Influence of Vindesine on the Lytic Phase of Mouse Natural Cytotoxicity Against Human Leukemic Cells

Abstract

Vindesine (VDS), a structural analogue of Vinca Alkaloids, was found to increase the NK-mediated cytolytic effects of mouse lymphocytes against human K562 target cells in a 18-hr assay. Pretreatment of effector or target cells with the drug did not affect substantially the NK reaction. The phenomenon has been detected using splenocytes of either congenitally a-thymic or conventional euthymic mice of different strains. Effector lymphocytes deprived of cells adherent to plastic surface or to nylon-wool column were still competent for drug-mediated increase of NK function. It is suggested that modification of the membrane make-up of effector or target cells re-versibly induced by VDS, would promote higher NK-mediated cytolytic effects.     

Natural Cytotoxicity to Murine Cytomegalovirus-Infected Cells Mediated by Mouse Lymphoid Cells: Role of Interferon in the Endogenous Natural Cytotoxicity Reaction

Lymphoid cells from unstimulated normal C57BL/6J mice were shown to lyse murine cytomegalovirus (MCMV)-infected syngeneic mouse embryo fibroblasts but not uninfected mouse embryo fibroblasts. This cytotoxicity by mouse effector cells was not restricted to MCMV-infected syngeneic cells since MCMV-infected xenogeneic rat heart fibroblasts were also lysed. Characterization of the effector cells mediating this cytotoxicity against MCMV-infected cells indicated that the effector cells are similar to described natural killer (NK) cells mediating lysis of tumor cells and virus-infected cells. Because of the described augmentation of NK activity by interferon, we examined the role of interferon in the NK reaction. Although low levels of virus-induced interferon were detectable in supernatants of MCMV-infected mouse embryo fibroblasts, no interferon was detectable in supernatants of MCMV-infected rat heart fibroblasts, a target significantly more sensitive to NK cytolysis than infected mouse embryo fibroblasts. We were able to augment the NK reaction against MCMV-infected cells by in vitro treatments with interferon. However, the amounts of interferon required for augmentation were significantly greater than the amounts generated by infected target cells. In vitro interferon-stimulated NK cells retained selective cytotoxic activity since they continued to remain incapable of lysing uninfected target cells. MCMV-infected rat heart fibroblasts induced more interferon and were also more susceptible to NK activity than MCMV-infected mouse embryo fibroblasts. In spite of this difference in interferon-inducing capacity, there was no augmentation of cytotoxicity of MCMV-infected mouse embryo fibroblasts when mouse splenocytes were cocultivated with both target cells. Finally, when production of interferon in the NK reaction was inhibited by the addition of actinomycin D, no reduction of NK activity was seen. Our findings suggest that native mouse NK cells can discriminate between MCMV-infected cells and uninfected cells, this ability leading to the selective lysis of the virus-infected cells. Furthermore, although we could demonstrate augmentation of NK activity by interferon, interferon activation of NK cells may not be a necessary precondition for the development of endogenous NK activity.     

Enhanced Cytotoxicity of Human Lymphocytes Against Rabies-Infected Cells by Rabies-Specific Antibodies

Human anti-rabies immune sera enhanced the in vitro cytotoxicity of human lymphocytes against rabies virus-infected green-monkey kidney cells. The immune sera were collected from patients immunized with rabies vaccine produced either in human diploid cells or in nervous tissue. Significant cytotoxicity was observed even with high serum dilutions, indicating that the K-cell assay might be a sensitive tool for detection of anti-rabies antibodies.     

Simultaneous Determination of the Concentration and Lytic Activity of Effector Cells that Mediate Natural and Antibody-Dependent Cytotoxicity

Cellular cytotoxicity reactions can be studied in a manner analogous to that used to measure enzyme activity. This approach yields two parameters; V_max the maximal rate of target cell lysis that can be achieved by the lymphocyte preparation tested, and K _M^app, the apparent Michaelis constant. By analogy to many enzyme-catalysed reactions, K _M^app values for cytotoxicity reactions have generally been interpreted in terms of dissociation constants for the interaction of receptor sites on effector cells with antigens on the target cells, In this paper we demonstrate that experimentally determined K _M^app values for natural or antibody-dependent cytotoxicity reactions are approximately equal to the concentration of NK or K. effector cells in the lymphocyte preparation tested. This result makes possible the simultaneous determination of both effector cell frequency and lytic activity in a given lymphocyte preparation.     

