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The role of enucleation in the treatment of ocular melanoma has been questioned by those who believe that it may increase circulating melanoma cells, increasing the incidence of metastases. Some aspects of the immune system are depressed by general anaesthesia, therefore this study was initiated to assess the effect of general anaesthesia on natural killer (NK) cell and interleukin-2 (IL-2)-activated cytotoxicity in 19 patients using 51Cr-labelled target cells (K 562 and SW 742). NK cytotoxicity was increased at induction and during the operation which was due to significant increases in female, but not male patients. At 1 week post-surgery, female patients had decreased NK cytotoxicity compared with males at this interval. NK cytotoxicity compared with males at this interval. NK cytotoxicity was increased intra-operatively when alfentanyl was used, and if the mean tumour diameter was > 15.0 mm the NK cytotoxicity was increased intra-operatively compared with that in patients with smaller tumours. IL-2-stimulated cytotoxicity was reduced at 1 day post-surgery in females only. There was an increased IL-2-stimulated cytotoxicity at induction when propofol was used and IL-2-stimulated cytotoxicity was reduced at 1 week post-operatively if the patient's mean tumour diameter was > 15.0 mm. If NK cell and lymphokine-stimulated cell cytotoxicity are involved in limiting metastatic disease, the patient' sex and tumour size and the anaesthetic agent used may influence the survival rates following surgery.

We have evaluated the severe combined immunodeficient (SCID) mouse as an in-vivo model for the study of non-Hodgkin's lymphomas (NHL). Characterization of the immune system of the animals in our SCID mouse colony was carried out to assess the numbers of lymphoid cells present, to determine natural killer (NK) cell activity as a function of age and to examine the histology of the lymphoid organs. In this study four human NHL established cell lines (Daudi, Namalwa, U937, MC116), lymphoma cells from four fresh NHL biopsies and normal peripheral blood mononuclear cells (PBMC) and bone marrow cells were investigated, after intraperitoneal injection into the mice. The presence of the human NHL cells in the peritoneum and spleen was assessed by FACS analysis. The colonization potential was investigated in a range of tissues by polymerase chain reaction (PCR) amplification of human repetitive sequences. These studies revealed clear differences in the abilities of the NHL cell types to colonize the SCID mice. Namalwa, Daudi and U937 cells demonstrated the highest efficiency of colonization and readily formed tumours, whereas MC116, the NHL biopsy cell populations and the non-malignant lymphoid cells showed little ability to survive and colonize other tissues in the SCID mice. Whole body irradiation of the SCID mice appeared to improve the survival of human PBMC, NHL biopsy cells and MC116 cells in the peritoneum, but had little effect on their colonization potential. The significance of these studies is discussed.

The mode of cytotoxic action employed by cytolytic lymphocytes remains unclear, with the possibility of several mechanisms being utilized dependent upon the activation state of the effector cell. In this work, the induction of apoptosis in target cells by 'killer' lymphocytes at differing states of activation has been studied. Although the cytotoxicity of natural killer (NK) cells and recombinant human interleukin-2 (rhIL-2) or interferon-alpha (IFN-alpha)-activated effector cells, against NK-sensitive target cells, was high, their cytotoxic action appeared to be mediated via differing pathways. Effector cells activated short term (4 hr) with rhIL-2 and those mediating rhIL-2 lymphokine-activated killer (LAK) activity after long-term (4 day) activation were found to induce the formation of sodium dodecyl sulphate (SDS)-insoluble apoptotic bodies in NK-sensitive target cells, as well as increasing the level of activity of the apoptosis related enzyme tissue transglutaminase, thus suggesting the induction of the apoptotic pathway as a means of effecting target cell death. Non-activated and short-term (4 hr) IFN-alpha-activated effector cells did not appear to utilize this pathway in the target cell as their means of cytotoxicity. Effector cells showing LAK activity were also cytotoxic towards NK-insensitive cells, and this cytotoxicity again appeared to be mediated via the apoptotic pathway.

Cryosurgery is a useful tool to treat unresectable liver carcinoma. In addition to the local effects of freezing, cryosurgery is thought to affect host immune response. We tested this by measuring natural killer (NK) cell cytotoxicity following cryosurgery of normal liver and an implanted liver tumour in the rat. Controls underwent sham cryosurgery. NK cytotoxicity was enhanced following cryosurgery of normal liver and liver tumour.

