on all days measured, but did not completely restore the defective NK cell activity. Serum from the patients did not suppress the NK cell activity of healthy mononuclear cells. The number of NK cells, identified as large granular lymphocytes (LGL), measured on days 1, 3, and 14 and at 6 weeks was not reduced in comparison to that of controls. Thus, the defective NK cell activity can be characterized as functional.     

In vitro natural killer and killer cell functions in uremia

In uremia, mitogen responses of lymphocytes are decreased as is their resistance to the immunosuppressive effect of steroids. We examined the functions of natural killer (NK) and killer (K) cells from uremic patients and their sensitivity to in vitro immunosuppression by methylprednisolone. NK- and K-cell activity was as high as that of control subjects, and remained so at different titrations of effector cells. The functions of NK and K cells from uremic patients tended to be more resistant to methylprednisolone than those of control NK and K cells. Thus, while uremia impairs T-cell functions and T-cell sensitivity to steroids in vitro, the cytotoxic effector cell functions which are not dependent on cell proliferation remain unaffected.     

Effect of human fibroblast interferon on natural killer cell activity: Stimulation in vitro and inhibition in vivo

Summary This paper describes the influence of human fibroblast interferon (IFN-) on the cytotoxic activity of natural killer cells (NK) in vitro and in vivo using the blood of healthy donors and myeloma patients. IFN- stimulates NK activity against all target cells tested in vitro in a dose-dependent way up to 250% of pretreatment values. At higher IFN concentrations, stimulation returned to baseline values. Stimulation was most pronounced in the lowest lymphocyte to target cell ratio. 1- to 2-h preincubation of effector cells with IFN was enough to achieve maximal stimulation. The effector cells of IFN-treated myeloma-patients, or patients with herpes zoster, showed a clear reduction of toxicity against all cells tested during the first infusion, as compared to the pretreatment values.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Wa 139/13-4)     

Combination chemo-immunotherapy: kinetics of in vivo and in vitro generation of natural killer cells and lymphokine-activated killer cells in the rat.

Rats received a single high dose of cyclophosphamide (Cy) (150 mg/kg), followed 48 h later (on day 0) by immunization with a T cell-dependent soluble antigen, ovalbumin in Freund's complete adjuvant (FCA). The effect of this treatment on lymphoid cell subpopulations in the spleen, natural killer (NK) cell and interleukin-2 (IL-2) induced lymphokine-activated killer (LAK) cell activity was examined. Cy (with and without ovalbumin) caused a large relative increase (by day 14) in splenic OX8+, OX19- cells with NK morphology. A marked relative increase in fresh NK cell activity was noted after Cy + ovalbumin, but not consistently after Cy alone. Elevated NK activity was Cy dose- and time-dependent, was evident within 7 days post Cy/ovalbumin and persisted for at least 28 days. Pooled splenic mononuclear cells (MNC), obtained 14 days after Cy/ovalbumin, lost all cytolytic activity against YAC-1 cells when cultured in the absence of human recombinant IL-2 (rIL-2). In contrast, similarly maintained cells from normal rats displayed NK activity higher than normal 'fresh' levels. Upon culture in medium containing 500 U/ml rIL-2, however, 'augmented' NK activity was equivalent, on a per-cell basis, in both normal and Cy/ovalbumin-pretreated groups. LAK activity generated in vitro (i.e. against NK-resistant target cells) was significantly lower in the latter group, and the overall yield of cells was reduced. By day 21 after Cy/ovalbumin, augmented NK activity was significantly greater than controls, on a per-cell and total culture yield basis. Moreover, LAK activity was now similar between groups. It is concluded that the chemotherapy/immunization protocol which we have used can greatly enhance NK activity in vivo and that these cells are responsive to induction of LAK activity by IL-2 in vitro.     

Effects of in vivo hyperthermia on natural killer cell activity, in vitro proliferative responses and blood mononuclear cell subpopulations.