Natural Killer-like Cytotoxicity of Human T-Cell Clones against Various Target Cells

MLC-T cells from two different donors were cultured after limiting dilution (0.3 cells/well) in medium containing T-cell growth factor. Clones showing lytic activity against K-562 in a preliminary screening were expanded and tested against various NK-sensitive target-cell lines (Chang, T-24, Daudi, and Molt-4) and phytohaemagglutinin blasts derived from the stimulator cells. Ten growing clones maintained their activity against K-562 and were able to lyse the other cell lines. A significant heterogeneity was nevertheless noticed in the lytic efficiency of the different clones against the various target cells. Taken together, our data indicate that single clones are able to lyse different NK-sensitive targets.     

Natural Cytotoxicity of Human Lymphocytes against Equine Target Cells in Vitro

Human lymphocytes displayed a frequent natural cytotoxicity (NK) in vitro against normal equine dermal fibroblasts (ED) and against equine tumour cells of a virus-containing cell line (Mc-1). Similarly, human normal sera contained antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) by normal human lymphocytes against the same target cells. Both NK and ADCC varied for different donors. For individual donors, however, cytotoxicity against the two target cells was significantly correlated both in NK and ADCC. For ED there was also a significant correlation between ADCC and NK activity. Both NK and ADCC showed some selectivity as assessed by cold target cell inhibition. Inhibition studies with Fab fragments of anti-human IgG established the involvement of immunoglobulins in the NK reaction. In this context, a marked and mainly immunoglobulin-dependent increase in both NK and ADCC activity against Mc-1 was observed in a laboratory worker frequently exposed to the target cells. The results indicate that variations of natural cytotoxicity in individual donors may sometimes be an indication of an ongoing spontaneous sensitization.     

Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

Effect of Human Seminal Plasma on the Lytic Activity of Natural Killer Cells and Presumptive Identification of Participant Macromolecules

ABSTRACT: Using the lytic activity of natural killer (NK) cells as an in vitro parameter, the immunoregulatory properties of human seminal plasma (SeP1) and participant macromolecules have been investigated. Significant (P < 0.05) suppression of NK cell activity by SeP1 and chromatographically separable fractions was demonstrated in association with high and low molecular weight (M_r) macromolecules. SePl suppression was retained after heating to 56°C for 30 min, and appeared to function at the level of the effector, rather than target cell. Physicochemical characterization of high and low M_r fractions provided presumptive identification of the participation of transglutaminase and prostaglandins as the principal molecules contributory to SeP1 immunosuppression.     

Kinetic Analysis of the Specificity of Human Natural Cytotoxicity

Distribution-free analysis of kinetic data for cellular cytotoxicity reactions enables the precise determination of kinetic parameters for the lysis of target cells by individual lymphocyte preparations. This report presents results obtained when this method was used to quantitate the inhibition of human natural cytotoxicity by unlabelled (cold) target cells. In agreement with previous studies, we found that the natural cytotoxicity of a given target cell can be inhibited by heterologous as well as homologous unlabelled cells; however, the strongest inhibition was usually produced when the unlabelled inhibitor cells were homologous to the labelled target cells. The general pattern of inhibition observed for the target and inhibitor cells tested in these experiments was competitive, and when inhibitor cells were homologous to the labelled cells, the observed increase in Kappm agreed with theoretical predictions based on equations derived previously. These results support earlier reports of limited but not absolute antigenic specificity by subsets of human NK cells. Moreover, while Kappm values for cytotoxicity reactions are not simply related to antigen binding, quantitative analysis of the relative inhibition of cytotoxicity by heterologous versus homologous unlabelled cells provides useful estimates of the relative affinity of the effector cell subset(s) that kill a given target cell for various NK target cells.     

Spontaneous Cytotoxicity by Monocyte-Enriched Subpopulations of Human Peripheral Blood Mononuclear Cells against Human or Mouse Anchorage-Dependent Tumour Cell Lines

Human peripheral blood mononuclear cells (PBMNC) were found to be cytotoxic for mouse or human anchorage-dependent target cell lines in a 48-72 h [¹²⁵I)iododeoxyuridine (IUDR) release assay. Unfractionated, adherent or nonadherent cells had significant levels of cytotoxicity, as did cells fractionated according to size into 'lymphocytes' or 'monocytes' by elutriation. Intermediate size cells, not enriched for monocytes, had high levels of cytotoxicity. In all fractions tested, including adherent populations, some cells with the morphology of large granular cells were observed. Treatment of all fractions with interferon (IFLrA, a purified. recombinant α-IFN) boosted cytotoxicity against four target cells lines. Treatment with lymphokines containing putative'macrophage-activating factor'(MAF) also enhanced cytotoxicity in fractions depleted of monocytes. Culture in fetal bovine serum enhanced cytotoxicity mainly in unfractionated and nonadherent PBMNC. These experiments indicated that N K-like cells can be appreciable contaminants in clutriator-purified monocyte-enriched or adherent cell populations and thereby contribute to observed cytotoxicity, particularly after pretreatment with IFN or other stimulatory factors.     

