Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity

Nonspecific cytotoxic cell (NCC) activity was assessed in the peripheral blood of four isogenic lines of rainbow trout (Oncorhynchus mykiss) which were derived by the chromosome set manipulation technique of androgenesis. In these fish, whose isogenicity was previously confirmed by multilocus DNA fingerprint analysis, NCC activity was studied by the release of ⁵¹Cr from YAC-1 targets. Two groups of trout (the homozygous Arlee 12 line and the heterozygous hybrid of the Arlee 63 and Arlee 12 lines) had significantly lower levels of NCC activity in peripheral blood than either outbred rainbow trout or other lines with Hot Creek or hybrid Arlee × Hot Creek ancestry. The low NCC activity in the Arlee line appears to be inherited as a recessive trait. Peripheral blood cells of the trout mediated lectin dependent cellular cytotoxicity (LDCC) with the addition of phytohemagglutirain to co-cultures of effector cells and YAC-1 cells. The low NCC activity in the peripheral blood of these fish is not due to a condition analogous to the NCC-deficient Chediak-Higashi syndrome of man or the beige mutation of mice.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). II. Parameters of target cell lysis and specificity

Fish nonspecific cytotoxic cells (NCC) lyse various transformed human B-cells (NC-37, P3HR-1) and erythroblastoid cells (K562) as well as mouse YAC-1 and P815 cells. Highest NCC activity was found in the anterior (head) kidney, but spleen cells and peripheral blood leucocytes (PBL) also demonstrated cytolytic abilities. Lysis of chromium-51 labeled target cells occurred rapidly and optimum cytolysis developed at either 16 degrees C or 26 degrees C incubation temperatures. Preincubation at temperatures of 4 degrees C or 37 degrees C for 4 hours reduced NCC cytotoxicity. Although catfish (Ictalurus punctatus) are extensively outbred, interfish NCC activities did not significantly vary at the optimum E:T ratios (160). The NCC target antigen specificities were partially determined by cold target inhibition (CTI) studies. YAC-1 and K562 did not produce significant CTI, however. These studies demonstrated the presence of a highly active cytotoxic cell which is widely distributed in fish lymphoreticular tissue. NCC kill divergent kinds of transformed cell types, and the target cell specificity for human transformed B-cells is different from the NCC target cell antigens on other human (K562) and on mouse (YAC-1 and P815) cells.     

Natural Killer Cells and Cytotoxic T Cells Induce DNA Fragmentation in Both Human and Murine Target Cells in Vitro

DNA fragmentation induced by cytolytic lymphocytes in human erythromyeloid cell line K562 and murine T lymphoma cell line YAC-1 was investigated by means of agarose gel electrophoresis. Murine natural killer (NK) and cytotoxic T (Tc) cells induced DNA fragmentation in YAC-1 cells, with the fragments being approximately multiples of 180 bp. More significantly, murine NK cells can induce a similar pattern of DNA fragmentation in human K562 cells. Therefore, cytolytic lymphocytes can induce apoptosis or programmed cell death in human target cells.     

Suppression of natural cytotoxic cell activity by social aggressiveness in Tilapia

Our earlier observations revealed that social stress causes drastic effects on different physiological mechanisms and degenerative changes in leucocytes. In this preliminary work, we analyzed the effect of social aggressiveness on functional activities of leucocytes emphasizing (NCC) activity in Tilapia. At 10 hr post stress induction, fish could be differentiated into three categories: 1) dominants; 2) subordinates; and 3) indeterminants. Results of NCC as indicated by 4 hr. 51Cr-release assay, demonstrated a significant suppression in cytotoxic reactivity in the subordinates and indeterminants compared to dominants. This suppression appears to be due to a decrease in the binding capacity of effector cells to YAC-1 target cells as indicated by the decreased number of conjugate forming cells. This binding process is a key event in activating the fish equivalent of NK cells in mammals. Decreased NCC-activity in stressed fish suggests that aberrations in cell mediated immunity result from social aggressiveness.     