This work was designed to test the hypothesis that elevations in body temperature of humans induce immunostimulation. Eight healthy volunteers were immersed in a water bath (water temperature 39.5 degrees C) for 2 h, during which their rectal temperature rose to 39.5 degrees C. On a later day they served as their own controls, being immersed into thermoneutral water (34.5 degrees C) for 2 h. Blood samples were collected before immersion, at body temperatures of 38 degree C, 39 degree C and 39.5 degree C, and 2 h after water immersion. The interleukin-2 (IL-2) enhanced natural killer (NK) cell activity (lysis per fixed number of mononuclear cells), as well as the proportion and total number of NK cells (CD16+ cells), increased significantly during hyperthermia compared with control values. The lymphocyte proliferative responses did not differ significantly between hyperthermia and thermoneutral conditions. The proportion of pan-T (CD3+) cells was maximally depressed 2 h after water immersion. The decreased proportion of CD3+ cells was mainly due to a decreased percentage of CD4+ cells (not significant). The proportion of B cells (CD19+ cells) did not fluctuate significantly, while a marked and significant increase in monocyte proportion (CD14+ cells) was found 2 h after hyperthermia. Two hours after hot water immersion the lymphocyte concentration declined while the neutrophil and monocyte concentrations were augmented. Induced hyperthermia causes significantly increased serum cortisol, plasma norepinephrine and plasma epinephrine concentrations compared to controls. It is possible that the altered immune functions induced by elevated body temperature can be ascribed to altered composition and function of blood mononuclear cells induced by elevated levels of stress hormones.     

Further studies of natural killer cell function in Chediak-Higashi patients.

Spontaneous natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) of blood lymphocytes against five human tumour cell lines (K562, Molt-4, HL-60, Chang, Daudi) and three mouse tumour lines (YAC, P815, RBL-5) were ten- to 100-fold lower than normal in six patients with Chediak-Higashi (CH) disease. NK and ADCC were defective at 4 hr, and less so at 18 hr. The NK activity in normals and CH patients was mediated in part by FcR+, E- effector cells. ADCC against human erythrocytes was normal in CH patients, as were lectin-dependent cytolysis and mixed lymphocyte proliferative responses. Phagocytosis of antibody-coated ox erythrocytes was normal in CH patients as well. These observations confirm that the CH syndrome is associated with a profound and selective defect in NK and ADCC activity against tumour cells, whereas other mononuclear cell-mediated functions are normal.     

Determination of natural killer cell function by flow cytometry.

Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes that mediate non-major histocompatibility complex-restricted cytotoxicity of foreign target cells. The "gold standard" assay for NK cell activity has been the chromium release assay. This method is not easily performed in the clinical laboratory because of difficulties with disposal of radioactive and hazardous materials, short reagent half-lives, expense, and difficulties with assay standardization. We describe a flow cytometric assay for the clinical measurement of NK cell activity. This study compared the chromium release assay and the flow cytometric assay by using clinically relevant specimens. There were no significant differences between the two assays in the measurement of lytic activity for 17 peripheral blood specimens or in reproducibility in repeated samplings of healthy individuals. We also established a normal range of values for NK activity in healthy adults and identified a small cluster of individuals who have exceptionally high or low levels of NK activity. The flow cytometric assay was validated by testing specimens from subjects expected to have abnormally low levels of NK activity (pregnant women) and specimens from healthy individuals in whom the activity of NK cells was enhanced by exposure to interleukin-2 or alpha interferon. Treatment with these agents was associated with a significant increase in NK activity. These results confirm and extend those of others, showing that the flow cytometric assay is a viable alternative to the chromium release assay for measuring NK cell activity.     

