Effect of enkephalins and endorphins on cytotoxic activity of natural killer cells and macrophages/monocytes in mice

The effect of [Met5]enkephalin, [Leu5]enkephalin, proenkephalin, dynorphin-(1-17) or beta-endorphin on the cytotoxic (51Cr release assay) activity of natural killer cells and macrophages/monocytes was studied in mice. It was found that a single i.p. injection of [Met5]enkephalin, [Leu5]enkephalin, beta-endorphin, dynorphin or proenkephalin as well as repeated treatment with both enkephalins increased natural killer cell activity. In vitro only [Met5]enkephalin stimulated natural killer cells. Opioid peptides did not affect the cytotoxic activity of macrophages/monocytes. In addition to functional alterations, both enkephalins and beta-endorphin increased the percentage of cells with natural killer phenotype. The results of this study suggest that the increase in natural killer cytotoxicity after opioid peptides injected once or for 14 days may result from an increased number of natural killer cells in the spleen.     

Dietary quercetin, immune functions and colonic carcinogenesis in rats

Rats fed 100 mg/kg quercetin (QUE) daily for 7 weeks had significantly enhanced natural killer cell activity compared to their vehicle (VEH)-fed control. In contrast, rats fed 100 mg/kg QUE and treated with the colon carcinogen, azoxymethane had significantly reduced natural killer cell activity compared to their VEH-fed azoxymethane-treated control. There was no significant difference in natural killer cell activity between the two control groups. Antibody production and delayed-type hypersensitivity were not altered by QUE feeding in any treatment group. In vitro exposure of splenic natural killer cells to 1mM QUE significantly decreased natural killer cell cytotoxicity. Lower QUE concentrations produced a non-significant reduction in natural killer cell activity that was restored to control values at 1 x 10(-13)M QUE. The distribution, multiplicity and total number of colonic preneoplastic lesions, aberrant crypt foci, was not significantly different in the QUE-fed azoxymethane-treated rats when compared to azoxymethane-treated vehicle-fed rats at the conclusion of 7 week feeding period. We found no correlation between immune function and development of preneoplastic colon lesions in this study.     

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The relationship between radical production and natural killer cytotoxic factor (NKCF) release via canine natural killer (NK)-mediated cytotoxic mechanism was examined. Radical production and NKCF release was induced in NK cells stimulated with either dead target cells, or their cytoplasmic membranes, as well as live target cells. Canine NKCF evoked target cell lysis but did not induce radical production. Radical production was inhibited by the addition of Tiron or n-propyl gallate, whereas NK-mediated cytotoxicity and NKCF release were only inhibited by the addition of n-propyl gallate. These results suggested that radical production and NKCF release may be induced by the contact and binding of NK cells to the target cell cytoplasmic membrane. Therefore, the release of NKCF from NK cells attached to the target cell cytoplasmic membrane may be associated with the production of radicals, especially hydroxyl radicals.     

Natural killer activity of Wistar rat spleen cells: blocking effect of homologous sera

The natural killer (NK) activity of lymphocytes from Wistar rats against mouse target cells wa studied using the 24 h microplate assay. The cytotoxic activity of NK cells was blocked in the presence of sera from adult rats and man, but not in the presence of sera from adult rats and man, but not in the presence of sera from cattle, rabbits, mice or newborn rats. The blocking serum factor was non-dialysable and inactivated by trypsin. In addition, data were obtained indicating that several continuous cell lines of mice differ significantly in sensitivity to Wistar rat spleen cells.     

Immune response in mice that lack the interferon-gamma receptor

Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.     