Cytotoxic lymphocytes are either MHC-restricted (cytotoxic T-cells) or nonrestricted (natural killer NK-cells), although cells of the monocyte/macrophage lineage are also cytotoxic, and lymphocytes or phagocytic cells expressing Fc-receptors for immunoglobulin can function as antibody-dependent killer cells (referred to as antibody-dependent cellular cytotoxicity: ADCC). Antigen-specific T-lymphocytes recognise their target antigen in the context of MHC class I components, focusing their attack only against those cells expressing the relevant antigen specificity on their cell surface. A more primitive and alternative mechanism exists whereby NK-cells, classified as large granular lymphocytes (LGL), are able to kill in a non-specific manner, not requiring prior sensitisation to antigen. Both antigen-specific T-cells and LGL mediate their cytotoxicity through the release of cytotoxic molecules at the target-effector cell interface. LGL also have a regulatory role in the immune system through the release of cytokines, and can be distinguished from T-lymphocytes by the expression of distinct phenotypic markers (CD16+, CD56+) and they lack CD3 antigen expression or rearranged alpha/beta or gamma/delta T-cell receptor gene products. Cytotoxic activity is positively regulated by interleukin-2 (IL-2) and interferon (IFN), whilst prostaglandins and transforming growth factor-beta (TGF beta) diminish activation and effector pathways. Cytotoxicity mediated by NK- and T-cell populations are principally involved in the defence against microbial infections and neoplasia; the abrogation of cytotoxicity either by direct interaction of 'suppressor factors' with effector cells, or indirectly by reducing cytokine production can inevitably lead to the proliferation of the disease.

Natural killer (NK) cell activity was observed in the peripheral blood and spleen of 8- to 10-week-old Syrian golden hamsters, but not in the bone marrow or thymus. Low, but significant, levels of cytotoxicity were also observed in mesenteric and axillary lymph nodes and cells harvested from the peritoneum. Cytotoxicity, in a 4-hr 51Cr-release assay, was found to be nylon wool non-adherent and was significantly reduced by treatment with trypsin or incubation at 37 degrees C for 18 hr. Natural cytotoxicity was shown to be low at 1 week of age, but increased to a maximum at 8 weeks and was maintained into old age. Correlation was observed between peripheral blood cytotoxicity and the presence of large, often granular, lymphocytes following fractionation of effector cells by Percoll discontinuous gradient separation. These findings are compared with previous studies in hamsters, where the results of longer-term cytotoxicity tests differ in some respects to those of the present study.

In vitro tests of immune reactivity in patients with transitional cell carcinoma (TCC) of the urinary bladder were compared with the clinical and histological diagnosis. Response of peripheral blood lymphocytes to the mitogen phytohaemagglutinin (PHA) tended to be reduced in patients with more advanced disease, although the difference between patients with advanced disease and those with early disease or controls was not statistically significant (P greater than 0.05). Assessment of natural killer (NK) cell activity in the same patients showed no change in levels of reactivity at any stage of the disease. There was no difference between the NK cell activity of patients with TCC of the bladder and normal controls.

Using whole blood from normal subjects, we have observed natural killing of K562 cells in a 4 h 51Cr-release assay comparable with that shown by separated PBMC and whole blood depleted of serum components. Separated plasma was not toxic towards K562 targets, and failed to potentiate the level of PBMC cytotoxicity through ADCC. The presence of red blood cells did not influence natural killing. The natural cytotoxicity of whole blood was augmented by interferon and depressed by prostaglandins E1 and E2. Studies with appropriate control blood fractions show that cytotoxicity tests with whole blood provide results reflecting natural cell-mediated cytotoxicity.

Natural cytotoxicity in the rat was assessed against solid tumors and normal and embryonic rat monolayer culture lines, and the results were compared with rat natural killer activity towards syngeneic, allogeneic, and xenogeneic lymphoma targets. The targets tested showed a very wide range of susceptibility to lysis by peripheral blood and spleen effector cells in both 4- and 18-hr cytotoxicity assays. Cytotoxicity toward the mouse lymphoma, YAC-1, and rat sarcoma LT1 targets was relatively high in a 4-hr assay, compared with that toward the other cell lines used in the study. At 18 hr, increased killing was seen against YAC-1, LT1, SP3 (rat pheochromoblastoma), F2304 (rat embryonic cells), and normal rat kidney cells, while a significant increase in susceptibility with time was not observed with other rat targets: W/Fu-T (sarcoma); ERTh/V-G (sarcoma); R35 (mammary tumor); and W/Fu-P21 (embryo fibroblasts) targets. The cytotoxicity against all of the targets tested was not age restricted and was potentiated by rat interferon. Natural cytotoxic reactivity was seen with effector cells from the blood, spleen, and peritoneal cavity of both W/Fu (euthymic) and Rowett nude (congenitally athymic) rats. Cytotoxic effector cells from the blood were present in the low-density fractions recovered from discontinuous Percoll density gradients and, as shown previously for lymphoma target cells, a strong correlation was observed between the killing of embryonic, normal, and solid tumor targets and the presence of large granular lymphocytes. These results indicate that the naturally cytotoxic rat effector cells for normal fibroblast and bone marrow targets, lymphomas-leukemias, embryonic cell lines, and solid tumor targets are all included in the large granular lymphocyte subpopulation.