Exercise-induced alterations in natural killer cell number and function

To study the effects of exercise on natural killer (NK) cell number and activity (NKCA) healthy male (n = 32) and female (n = 32) subjects were randomly assigned to an exercise or control condition. Exercise involved a continuous incremental protocol consisting of cycling for three periods of 6 min at work rates corresponding to 55%, 70% and 85% peak oxygen uptake ( ). Blood samples were drawn at baseline, at 6 min, 12 min and 18 min during exercise, and at 2 h following completion of exercise. Relative to both baseline and control conditions, exercise resulted in an increase in the number of circulating lymphocytes. The proportion of T cells (CD3⁺) and B cells (CD19 ⁺) significantly decreased, and NK cells (CD3^–CD16⁺CD56⁺) increased throughout exercise. NKCA increased (P < 0.001) during the initial 6 min of exercise with no further changes observed, despite increases (P < 0.001) in the number and proportion of circulating NK cells during exercise at 70% and 85% . Plasma epinephrine and norepinephrine increased (P < 0.001) above baseline at 12 min and 18 min. The changes in NK cell number and function were independent of gender. The results indicate that short-duration low-intensity exercise can significantly increase NK cell number and activity. However, alterations in NK cell number are not accompanied by changes of a similar magnitude in NKCA.     

Effect of isoprinosine on natural killer cell activity of blood mononuclear cells in vitro and in vivo

Addition of isoprinosine to cultures of blood mononuclear cells was shown to inhibit natural killer (NK) cell activity against the K562 myeloid cells and melanoma cells. This appeared to be due to inhibitory influences of monocytes in that after removal of adherent cells isoprinosine appeared to stimulate NK activity. Similar effects were noted on T cells separated by E rosette procedures. Administration of isoprinosine in vivo had variable effects on NK activity during drug administration but there was a significant increases in NK activity 3 days after cessation of the drug. These changes in NK activity may reflect different threshold levels of the drug on suppressor and NK cell populations and the relative proportions of different lymphocyte populations in individual subjects. Further studies with a wider range of drug doses and more frequent monitoring of responses are required to further evaluate the effect of isoprinosine on NK activity.     

Assessment of physiologic natural killer cell cytotoxicity in vitro

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.     

Immunocytochemical determination of the role of alveolar macrophages in endotoxin processing in vitro and in vivo.

Endotoxin (lipopolysaccharide or LPS) inhalation has been implicated in increased pulmonary edema, most likely due to activation of an inflammatory response. The purpose of this study was to determine the cell types in the lung responsible for binding inhaled lipid A from Enterobacter agglomerans LPS. Five-hour exposures of aerosolized lipid A resulted in measurable pulmonary edema in hamsters, as determined by the accumulation of lung water. Immunocytochemistry was used to localize the inhaled lipid A in the cell types in the lung. Alveolar macrophages had decreased levels of lipid A as compared to unexposed controls, suggesting a possible metabolism by the macrophages. In vitro exposure of macrophages to lipid A resulted in a time-dependent clearance of lipid A which was inversely related to its concentration. Alveolar macrophages thus appear to be responsible for the removal of inhaled lipid A in this model and may initiate the physiological events which bring about pulmonary edema.     

The natural killer gene complex: a genetic basis for understanding natural killer cell function and innate immunity

Summary: The natural killer gene complex encodes proteins, some of which are structurally unrelated, that impact on NK-cell function. Detailed analyses have indicated that these molecules are involved in NK-cell recognition, activation, and inhibition. The importance of this genomic region is highlighted by studies indicating that NKC-associated genes significantly influence NK cell-mediated innate host defense against life-threatening pathogens and that the NKC is conserved among diverse species. Thus, further elucidation of the NKC and its gene products will provide a genetic basis for understanding innate immunity and NK-cell activity at the molecular level.     

Bacterial activation of human natural killer cells: role of cell surface lipopolysaccharide.