用~3H-TdR释放法测量细胞介导的细胞毒功能

本文用 ~3H-胸腺嘧啶核苷(~3H-TdR)标记多种靶细胞(YAC-1、P815、LAC-1、小鼠淋巴母细胞)与不同效应细胞培养4或18小时,再用胰酶和DNA 酶处理靶细胞,收集细胞,用液体闪烁微杯法测定小鼠特异性和非特异性淋巴细胞介导的细胞毒试验,均获得良好结果。与~(51)Cr释放相比,~3H-TdR 方法具有自然释放率低(<10％),特异性释放率高的特点。     

Augmented Followed by Suppressed Levels of Natural Cell-Mediated Cytotoxicity in Mice Infected with Toxoplasma gondii

The cytotoxic activity of effector cells from mice infected with Toxoplasma gondii was tested in a 4- to 5-hr (51)Cr release assay, using RL 0001000 0011100 0101010 0001000 0001000 0011100 0100010 1000001 1000001 1000001 0100010 0011100 1 and YAC-1 target cells. They showed enhanced cytotoxicity with a peak on the 3rd day postinfection followed by suppression with a peak on the 12th day. The cytotoxicity seemed to be exhibited by natural killer (NK) cells because: (i) pretreatment of the effector cells with antiasialo GM(1) or antiasialo GM(2) plus complement abolished the cytotoxic activity; (ii) the altered cytotoxicity levels were also induced in nude mice; and (iii) the activity was elicited by nonadherent-nonphagocytic cells. The alteration occurred simultaneously in various lymphoid organs with a similar profile. Neither spleen nor bone marrow cells of 12-day-postinfected mice inhibited NK activity of uninfected mice. Culture fluids of the infected mouse spleen and bone marrow cells did not affect the normal mouse NK activity. The proportion of effector cells capable of binding to target cells was constant during the infection. There was no positive correlation between NK activity and serum interferon level; i.e., interferon was detected in the serum of 12-day-postinfected mice but not in that of 3-day-postinfected or uninfected mice. Passively administered interferon or polyinosinic-polycytidylic acid could not restore the suppressed NK activity of 12-day-postinfected mice. Moreover, in vitro treatment of spleen cells from 12-day-postinfected mice with interferon failed to restore the suppressed NK activity. These results suggested that after toxoplasma infection, defective sensitivity to interferon was induced in NK precursor cells, and differentiation to functionally active NK cells might be blocked.     

Inhibition of SK cell activity in frogs by certain drugs and sugars

We present similarities between mammalian natural killer (NK) cells and (anuran amphibian) frog spontaneous killer (SK) cells. A cytotoxic assay utilizing allogeneic erythrocytes as target cells was used and lysis assessed by measuring release of hemoglobin. SK effector cells, just as mammalian NK cells, are not sensitive to cycloheximide nor most simple sugars (50 mM glucose, glucose-6-phosphate, galactose, fucose, mannose). However, SK activity is inhibited by chloroquine, colchicine and mannose-6-phosphate. When SK cells were co-incubated with mammalian tumor cells, they were able to lyse only the NK-sensitive target YAC-1, but not other mammalian tumor cell targets including K562, Molt-4, Raji, P815 and EL4. Lysis of YAC-1 cells was also inhibited by colchicine and chloroquine. These results allow speculation on the evolution of cell mediated cytotoxicity since natural cytotoxic cells are present in ectothermic vertebrates.     