Depressed Natural and Lectin-Dependent Cell-Mediated Cytotoxicity Against Adherent Hep-2 Cells in Patients with Systemic Lupus Erythematosus

Natural cell-mediated cytotoxicity /NCMC/ was evaluated using human adherent 3H-thymidine-prelabelled HEp-2 epipharynx carcinoma cells as targets at 50:1 effector-target cell ratio in a 24 hr assay. For lectin-dependent cell-mediated cytotoxicity /LDCC/ studies cultures contained also 25/Ug/ml concanavalin A /Con A/. Peripheral blood mononuclear cells /PBMC/ of nine patients with active systemic lupus erythematosus /SLE/ failed to exert NCMC or LDCC against HEp-2 targets. In contrast, an increased adherence /decreased detachment from the monolayer/ of HEp-2 target cells was observed in the presence of PBM3 from SLE patients that was further promoted by the addition of Con A during LDCC assay.     

Depressed Natural and Lectin-Dependent Cell-Mediated Cytotoxicity Against Adherent Hep-2 Cells in Patients with Systemic Lupus Erythematosus

Natural cell-mediated cytotoxicity /NCMC/ was evaluated using human adherent 3H-thymidine-prelabelled HEp-2 epipharynx carcinoma cells as targets at 50:1 effector-target cell ratio in a 24 hr assay. For lectin-dependent cell-mediated cytotoxicity /LDCC/ studies cultures contained also 25/Ug/ml concanavalin A /Con A/. Peripheral blood mononuclear cells /PBMC/ of nine patients with active systemic lupus erythematosus /SLE/ failed to exert NCMC or LDCC against HEp-2 targets. In contrast, an increased adherence /decreased detachment from the monolayer/ of HEp-2 target cells was observed in the presence of PBM3 from SLE patients that was further promoted by the addition of Con A during LDCC assay.     

Increased Susceptibility of Periodate-Treated Tumour Cells to Human but Not to Mouse or Rat Natural Killer Cells

Treatment of intact cells in the cold with low concentrations (1 mM) of sodium meta periodate (PI) selectively oxidizes the surface-exposed sialic acid residues to the corresponding aldehydes. Such treated tumour cells show greatly enhanced sensitivity to lysis by fresh human NK cells but not to mouse or rat NK cells. Reduction of the PI-treated cells with sodium borohydride (NaBH4) reduced their NK sensitivity to that of untreated cells. In target conjugate formation assays PI-treated tumour cells displayed a higher binding capacity than control cells or PI+NaBH4-treated cells to both mouse and human effector cells. Neuraminidase treatment of K562 and Molt-4 increased target susceptibility to human NK cells but not to mouse, whereas the susceptibility of Yac-1 cells was left unchanged using both human and mouse effector cells. The same pattern of reactivity is shown in the target binding assay. These findings indicate that subtle molecular changes in the surface-exposed carbohydrates of target cells might have a fundamental impact on their sensitivity to lysis by NK cells from certain species, and that in cross species effector-target combinations a higher binding capacity is not sufficient for increased lysis to occur.     

Generation of Inositol Phosphates during Triggering of Cytotoxicity in Human Natural Killer and Lymphokine-Activated Killer Cells

We investigated early molecular mechanisms involved in the triggering of cytolytic responses in natural killer (NK) and lymphokine-activated (LAK) cells. When NK or LAK cells were conjugated to the sensitive target cells K562, an increased formation of both inositol monophosphate (IP1) and inositol trisphosphate (IP3) was detected. Target cells like Raji or Jok-1, which form conjugates with NK cells but are insensitive to NK lysis, did not elicit IP1 formation. Treatment of NK cells with interleukin 2 increased the basal turnover of inositol phosphates and enhanced the phosphatidyl inositol breakdown upon confrontation with sensitive targets. These finding indicate that hydrolysis of phosphatidyl inositols is associated with the signal which triggers the cytolytic response in NK and LAK cells. These events therefore constitute an early marker of the cytolytic activation.     