In vitro and clinical effect of pentoxifylline on natural killer cell activity

Pentoxifylline used in the treatment of vascular diseases has also some immunological effects. Among of these effects the influence of pentoxifylline on the natural killer cell activity was studied. In in vitro experiments the human natural killer cell cytotoxicity was examined in the presence of pentoxifylline. In our clinical trial we investigated the topic whether this drug has an immunomodulatory effect while administering it for different periods. The natural cytotoxicity of macroangiopathic patients treated with pentoxifylline was compared with the values of healthy controls and patients suffering from vascular disease and obtaining no pentoxifylline therapy. Sixty two macroangiopathic patients and twenty healthy controls were investigated altogether. In the in vitro natural killer cell reaction we found that the pentoxifylline was able to suppress the cytotoxicity at any time of the reaction. The influence of pentoxifylline on natural killer cell activity was neither due to the expression of intercellular adhesion molecule-1, nor to the alteration of membrane fluidity. However, this drug significantly (p < 0.05) decreases the apoptosis of target cells. The natural killer cell activity of patients with chronic pentoxifylline therapy lasting for more than a year was proved to be significantly (p < 0.005) lower. The presence of vascular disease does not influence the natural killer activity by itself. Concluding our results we can state that the suppressing effect of pentoxifylline on natural killer cell activity needs to be taken into consideration in case of clinical diseases where this drug is administered chronically.     

Anti-tumor Immune Response Mediated by Newcastle Disease Virus HN Gene

Hemagglutinin-neuramidinase(HN) is one of the most important surface structure proteins of the Newcastle disease virus(NDV). HN not only mediates receptor recognition but also possesses neuraminidase(NA) activity,which gives it the ability to cleave a component of those receptors, NAcneu. Previous studies have demonstrated that HN has interesting anti-neoplastic and immune-stimulating properties in mammalian species, including humans. To explore the application of the HN gene in cancer gene therapy, we constructed a Lewis lung carcinoma(LLC) solid tumor model using C57BL/6 mice. Mice were injected intratumorally with the recombinant adenovirus expressing HN gene(Ad-HN), and the effect of HN was explored by natural killer cell activity assay, cytotoxic lymphocyte activity assay, T cell subtype evaluation, and Th1/Th2 cytokines analysis. The results demonstrate that HN not only can elicit clonal expansion of both CD4+ and CD8+ T cell populations and cytotoxic T lymphocyte(CTL) and killer cell response, but also skews the immune response toward Th1. Thus, vaccination with Ad-HN may be a potential strategy for cancer gene therapy.     

Impaired release of natural killer cytotoxic factor(s) by peripheral blood lymphocytes in patients with chronic LGL-proliferative disease

BACKGROUND: Chronic LGL-proliferative disease (LGL-PD) is a clonal expansion of cells with large granular lymphocyte (LGL) morphology. In most cases, proliferating cells express both suppressor/cytolytic T-cell and natural killer (NK) cell surface markers, but other cell phenotypes may be observed. LGL-PD lymphocytes have been found to lack or show very low natural killer cell activity (NKa). The aim of the present paper is to investigate the underlying mechanisms responsible for impaired NKa in a homogeneous group of five selected LGL-PD patients with a CD3+, CD8+, CD57+ cell phenotype. RESULTS: In all patients, the expanded cell population expressed very low NKa against K562 cell targets, but this increased significantly with recombinant human interleukin-2 (rhIL-2) and phytohemagglutinin (PHA) activation. Recombinant human alpha-interferon (rhIFN-alpha) had no significant effect on NKa. Cells displayed normal tumor cell binding capacity but failed to release sufficient amounts of functionally active natural killer cytotoxic factor(s) (NKCFs) upon interaction with the NK-sensitive K562 cells targets. However, they did release soluble cytolytic molecules against K562 cells upon activation with PHA. CONCLUSIONS: Our findings provide evidence that the defective NKa in LGL-PD patients with the aforementioned phenotype is probably due, at least in part, to the inability of expanded lymphocytes to release NKCFs upon interaction with NK-sensitive cell targets. Since recognition of target cells by patient lymphocytes is not disturbed and the cells are capable of producing NKCFs upon activation with PHA, it is probable that the cause of this abnormality is located at the level of the activation signal provided by the stimulatory target cells. Studies in subcellular level are certainly needed for a more precise determination of the underlying defect.     

A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.     