Miniaturization of the standard 51Cr release assay for long term follow-up of NK activity of individual mice

Two modifications for miniaturizing the standard 51chromium release assay have been described. Using peripheral blood lymphocytes (PBL) as effectors and very few target cells, this permits longitudinal studies on NK cell activity of individual mice to be performed. The modifications are based on methods to increase the chromium uptake by the target cell. Firstly, chromium uptake by the YAC-1 target cells was enhanced to such an extent that appreciable activity could be detected against as little as 750 cells/well at an E/T ratio of 50:1. Secondly, the uptake was increased even further by labelling the target cells in the presence of isotonic sucrose, thus permitting the use of only 125 target cells/well at an E/T of 25:1. The long term pattern of NK activity in individual BALB/c mice was studied by repeated bleedings over a period of 8 months. Considerable fluctuations with time were observed in the pattern of NK activity of individual mice. However, the NK activity averaged over a large number of mice did not show a significant decrease with age. These methods may allow the study of the long term pattern of NK activity in individual mice and the response of this activity to physiological and environmental factors.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). IV. Target cell binding and recycling capacity

The morphology of nonspecific cytotoxic cells (NCC) was identified. NCC were purified by target cell conjugate formation and density gradient separation. NCC are monocyte-like. They have reniform nuclei and a low nucleus/cytoplasm ratio. Cytoplasmic granules were not seen after giemsa staining. Scanning electron microscopy demonstrated moderate surface villi and target cell attachment occurred via long membraneous filament-like surface projections extending to the target cell membranes. Transmission electron microscopy of effector:target cell conjugates revealed membrane contact areas without fusion or fragmentation. The nucleus of the NCC had accentuated peripheral chromation and a prominent Golgi apparatus; the cytoplasm contained osmiophilic granules. Michaelis-Menten and Lineweaver-Burk transformation of target cell binding revealed a Vmax of 11-15,000 and a Km of 40,000. The percentage of NCC bound to target cells was 16-18%. Results of these studies were combined with the conjugate experiment to obtain an estimated percentage of active NCC (5-7%). A maximum recycling capacity of .16-.30 indicated that once attachment by NCC to the target cell occurred (and a lethal signal delivered by an effector cell), either the NCC did not recycle or a long lag period was required to restore its cytotoxic capability.     

In vitro binding of natural killer cells to Cryptococcus neoformans targets.

Nylon wool-nonadherent splenic cells from 7- to 8-week-old CBA mice were further fractionated on discontinuous Percoll gradients. Enrichment of natural killer (NK) cells in Percoll fractions 1 and 2 was confirmed by morphological examination, by immunofluorescent staining, and by assessing the cytolytic activity of each Percoll cell fraction against YAC-1 targets in the 4-h51Cr release assay. Cells isolated from each Percoll fraction were tested for growth-inhibitory activity against Cryptococcus neoformans, a pathogenic yeastlike organism, by using an in vitro 18-h growth inhibition assay. The results showed that NK cell enrichment was concomitant with enrichment of anti-Cryptococcus activity in Percoll fractions 1 and 2. Cells from NK cell-rich fractions formed conjugates with the mycotic targets similar to the conjugates reported in NK cell-tumor systems. In addition, the percentage of effector cell-Cryptococcus conjugates was directly proportional to the level of the C. neoformans growth-inhibitory activity of the effector cells used. Scanning electron microscopy of the effector cell-Cryptococcus conjugates showed direct contact between the effector cells and the cryptococcal targets. An immunolabeling method combined with scanning electron microscopy was used to demonstrate that the effector cells attached to C. neoformans were asialo GM1 positive and, therefore, had NK cell characteristics.     

Functional and phenotypic characteristics of bovine natural cytotoxic cells

Bovine natural cytotoxic (NC) cell activity of peripheral blood leukocytes (PBL) was studied in a chromium release assay, using xenogeneic tumor cells (YAC-1, P815) and herpes virus-infected bovine fibroblasts. The activity pattern resembled that of murine natural killer cells, in that YAC-1 cells were readily lysed, whereas P815 cells were resistant. However, 10-16 h of incubation were usually required to give appreciable cell lysis. Low, but consistent NC-activity was expressed against virus-infected fibroblasts. Using various biophysical and biochemical cell-separation methods, it was found that the effector cell active against both the xenogeneic tumor cells and virus-infected fibroblast belonged to the mononuclear phagocyte system. The indications are that at least two subpopulations of the blood monocytes, differing perhaps in maturation or clonal derivation, express NC-activity.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). I. Optimum requirements for target cell lysis