Due to the current interest in natural killer (NK) cells as a host defence mechanism against neoplasia, we have investigated the susceptibility of short-term cultures of human ocular melanoma cells to spontaneous and interferon (IFN)-augmented natural cytotoxicity. Cultures of ocular melanoma cells were readily established and identified as bipolar and multipolar pigmented melanoma cells. In short-term cytotoxicity assays these cell lines demonstrated a weak susceptibility to spontaneous human PBL natural cytotoxicity, and increased killing was observed using IFN-augmented cytotoxic effector cells. These findings may be pertinent in relation to the role of NK cells in vivo.

We have identified two components of human seminal plasma which suppress natural killer (NK) cell activity in vitro. Dialysis and gel filtration experiments have shown both components to be of low molecular weight. The first will suppress NK cells following a short period of pretreatment, but this suppression is dependent upon the presence of bovine serum in the medium and is directly related to a loss of cell viability as measured by trypan blue dye exclusion. We suggest that this molecule is a polyamine. The second factor will not suppress NK activity following pretreatment of lymphocytes, but is a potent suppressor when added for the duration of the assay. This suppression is completely bovine serum independent, unrelated to toxicity and appears to be mediated by prostaglandin E2. The relevance of these results to a clinical situation is discussed.

A substantial proportion (44%) of peripheral blood lymphocyte samples from 41 patients with malignant lymphoma have been shown to have depressed or undetectable levels of natural cytotoxicity against the leukaemic cell line K562 in a 4-hr [51Cr]-release assay. No correlation was found between low levels of natural killer (NK) cell activity and either the age of the patients, total or differential white blood counts, or the type or stage of disease. Furthermore, pre-treatment of lymphocytes with human lymphoblastoid (Namalva) interferon failed to enhance NK levels in 5/11 patients with Hodgkin's disease and 5/8 patients with non-Hodgkin's lymphoma, and was in contrast to the response of control peripheral blood lymphocytes assayed under the same test conditions. The lack of responsiveness to interferon of peripheral blood NK cells from lymphoma patients was not wholly associated with those patients shown to have low levels of spontaneous NK activity.

The influence of intact influenza virus and purified detergent solubilized haemagglutinin (HA) subunits from these viruses on human natural killer (NK) cell activity was examined. Effector cells incubated with whole influenza virus for 18 hr initiated the production of alpha interferon which was associated with the enhancement of NK cell activity. In contrast, purified influenza virus HA suppressed NK activity in a dose-dependent manner, when added at the onset of the cytotoxicity assay, or when used to pre-treated effector cells prior to assay for cytotoxicity against K562 target cells. Effector cells exposed to influenza HA for 90 min, washed and re-incubated in fresh medium for up to 18 hr, failed to regain their cytotoxicity. Suppression of NK cell cytotoxicity could not be ascribed to direct toxicity of HA preparations or residual detergent and preservative in these preparations. The augmented cytotoxicity of activated human effector cells was also susceptible to suppression by virus HA, and pretreatment of human PBL effector cells with HA for 90 min, prior to exposure to human alpha interferon caused NK effector cells to become refractive to the enhancing effects of HIFN. That direct interaction between influenza virus HA and effector cells was a requirement for suppression of activity was shown in experiments using Bromelain-released influenza HA, which would not be expected to bind to cells and which failed to suppress NK cell activity.