Functional studies of human decidua in spontaneous early pregnancy loss: effect of soluble factors and purified CD56+ lymphocytes on killing of natural killer- and lymphokine-activated killer-sensitive targets

Inappropriate leukocyte activation and disturbance of the delicate cytokine balance within the uterus during early human pregnancy may initiate spontaneous abortion. The purpose of the present study was to assess whether decidual soluble factors from women suffering sporadic spontaneous early pregnancy loss could enhance the cytotoxic activity of peripheral blood lymphocytes (PBL), and to investigate the lytic activity of endometrial granulated lymphocytes (eGL) purified from decidua against natural killer (NK)- and lymphokine-activated killer (LAK)-sensitive targets. Decidual cell culture supernatants from sporadic spontaneous abortion cases did not have any effect on PBL cytotoxic activity against the NK-sensitive target cells K562. Endometrial GL purified from decidua of spontaneous aborters were unable to lyse the LAK-sensitive target cells Raji and, in contrast with eGL from decidua of first-trimester therapeutic aborters, approximately 50% of the cases also failed to kill K562 cells. These results do not provide evidence to implicate either cell-mediated or cytokine-mediated cytolytic mechanisms in early spontaneous pregnancy loss, thus strengthening the possibility that other damaging effects are operating. Nevertheless, the deficient cytotoxic activity in a proportion of spontaneous abortion decidua merits further investigation.     

Inhibition of natural killer cell activities from normal donors and AIDS patients by envelope peptides from human immunodeficiency virus type I

Recombinant and synthetic peptides derived from the human immunodeficiency virus type I (HIV-1) genome corresponding to portions of the envelope (env) and internal core protein (gag) were examined for their immunoregulatory effects on the natural killer (NK) cell activity of lymphocytes from healthy donors and from patients with the acquired immunodeficiency syndrome (AIDS). Two recombinant peptides (env-gag and Env 80-DHFR) and three chemically synthesized peptides (env 487-511, env 578-608 and env 647-659) were used. Normal lymphocytes precultured for 24 to 72 hrs. with either env-gag, env 487-511, or env 647-659 at 5 and 50 ng/ml concentrations which significantly stimulated lymphocyte proliferation, produced significant suppression of NK activities. Two control peptides, one derived from the E. coli vector used to clone the HIV env-gag fusion peptide and another, a non-HIV-1 viral antigen (rubeola virus) did not produce any observable effect on NK activity of normal lymphocytes demonstrating the specificity of the reaction. Env-gag peptide also inhibited the NK activities of Percoll-separated, NK-enriched large granular lymphocytes. In target binding assays, lymphocytes precultured with env-gag significantly suppressed the target binding capacity of effector cells and produced significantly lower levels of natural killer cytotoxic factor (NKCF). In kinetic studies, lymphocytes from normal donors preincubated with env-gag for 24 to 72 hrs. produced significant inhibition of their NK activity and an even greater inhibitory effect on NK activities was observed when lymphocytes from AIDS patients were preincubated with HIV peptides. Thus HIV-1 peptides, which we previously demonstrated could regulate B- and T-lymphocyte activities, are also capable of regulating the NK activities of lymphocytes from HIV-1-infected and normal individuals.     

Perforin-secreting killer cell infiltration in the aortic tissue of patients with atherosclerotic aortic aneurysm

Cell-mediated immunity has been implicated in the pathogenesis of vascular cell injury in patients with atherosclerotic aortic aneurysms. To clarify the immunologic mechanisms involved, we examined the expression of a cytolytic factor, perforin, in infiltrating cells from aortic tissue samples taken from 6 patients with atherosclerotic aortic aneurysms. Immunohistochemical studies showed that the infiltrating cells consisted mainly of macrophages, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and T helper cells, and that perforin was expressed in NK cells and CTLs. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly on to the surface of arterial vascular cells. These findings provide the first direct evidence that some of the infiltrating cells in the aortic tissue consist of killer cells, and strongly suggest that these killer cells, especially NK cells and CTLs, may play a critical role in the vascular cell injury caused by atherosclerotic aortic aneurysm by releasing perforin.     