Failure to Detect Complement-Mediated Antibody-Dependent Cytotoxicity Against Human Orbital Tissue Cells in the Serum of Patients with Thyroid-Associated Ophthalmopathy

Antibodies which are cytotoxic to human eye muscle cells and orbital fibroblasts in antibody dependent cell-mediated cytotoxicity (ADCC) are routinely detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In the present study sera from patients with TAO, most of which were shown to be positive in ADCC against eye muscle cell targets, were tested for complement-mediated antibody-dependent cytotoxicity (CMAC) against human and pig eye muscle cells, human (abdominal) skeletal muscle cells and human orbital fibroblasts, in ⁵¹ Cr release assays. Several different assay protocols and complement sources were used and patient and age, sex matched normal sera compared. In preliminary studies tests were always negative when eye muscle cells, other skeletal muscle cells, or orbital fibroblasts were used as targets, regardless of the complement source, or concentration, or assay conditions used. When larger numbers of patients and normals were tested in a single assay mean (± SE) % specific lysis for patients with TAO was not significantly different from that for normals for either eye muscle cells or other skeletal muscle cells and taking the upper limit of normal as mean + 2 SD for the normals, tests were positive in no patient with either target. On the other hand ADCC tests were positive in 57 % of the same sera tested with the same eye muscle cell targets. When human thyroid cells were used as targets, tests were positive in 10 of the 14 patients tested and monoclonal antibodies, in enzyme-linked immunosorbent assay, reactive with eye muscle antigens gave positive lysis of eye muscle cells.

Although eye muscle reactive autoantibodies have been well described in TAO, particularly IgG₃ subclass antibodies against a 64 kDa membrane antigen, their significance in the pathogenesis of the eye muscle component of this disorder is unclear. In contrast to thyroid cells where microsomal, and perhaps other membrane reactive antibodies, are cytotoxic in both ADCC and CMAC, antibodies associated with TAO appear to be cytotoxic only in ADCC. Although the reason for this discrepancy is unknown one possible mechanism is failure of IgG₃ subclass antibodies, which are usually cytotoxic, to activate complement when the target antigen is in too low a density on the cell surface.     

Self-Induction of Defense Against Tumor Necrosis Factor Cytotoxicity in Tumor Cells and Normal Cells

Investigation on the effect of TNF on RNA and protein synthesis by tumorigenic and normal cell lines showed their synthesis in tumor cells to be increased at 12 h and to peak at 24 h of incubation with TNF, while that in normal diploid fibroblast (HEL) cells was apparently unaffected by the presence of TNF. The increase correlated with cell susceptibility to cytotoxic effect by TNF. Artificial inhibition of either RNA or protein synthesis by L-M cells. by addition of artinomycin D or cycloheximide, increased the cytotoxic effect of TNF and thus suggested that the elevated RNA and protrin synthesis is related not, to the cytotoxic reaction itself but rather to a defense mechanism. Similar incubaticin of HEL cells with TVF in the presence of either inhibtor resulted in the occurrence of cytotoxicity not observed with TNF alone, thus suggesting the existence of a defense mechanism in normal, TNF-resistant rolls which is absent, or greatly wealtend in tumor cells.     

Selective Cytotoxicity of Manganese Nanoparticles against Human Glioblastoma Cells

Bulletin of Experimental Biology and Medicine - Toxicity of different types of manganese nanoparticles against glioblastoma U-87MG and U-251 cells and normal human cells was studied using MTT test....     

Are Granulocytes the Main Effector Cells of Natural Cytotoxicity in Chickens?

Purified peripheral blood granulocytes from chicken were tested for cytotoxic activity against two types of virus-transformed chicken cell line, LSCC-H32 and LSCC-RP9. Strong cytotoxicity could be demonstrated, as measured in a 4-hr 51Cr-release assay, especially against the fibroblastoid LSCC-H32 cells. The degree of cytotoxicity was dependent on the E:T ratio. Normal CEF cells were completely resistant to the cytotoxicity. No cytotoxicity of human granulocytes could be observed against a variety of adherent and non-adherent target cells, as measured by the same microcytotoxicity technique. The priority of granulocytes in the natural cytotoxicity in the avian system is, therefore, suggested.     

Effects of Theophylline on Human Natural Killer Cells

Theophylline has been shown previously to inhibit a number of cellular immune functions of granulocytes and T-lymphocytes. In the present report, we demonstrate that theophylline, in a dosedependent fashion, suppresses human natural killer (NK) cell activity in vitro. To determine if theophylline produces quantitative or qualitative alterations in NK cells in vivo we quantitated peripheral blood NK cells with three monoclonal antibodies and FACS analysis and measured NK cytolytic activity in eight normal volunteers who took theophylline for eight days. No change was noted in the number or cytolytic activity of NK cells over the eight days of monitoring. We conclude that theophylline does not alter NK cells in vivo when given in therapeutic doses.