Culture of human peripheral blood lymphocytes with gram-negative bacteria associated with periodontal disease caused a rapid increase in the cytotoxic potential of natural killer (NK) cells. The NK cells were activated to kill NK-resistant targets, the peak cytotoxicity occurring on day 1 of culture. The addition of anti-Tac, anti-CD3, or anti-OKT-11 antibodies to block activation via the interleukin-2 (IL-2), T-cell, or E rosette receptors had a minimal effect on this inductive process. Anti-IL-2 antiserum was effective in blocking a significant amount, but not all, of the cytotoxicity in bacterium-activated cultures. Modest IL-2 production (5 to 6 National Institutes of Health units) was measured in lymphocyte cultures activated by bacteria, but proliferation was not induced during a 1-week period. When polymixin B sulfate was added to bind and block lipopolysaccharides, bacterium-induced cytotoxicity was completely abrogated for all activating bacteria. In addition, when culture supernatants from Actinobacillus actinomycetemcomitans were tested, activation still occurred. However, again, this activation was totally inhibited by polymixin B sulfate. Monocytes were also activated by bacteria to produce tumor necrosis factor (TNF). To exclude the possibility that TNF was responsible for cytotoxicity, an antiserum to TNF was added to cocultures of bacteria and lymphocytes with adherent cells removed. The antiserum had no effect on the inductive process. In addition, exogenous TNF did not kill M14 targets. These results suggest that bacterial cell surface lipopolysaccharides provide a major activation signal for NK cells to enhance cytotoxicity.     

Role of natural killer cell function in dendritic cell-based vaccines

Recent studies have elucidated the functional links between natural killer (NK) cells and, demonstrating the reciprocal activation of these cell types through NK-DC interactions. The subsets of cells and molecular pathways involved in such interactions have been defined, and the possible anatomical sites of these interactions have also been reported. Murine experiments have demonstrated that injection of mature DCs induces rapid recruitment of NK cells to lymph nodes and that these NK cells provide interferon-gamma for Type 1 priming. Thus, there is an increasing body of in vivo evidence indicating that NK-DC interactions during the early phase of innate immunity can impact the quality and magnitude of the subsequent adaptive immune response. Importantly, these studies imply that NK cells might not serve merely as cytotoxic lymphocytes combating viral pathogens and malignant tumors, but must also be considered as important immunoregulatory cells with a significant influence on adaptive immunity. In contrast to the large volume of knowledge obtained through basic research, there is a relative paucity of information regarding NK cell function in adaptive immunity from clinical trials, as few DC vaccine studies have attempted to evaluate the nonspecific, yet potentially clinically relevant, NK response to immunization. In this article, the authors will review studies focusing on NK-DC interactions and highlight the most recent clinical findings relating to the potential role of NK cells in DC-based vaccine therapy.     

Effects of pertussis toxin treatment on human natural killer cell function.

Membranes from highly purified natural killer (NK) cells were ADP ribosylated by treatment with pertussis toxin (PTX). PTX treatment resulted in a single band of 32P incorporation at M(r) 41,600. PTX treatment of NK cells diminished their ability to lyse K562 tumour cells by about 50%. However PTX treatment had no measurable effect on cAMP levels in NK cells. PTX pretreatment also had no effect on the ability of target cells to induce phosphoinositide turnover or on the ability of the NK cells to conjugate with the K562 tumour cells. Movement toward the chemoattractants interleukin-2 (IL-2) and formylmethionylleucylphenylalanine (FMLP) was significantly inhibited indicating that a PTX substrate in NK cells may be involved in the transduction of signals which are involved in cell motility.     

Natural killer cells. In vitro and in vivo.

A nonadherent, nonphagocytic mouse cell found in lymphoid organelles, but lacking characteristic surface markers of mature lymphocytes, is capable of lysing a wide spectrum of tumor cells but shows little cytolytic activity toward normal cells. This cytotoxic cell, termed a "natural killer" (NK) cell, shows a marked capacity to lyse lymphomas (syngeneic, allogeneic, or even xenogeneic) to the effector cell source. Its activity is inhibited by a variety of pharmacologic agents, eg, cytochalasins, cAMP-"active" drugs, and colchicine, over the same dose range at which these drugs inhibit other cytotoxic cells. We have no evidence that NK cell "specificities" are clonally distributed. Two sets of evidence are presented which suggest that the same NK cell population is responsible for lysing a variety of tumor target cells. Preliminary evidence suggests that modulation of NK cell levels in vivo is correlated with resistance to challenge with a syngeneic tumor, inferring that NK cells may play a salient role in host defenses against neoplasia.     