TGF-beta mediates natural suppressor activity of IL-2-activated lymphocytes

In addition to generating cells with non-MHC-restricted cytotoxic activity that is characteristic of lymphokine-activated killer (LAK) cells, in vitro cultures of lymphocytes with relatively high concentrations of IL-2 generate cells that simultaneously exhibit two distinct types of suppressor activities: veto, the ability of cells to specifically suppress generation of allo-CTL against their own histocompatibility Ags; and natural suppression, the ability of these same cells to nonspecifically suppress the generation of allo-CTL against both their own and unrelated cell surface Ags. In contrast to veto, which is known to require cell-cell contact between veto-active cells and precursors of CTL, natural suppression is known to be mediated by soluble factors. To identify and characterize suppressor factors that might mediate the natural suppressor activity of IL-2-activated lymphocytes, murine spleen cells were cultured with 1000 U/ml IL-2, and, after varying periods of incubation, their LAK cytolytic activity and natural suppressor activity was determined and cell supernatants were collected and tested for their effects on mixed lymphocyte culture-induced generation of allo-CTL. Like the IL-2-activated lymphocytes themselves, supernatants of these cells nonspecifically inhibited mixed lymphocyte culture-induced generation of allo-CTL. Rabbit anti-TGF-beta specifically neutralized the suppressive effects of both LAK cell supernatants and the IL-2-activated lymphocytes themselves. These findings indicate that TGF-beta is the primary mediator of the natural suppressor activity of IL-2-activated lymphocytes.     

Direct cellular immunomodulation produced by diacetylmorphine (heroin) or methadone

1. Abuse of the narcotic drug diacetylmorphine (heroin), as well as methadone, a drug for treating heroin addiction, has been associated with alterations in immune function in humans. The current study was performed to assess the direct (in vitro) immunomodulatory effect of exposure to these drugs, in view of the very limited studies reported thus far on this effect. 2. Murine splenocytes or peritoneal macrophages were cultured in vitro at concentrations of 0.0001-100 microM heroin or methadone. B-cell function was assessed by quantitating cellular proliferation in response to stimulation with an antigen analog; T-cell regulatory function was assessed by culturing splenocytes with or without drugs in the presence of anti-CD3 antibody and subsequently quantitating cytokine production; and T-cell effector function was evaluated by culturing lymphocytes with or without drugs during a 5-day induction culture followed by assessment of specific cytotoxic T-lymphocyte (CTL) activity. Natural immunity was assessed by quantitating basal and IL-2 augmented natural killer (NK) cell function, and macrophage function was assessed by cytokine production. 3. In vitro exposure to heroin resulted in decreased B-cell proliferation at concentrations of 1-100 microM, and methadone had a similar effect at concentrations of 0.1-100 microM. 4. Production of IL-2 was suppressed by 0.1-100 microM of heroin, whereas exposure to methadone appeared to result in a generalized modulation, with suppression of IL-2 at most concentrations. In contrast, IL-4 production was only affected at the 100 microM concentration of both drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced immunosuppression in A/J mice by treatment with nonsteroidal anti-inflammatory drugs

BACKGROUND: We have previously reported that nonsteroidal anti-inflammatory drugs inhibit lung tumorigenesis induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. PURPOSE: The aims of this study were to determine if NNK suppresses humoral (i.e., antibody) and cellular immune responses in mice and if nonsteroidal anti-inflammatory drugs could attenuate these immune responses. METHODS: Female A/J mice (7-8 weeks old) were fed nonsteroidal anti-inflammatory drugs starting 2 weeks before the beginning of NNK treatment (9.1 mg per mouse in total) and continuing through the 7 weeks of NNK treatment. Eight groups (two control groups and six experimental groups) of 10 mice each were used per experiment. Animals in the two control groups received the same diet and water as animals in the six experimental groups; one control group received no nonsteroidal anti-inflammatory drugs or NNK and the other control group received only NNK. The primary humoral and cellular immune responses to the various treatments were assayed by the plaque-forming cell technique and by measurement of natural killer cell cytotoxic activity, respectively. At the end of each experiment, the animals were killed, blood was collected, plasma was prepared, and levels of the immune system modulator prostaglandin E2 were measured. RESULTS: NNK treatment inhibited the plaque-forming cell response by approximately 50%; this inhibition was attenuated by treatment with sulindac or acetylsalicylic acid (P = .0001 for both). In contrast, treatment with naproxen, which had no chemopreventive (i.e., tumor inhibitory) efficacy, further increased by 26% (P = .05) the immunosuppressive effect of NNK. The cytotoxic activity of splenic natural killer cells against YAC-1 cells was reduced by 60% (P = .002); treatment with acetylsalicylic acid (254 mg/kg of diet) reduced the NNK-induced natural killer cell cytotoxicity inhibition by 50% (P = .02), whereas the administration of the specific cyclooxygenase-2 inhibitor NS-398 (7 mg/kg of diet) resulted in an almost complete recovery (approximately 95%, P = .04) of natural killer cell activity. The prostaglandin E2 plasma concentration was approximately 100% greater in NNK-treated mice than in untreated mice. Treatment of the mice with nonsteroidal anti-inflammatory drugs attenuated this elevation (from approximately 25% to 100%), and NS-398 (7 mg/kg of diet) was the most effective (100%). CONCLUSIONS AND IMPLICATIONS: The ability of various nonsteroidal anti-inflammatory drugs to inhibit NNK-induced carcinogenesis appears to be directly related to the ability of these drugs to inhibit NNK-induced immunosuppression. Our results suggest that the chemopreventive effect of nonsteroidal anti-inflammatory drugs may be mediated through the modulation of prostaglandin E2 synthesis.     