Nonspecific cytotoxic cells (NCC) obtained from the head (anterior) kidney of fish (Ictalurus punctatus) lyse human transformed B-cell targets. Lysis depended on direct cell-cell contact. Fish size, age, environmental holding temperatures, and lytic reaction conditions such as osmolality and optimum effector:target cell ratios were optimized. Experiments to characterize optimum kinetics demonstrated highly efficient killing after two hours incubation. This rapid cytolysis was further studied by determining NCC activity against appropriately labeled target cells after 30, 60, 90 and 120 minutes of cocultivation. At 160:1 (E:T) greater than 40% of the 5 hour percent specific release value was produced after 30 minutes. After 90 minutes, more than 90% of total percent specific release was observed. At least one mechanism of regulation of NCC killing was described. In the presence of normal (homologous or heterologous) catfish serum (CFS), essentially no NCC activity was observed. This suppression was reversible by preincubation in 10% fetal bovine serum (FBS). NCC "activation" by preincubation in 10% FBS was time-dependent (at least four hours was required to generate significant lysis). NCC activation could be reversed by treating potentially lytic cells with supernatants containing dissociated CFS. In addition, reversible activation could be demonstrated by treating potentially lytic effector cells with CFS to produce suppression. Regulation occurred at the effector cell level because treated target cells did not suppress NCC activity. These data demonstrate a population of nonspecific effector cytolytic cells that potentially represent a phylogenetic precursor to mammalian natural killer cells.     

Nonspecific cytotoxic cells in fish ( ) IV. Target cell binding and recycling capacity

The morphology of nonspecific cytotoxic cells (NCC) was identified. NCC were purified by target cell conjugate formation and density gradient separation. NCC are monocyte-like. They have reniform nuclei and a low nucleus/cytoplasm ratio. Cytoplasmic granules were not seen after giemsa staining. Scanning electron microscopy demonstrated moderate surface villi and target cell attachment occurred via long membraneous filament-like surface projections extending to the target cell membranes. Transmission electron microscopy of effector: target cell conjugates revealed membrane contact areas without fusion or fragmentation. The nucleus of the NCC had accentuated peripheral chromation and a prominent Golgi apparatus; the cytoplasm contained osmiophilic granules.Michaelis-Menten and Lineweaver-Burk transformation of target cell binding revealed a Vmax of 11–15,000 and a Km of 40,000. The percentage of NCC bound to target cells was 16–18%. Results of these studies were combined with the conjugate experiment to obtain an estimated percentage of active NCC (5–7%). A maximum recycling capacity of .16–.30 indicated that once attachment by NCC to the target cell occurred (and a lethal signal delivered by an effector cell), either the NCC did not recycle or a long lag period was required to restore its cytotoxic capability.     

Virus-Dependent Cellular Cytotoxicity in Vitro

When human peripheral blood lymphocytes were incubated with 51Cr-labelled tissue culture cells (T24 bladder carcinoma cells or Chang liver cells), their natural cytotoxicity (NK) usually stopped after 8 h of incubation. The 51Cr release induced by lymphocytes treated with small amounts of live or ultraviolet-inactivated mumps virus was strongly enhanced and lasted longer. When the lymphocytes were fractionated by Percoll gradient centrifugation, the highest NK activity was found in the low-density fraction enriched in large granular lymphocytes, whereas that of the T-cell-enriched high-density fractions was low. In contrast, the virus-dependent cellular cytotoxic (VDCC) activity was more evenly distributed between these fractions. However, there was a difference between the target cells in that the T24 cells were more susceptible to the cytotoxicity of lymphocytes in the high-density fractions than the Chang cells. Studies of Percoll fractions in the single-cell agarose assay showed that virus treatment increased the proportion of both target binding cells and killer cells in all fractions. Moreover, in the high-density fractions the increase in the number of killer cells was greater than that in binding cells, suggesting that the enhanced target cell killing induced by the virions reflected both increased binding and effector cell activation. Surface marker analysis of unfractionated lymphocytes indicated that the number of T3+ effector cells was greater than that of the HNK-1+ effector cells, regardless of whether the lymphocytes were treated with virus or not. However, for both NK and VDCC, the T3 to HNK-1 distribution ratio on the effector cells was 5-8:1 for T24 and 2:1 for Chang. Taken together, the results indicate that both NK and VDCC effector cells are phenotypically heterogeneous and that the target cells may play an active role in the recruitment of those effector cells that are most efficient in that system. The enhancement of lymphocyte cytotoxicity primarily reflects effector cell recruitment.     