Selenium and the immune response: 2. Enhancement of murine cytotoxic T-lymphocyte and natural killer cell cytotoxicity in vivo

An inverse correlation between cancer incidence and dietary intake of the trace mineral element selenium has been well established in epidemiological and experimental studies. The mechanisms for this chemoprotective effect are unresolved. Much attention has been focused on the antiproliferative effects of selenium on various normal and neoplastic cell types. However, dietary selenium supplementation can also enhance the expression of various humoral and cellular immune responses. In examining the effects of dietary selenium on cell-mediated immunity in mice, we observed that selenium supplementation caused the enhanced expression of spontaneous natural killer (NK) cytotoxicity in spleen cells and of specific cytotoxic T-lymphocyte (CTL) cytotoxicity in peritoneal exudate cells (PEC). NK activity of spleen-cell suspensions from selenium-supplemented mice increased an average of 70% over that of the control group (basal diet). Cytotoxic activity of PEC from mice injected with tumors intraperitoneally peaked earlier in selenium-supplemented animals, and the appearance of cells staining positively for Thy 1.2 surface antigen in selenium-supplemented animals also preceded the values observed in control animals. We propose here that enhancement of in vivo cytotoxic mechanisms, is likely to act synergistically with tumor growth inhibition in the reduction of tumor incidence associated with selenium intake.     

树突状细胞对自然杀伤细胞功能调控的研究进展

树突状细胞（DC）和自然杀伤细胞（NK）分别在固有免疫和适应性免疫中发挥着关键作用,二者之间还存在着复杂的交互作用.就DC对NK细胞功能的影响而言,前者可以通过膜表面分子直接激活静息的NK细胞,也可以在趋化因子的作用下将NK细胞招募至炎症部位或次级淋巴结,通过分泌可溶性细胞因子促进NK细胞活化、增殖,增强产生IFN-γ的能力,提高细胞毒活性,进而增强其抗病毒、抗肿瘤等效应.DC对NK细胞功能调控的研究在感染、肿瘤和免疫排斥等的防治中具有重要意义.     

Interference of hepatitis B virus surface antigen with natural killer cell function.

The influence of purified hepatitis B virus surface antigen (HBsAg) preparations or of supernatants derived from PLC/PRF/5 cell line (which produces HBsAg) on human natural killer (NK) activity was examined. Lymphocytes pre-incubated with HBsAg and subsequently washed showed a significant decrease in NK cytotoxicity against K-562 target cells. This effect was reversible and dose-dependent. In addition, pre-incubation with either HBsAg or PLC/PRF/5 supernatants inhibited in a reversible manner lymphocyte--K-562 conjugates and the binding of B73.1 monoclonal antibody (MoAb), which recognizes Fc receptors on NK cells. This effect was not observed with HNK-1, T3, T4, T6, T8 and T11 MoAb. HBsAg was non-toxic to lymphocytes, and ineffective with K-562 target cells. Beta-interferon did not modify HBsAg-mediated inhibition, when added either before or during the contact with HBsAg. Moreover, no modification was observed when neutrophils (at various neutrophil:lymphocyte ratios) were added, even though HBsAg is known to stimulate neutrophils to produce oxygen radicals which may modulate NK activity. We speculate that HBsAg produces these effects by reacting into receptor sites (possibly Fc receptor sites) on NK cell membrane. The overall significance of our results in relation to hepatitis and hepatocellular carcinoma is discussed.