Long-term stimulant treatment of children with attention-deficit hyperactivity disorder symptoms. A randomized, double-blind, placebo-controlled trial

In this study we evaluated the effect of long-term melatonin (MEL) treatment on the cytotoxic activity and number of natural killer (NK) cells and the proliferative response of spleen lymphocytes to phytohemagglutinin (PHA) or interleukin-2 (IL-2) in old mice. Seventeen-eighteen month-old Balb/c mice were supplemented with MEL (40-50 microg/day/mouse) and sacrificed after eight months. The MEL supplementation was unable to recover the low levels of both endogenous and IL-2-induced NK cell activity found in old untreated mice. Also the NK cell number was unaffected by MEL treatment. The spleen lymphocyte proliferative response to both PHA and IL-2 was not different in old MEL-treated compared to old untreated mice. These results indicate that long-term MEL supplementation does not recover the age-related deterioration of NK cell activity and lymphocyte proliferative capacity.     

SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors

Activation of immune system cells via antigen-, Fc-, or natural killer cell-triggering-receptor stimulation is aborted by co-engagement of inhibitory receptors. Negative signaling by killer cell inhibitory receptors and related receptors depends on the Src homology 2 (SH2)-containing protein tyrosine phosphatase SHP-1. Using a combination of direct binding and functional assays, we demonstrated that the SH2 domain-containing leukocyte protein 76 (SLP-76) is a specific target for dephosphorylation by SHP-1 in T cells and natural killer cells. Furthermore, we showed that tyrosine-phosphorylated SLP-76 is required for optimal activation of cytotoxic lymphocytes, suggesting that the targeted dephosphorylation of SLP-76 by SHP-1 is an important mechanism for the negative regulation of immune cell activation by inhibitory receptors.     

Inhibition of natural killer cell activity in mice treated with tobacco specific carcinogen NNK

Among the different chemicals present in tobacco and tobacco smoke, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen. In the present study the immunosuppressive effect of NNK was investigated in laboratory animals by analyzing the antitumor immune responses. Mice of B6C3F1 strain were treated with different doses of NNK by IP and assayed for natural killer cell activity by the lysis of 51Cr-labeled YAC-1 lymphoma cells. The control mice received physiological saline. The results showed a significant inhibition of natural killer cell activity in the spleen cells of mice treated with 100 or 250 mg/kg NNK. In contrast to the high-dose NNK group, treatment of mice with lower doses of NNK like 10 or 50 mg/kg had no significant effect on the natural killer cell activity. In addition to spleen, the natural killer cell activity was also suppressed in the hilar lymph nodes and lung cells of NNK-treated mice. The clearance of 125I labeled YAC-1 tumor cells was also reduced from the lungs of mice injected with NNK. Further, the metastatic potential of B16F10 melanoma cells was significantly higher, as evidenced by the increased lung tumor nodules in the high-dose NNK-treated mice. The decreased antitumor immune response in the carcinogen-treated mice was not due to a decrease of NK cells, because flow cytometric analysis indicated no change in the frequency of NK 1.1+ cells between control and treated animals. However, there was an increased plasma cortisone levels in the carcinogen-treated mice compared to control animals. Injection of mice with poly I:C or interleukin-12 was able to restore natural killer cell activity in the tobacco carcinogen-treated mice.     

Direct activation of human CD8+ cytotoxic T lymphocytes by interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8+ T cells present after 28 days in the induction culture. When purified CD8+ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8+ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.