The effect of recombinant human interleukin 4 (rhIL-4) on the induction in vitro of human lymphokine activated killer cell (LAK) activity was investigated. Peripheral blood mononuclear cells (PBMC) from normal healthy donors were incubated for 4 days with or without recombinant human interleukin-2 (rhIL-2) in the presence or absence of rhIL-4. LAK activity was measured against the NK-resistant colon adenocarcinoma cell line SW742, and NK mediated cytotoxicity was determined using NK sensitive K562 cells. Unlike previous reports using mouse effector cells, rhIL-4 neither induced LAK activity nor augmented the cytotoxic response induced by rhIL-2. In four out of six experiments there was a significant reduction of rhIL-2 induced LAK in the presence of rhIL-4, accompanied by a reduction of Tac antigen expression by rhIL-2 activated cells. Recombinant hIL-4 failed to influence the effector phase of the activated PBMC against SW742 or K562 targets.

The influence of human seminal plasma (SP) and whole semen (S) on the expression of natural cytotoxicity by human peripheral blood mononuclear cells (PBMNC) was examined. Marked suppression of natural cytotoxicity against K562 targets was observed when effectors were pre-treated for 1 h with SP or S diluted up to 1:400. Abrogation of cytolytic activity by SP was not the result of direct lymphotoxicity, although a reduction of approximately 50% in the number of target binding cells was observed. In addition, the cytotoxicity of interferon (alpha-IFN, beta-IFN, gamma-IFN) and interleukin 2 (IL-2) activated human PBMNC was suppressed by components present in human SP, although IL-2 activated human PBMC were relatively resistant to suppression compared with other effector (spontaneous or activated) populations. Following 1 h exposure to SP, PBMNC failed to recover more than 25% of their initial cytotoxic potential upon further in vitro incubation (18 h) in the absence of SP. However, both interferon and IL-2 caused an increase in the cytotoxicity of these populations, in some instances to the level obtained with control, IFN or IL-2 activated PBMNC. The biological significance of SP as an inhibitor of immune function under experimentally defined conditions is discussed in relation to its possible role in vivo.

The influence of interferon alpha and gamma alone or in combination on the augmentation of human natural cytotoxicity was studied. Treatment of peripheral blood lymphocytes with IFN- alpha led to a rapid augmentation of NK activity, in contrast to IFN-gamma where target cell killing was observed only following 18 hrs exposure of lymphocytes to IFN-gamma. The results of the single cell assay paralleled those obtained using the Chromium release test, but neither interferon type caused an increase in the number of target binding lymphocytes. The combined effect of IFN-alpha and IFN-gamma in stimulating human natural cytotoxicity demonstrated individual lymphocyte responses to be variable. Exposure of lymphocytes to IFN-alpha and IFN-gamma for 18 hrs prior to assay for cytotoxicity usually decreased the level of cytotoxicity compared with control values, whereas other treatment regimes gave an additive and sometimes synergistic effect. Only treatment with IFN-alpha for 18 hrs and IFN-gamma for one hr produced a synergistic response in the majority of individuals tested. We conclude from this study that individual responses to IFN-alpha and IFN-gamma alone or in combination are variable and dependent upon timing of exposure of lymphocytes to individual interferon types, and possibly reflects the donor status at the time of sampling.

Endogenous NK activity was measured in ALL patients and compared with child and adult control values. ALL children undergoing maintenance chemotherapy showed significantly lower NK activity than control groups; however, patients off treatment and in remission expressed cytotoxicity within the normal range of the control groups. The expression of the HNK-1 marker in the ALL children was not significantly different from child controls, although ALL patients failed to show the same correlation between cytotoxic activity and HNK-1 expression. The target binding capacity of PBLs from ALL children was significantly greater compared with controls but did not correlate with NK activity. In addition plasma from ALL patients was not inhibitory for NK cytolytic activity, suggesting an innate defect in cytotoxicity mediated by NK cells rather than a plasma inhibitory factor. Patients failing to display NK activity against K562 target cells appeared to mediate killing of measles virus infected (Raji) targets.

Modulation of NK activity of PBLs from ALL patients was studied following exposure to IFN-alpha, staphylococcal protein A and interleukin-2. Only 52% of ALL patients responded to IFN-alpha stimulation, where the majority of controls showed positive enhancement of NK activity. Protein A failed to cause a significant stimulation of ALL patient PBLs whereas all controls showed a positive response. The majority of ALL patient and child control PBLs were however able to produce significant levels of IFN-gamma (protein A stimulation) and IFN-alpha (Sendai virus stimulation), although significantly more of both types of interferon could be induced in adult PBL samples. The ability of IL-2 to activate NK activity of ALL PBL samples showed a similar trend to IFN-alpha stimulation; thus, not all ALL patients showed positive augmentation of NK activity upon IL-2 stimulation. It is clear from these results that interferons and IL-2 may not necessarily lead to activated NK cytolytic activity, and in the present study approx. 50% of ALL patients failed to respond to lymphokine stimulation.