Comparative natural killer cell activities of thymic, bursal, splenic and intestinal intraepithelial lymphocytes of chickens

The natural killer (NK) cell activities of spleen, thymus, bursa, peripheral blood and gut intraepithelial lymphocytes (IEL) from FP and SC chickens were investigated in 4-hr and 16-hr 51Cr release assays. Target cells were 4 different tumor cell lines derived from either an avian leukosis tumor transplant (LSCC-RP9, LSCC-RP12) or from Marek's disease lymphomas (MDCC-MSB-1, MCDD-CU36). Great variability in cytotoxic potential was observed among NK cells of different lymphoid organs. NK cell cytotoxicity varied depending upon the type of effector cells, type of target cells, the ratio of effector to target cells, and the age and genetic background of chickens. Substantial levels of NK cell activity were detected in spleen and gut IEL of SC chickens in a 4-hr assay. In contrast, the NK cytotoxicity in gut IEL of FP chickens was not detectable until 16 hr after incubation. The ranges of target cell specificity demonstrated by IEL, spleen, thymus and bursa NK cells were similar to one another and, in general, the level of cytotoxicity increased with incubation time. Thymus and bursa NK cell activity of both SC and FP chickens was not detectable in a 4-hr assay but substantial NK cell activity was demonstrated in a 16-hr assay. The results of the present study demonstrate that various lymphoid organs of chickens, such as spleen, thymus, bursa, and gut intraepithelium, contain subpopulations of cells that can mediate spontaneous cytotoxicity.     

Epstein-Barr virus (EBV)--lymphoid cell interactions. II. The influence of the EBV replication cycle on natural killing and antibody-dependent cellular cytotoxicity against EBV-infected cells.

We investigated the influence of the Epstein-Barr virus (EBV) replication cycle on natural killing (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) against EBV-infected cells. Peripheral blood lymphocytes from healthy EBV-seropositive and -seronegative donors were separated on Ficoll-Hypaque gradients and used as effector cells in the standard 51Cr release assay to measure NK and ADCC. EBV-genome positive RAJI and DAUDI cells superinfected with either the non-transforming P3HR-1 EBV or the transforming B95-8 EBV were used as targets. The results obtained show that most normal individuals have ADCC and NK activity against P3HR-1 EBV-infected RAJI cells. Both the cytotoxic activities increased with the proportional increase in effector/target (E/T) ratios, assay incubation time, dose of the infecting virus and the time of pre-infection with EBV. Moreover, the data obtained indicate that different immune mechanisms are effective at different stages of the virus replication cycle. During the early stages of virus replication, EBV-superinfected cells are more susceptible to ADCC than to NK, whereas in later stages the susceptibility to NK is increased significantly and appears to play a more dominant role. The nature of the target cells or the strain of EBV used to superinfect these targets did not influence their susceptibility to ADCC and NK activity; however some quantitative differences were found. Using metabolic inhibitors such as cytosine arabinoside, phosphonoacetic acid, actinomycin D, cycloheximide and puromycin, it was found that new DNA synthesis is not essential but some RNA and protein synthesis is necessary, late in the viral cycle, for the superinfected cells to become susceptible to NK and ADCC.     

A sensitive test for the detection of NK activity. Plasma clot clonogenic assay

Natural killer (NK) activity, which has been implicated in the immune response against viral infections and neoplasias, is currently evaluated by means of a chromium (51Cr) release assay. However, criticisms have been raised with regard to the reliability and reproducibility of the test. We have developed a different in vitro method for measuring NK activity, based on the inhibition of the target clone growth in plasma clot semisolid medium. This method overcomes the limitations inherent to the 51Cr release test and more closely mimics the in vivo situation. The inhibitory activity revealed by the cloning assay was always greater than the lytic activity in the 51Cr release assay. Moreover, effector/target ratios of 3:1 and 1.5:1 still produced clonal inhibition. B-CLL cells, used as effectors, showed no inhibitory activity and the Raji cell line employed as target was resistant in both techniques. Thus, the clonogenic assay appears to be more sensitive for the evaluation of low levels of NK activity, for basic studies on the effector/target interactions, for the evaluation of LAK cell activity, and in diseases in which an involvement of the NK compartment has been hypothesized.     

D variant of encephalomyocarditis virus (EMCV-D)-induced diabetes following natural killer cell depletion in diabetes-resistant male C57BL/6J mice

The involvement of natural killer (NK) cells in the development of diabetes in the normally resistant 9-10 week old C57BL/6J male mice by the D variant of encephalomyocarditis virus (EMCV-D) was examined. Inoculation of purified EMCV-D induced maximum NK cell activity in splenic cell populations on day 4 post-inoculation as determined by lysis of YAC-1 target cells in a standard 51chromium release microcytotoxicity assay. Selective depletion of NK cells by the administration of rabbit anti-asialo GM1 sera prior to challenging the C57BL/6J mice with EMCV-D, resulted in diminished splenic NK cell activity, increased EMCV-D viral titers in the pancreas, spleen, heart and brain, and the induction of diabetes in 60-80% of the mice. The data suggest that NK cells play a role in host protection against the diabetogenic EMCV-D.     

Natural killer activity of gut mucosal lymphoid cells in mice

Intraepithelial lymphoid cells (IEL) obtained from the mucosa of mouse small intestine were tested for natural killer (NK) activity in a 20-h ⁵¹Cr-release assay against YAC-1 and RL♂ 1 tumor cells. It was found that IEL possess a strong NK activity, higher than spleen cells, whereas lymphocytes from Peyer's patches (PPL) did not show any NK activity. The cytotoxic activity of IEL could be boosted by interferon (IFN-β) treatment, but no NK activity could be induced in PPL by in vitro IFN-β treatment. The characteristics of the NK effector cells present in the mouse intestine strictly resemble those of NK cells in the spleen. In fact, intestinal NK activity is not affected by depletion of adherent cells and only partially reduced by anti-Thy-1.2 antibodies plus complement. Moreover, the age dependency of NK cytotoxicity is identical for IEL and spleen. Finally, NK-insensitive target cells are not lysed by IEL.     

Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity

Interleukin 12 (IL-12)-activated NK1.1+TCRalpha beta+ (NKT2) and NK1.1+TCRalpha beta- (NK) cells exhibit cytotoxic activity against a wide variety of tumor cells in the absence of prior sensitization. Here we demonstrate that the integrin adhesion receptor LFA-1 (CD11a/CD18) regulates the cytotoxic activity of IL-12-activated NKT and NK cells against YAC-1 and EL-4 tumor cells. Differentiation in vivo and the expression of the cytolytic effector molecules perforin and Fas-L were comparable in both IL-12-activated NKT and NK cells from LFA-1-/ - and LFA-1+/+ mice. However, LFA-1-/-IL-12-activated NKT and NK cells showed impaired conjugate formation with target cells. These results provide the first genetic evidence for a role for an adhesion receptor in killing by IL-12-activated NK cells.