The natural killer (NK) cell susceptibility of hamster tumours exhibiting high and low levels of spontaneous metastasis in vivo was investigated. The parent cell line (HSV-2-333-2-26), which was weakly metastatic, and the NK-sensitive target line K562 were both sensitive to hamster natural killer cell activity. In contrast, 2 in vivo cloned sublines, designated met A and met B, exhibited a high level of spontaneous metastasis and were shown to be weakly or non-susceptible to hamster UK reactivity.

This study was instituted to determine the mechanism of enhanced natural killer (NK) lymphocyte activity during surgery. Natural cytotoxicity of whole blood to K562 target cells was assayed before anesthesia and during anesthesia and surgery in patients with benign and malignant gastrointestinal disease. Those patients with benign conditions and localized primary tumors showed enhanced NK lymphocyte cytotoxicity during surgery (p less than 0.025 and p less than 0.0025, respectively) but not patients with disseminated tumors. In patients with localized tumors, enhancement of NK lymphocyte cytotoxicity was an interferon-independent phenomenon but appeared to be related to a significant rise in the percentage of cells bearing the Leu 7 monoclonal antibody marker for NK cells (p less than 0.02). Exogenous leukocyte interferon caused further enhancement of NK cytotoxicity in patients with benign disease and some cancer patients. Enhancement of NK lymphocyte activity during surgery may be of significance in reducing tumor metastases by stimulation of natural cytotoxic mechanisms to circulating tumor emboli.

The calmodulin antagonist W7 and 4 of its analogues were examined for their ability to inhibit human NK cell mediated cytotoxicity. With the exception of one of these compounds, which is extremely hydrophobic, there was a good correlation between the ability of drugs to inhibit human NK antitumour cytotoxicity and calmodulin-dependent phosphodiesterase activity in vitro. The most potent of the compounds, 5-iodo-1-C8, an analogue of W7, has an IC50 of 3 microM upon biological and biochemical assay. This particular compound is both more potent and specific than the parent compound W7, is non-toxic to cells over the range used and is also capable of inhibiting the biological activity of NK cells upon pre-treatment of the effector cells, inferring the mechanism of NK cytotoxicity to be calmodulin dependent.

Fifty per cent of untreated malignant lymphoma patients were shown to have profoundly reduced levels of peripheral blood lymphocyte-natural cell mediated cytotoxicity (PBL-NCMC) when tested against the leukaemic cell-line K562; assessment of NCMC in unfractionated blood from a large number of these patients showed a comparable reduction in activity. High levels of NCMC were observed in some patients with stage III/IV non-Hodgkin's lymphoma (NHL), while Hodgkin's disease (HD) patients over 40 yr of age had normal levels. In both NHL and HD patients there was no correlation between NCMC and absolute lymphocyte count or lymphoma histology. Human interferon was seen to boost NCMC in the majority of NHL patients, but most HD patients were nonresponders. Almost all patients tested had normal number of target binding lymphocytes, and most had numbers of HNK-1+ cells within the control range. However, on exposure to IFN, the number of target binding lymphocytes increased in over half of the patients tested, with some patients showing an increase in HNK-1+ cells, in the majority of cases without enhancement of NCMC.

A series of Escherichia coli cloned influenza viral gene products were assayed for their ability to augment human natural cytotoxicity in overnight cultures (18 h) at 37 degrees C. Nylon wool nonadherent peripheral blood mononuclear cells (PBMC) proved responsive to stimulation by a number of cloned viral proteins, the most effective being the nonstructural (NS1) protein (but not NS2 protein) and haemagglutinin and matrix antigen components fused to the N-terminal 81 amino acid sequence of NS1. Furthermore, interferon (IFN) was generated in cultures in which enhanced cytotoxicity was detected and was identified as mostly IFN alpha (greater than 90%) with less than 10% IFN gamma contamination. The cell type responding to antigen stimulation was present in Percoll fractions enriched for large granular lymphocytes (LGLs); furthermore PBMC activated by NS1 protein fractionated in the low density Percoll fractions (LGL enriched). Using specific anti-IFN antisera, it was shown that IFN alpha but not IFN gamma was responsible for the enhancement of cytotoxicity. Interferon induction and activation of cytotoxicity could not be ascribed to the presence of contaminating bacterial products. These results suggest that a particular NS1 protein configuration is capable of activating human natural killer cells via the induction of IFN alpha.