Eero Saksela

Isolation of human NK cells by density gradient centrifugation

Sedimentation characteristics of human NK cells in discontinuous Percoll density gradients were studied. NK activity against the leukaemic cell line K-562 peaked in a single low density fraction mainly consisting of large granular lymphocytes previously shown to be the principal NK cells in human peripheral blood. Percoll density gradient centrifugation provides a useful tool for analysis of human NK cells and their relationship to other blood mononuclear cells.

Morphological and functional characterization of isolated effector cells responsible for human natural killer activity to fetal fibroblasts and to cultured cell line targets.

White cells of human blood (Oldham et al. 1973, Takasugi et al. 1973) and cells recovered from various lymphohematopoietic organs of experimental animals (Herberman et al. 1975a, Kiessling et al. 1975a) display spontaneous cytotoxic activity against various, mostly tumor, target cells in vitro. This cytotoxic activity, taking place in the absence of known presensitization, has been termed natural killer (NK) cytotoxicity. Because the natural killer phenomenon can most readily be demonstrated by using cultured tumor cells as targets, it has been suggested that the phenomenon may be linked to surveillance against cancer (Kiessling et al. 1975c, 1976a, Jondal et al. 1978). Numerous attempts have been made to characterize the effector cells responsible for the NK activity. Fractionation procedures utilizing different surface antigens, surface markers and functional properties of lymphohematopioetic cells have been employed (Herberman et al. 1975b, Kiessling et al. 1975b, Herberman et al. 1977, Kiuchi & Takasugi 1976, Jondal & Pross 1975, Vose & Moore 1977, West et al. 1977, Eremin et al. 1978, Bakacs et al. 1977,

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Abstract Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.

Human Natural Killer Cell Activity is Augmented by Interferon via Recruitment of ‘Pre‐NK’ Cells

Contact with various cell‐line targets increases the natural killer (NK) activity of human lymphocytes, Supernatants of such 20 h co‐cultures augment the NK activity of virgin lymphocyte populations, and the augmenting factor penetrates 0.2 mTm Millipore filters. The supernatants also contain interferon, and partially purified human leucocyte interferon increases NK activity when added to 20 h assays with 51Cr‐labelled K‐562 target cells. Potent anti‐interferon antiserum added to the co‐cultures inhibits the target‐cell‐induced augmentation phenomenon. Both the target‐cell contact and interferon‐induced augmentation affect a population of human lymphocytes, from which the ‘mature’ NK cells have been removed by adsorption‐elution using fetal fibroblasts as adsorbents. The activity of ‘mature’ NK cells is not enhanced, and we conclude that the augmentation is mediated by recruitment of ‘pre‐NK’ cells.

c-Myc induces cellular susceptibility to the cytotoxic action of TNF-alpha.

Tumor necrosis factor‐alpha (TNF) is a multifunctional cytokine which is cytotoxic for some tumor cells and transformed cells. The molecular mechanisms which render transformed and tumor cells sensitive to the cytotoxic action of TNF are unclear. We show here that an increased expression of the c‐Myc oncoprotein strongly increases cellular sensitivity to TNF cytotoxicity. In Rat1A fibroblasts, which are resistant to TNF, the addition of TNF with a concomitant activation of a hormone‐inducible c‐Myc‐estrogen receptor chimera (MycER) resulted in apoptotic cell death. Similarly, c‐Myc overexpression enhanced the sensitivity of NIH3T3 fibroblasts to TNF‐induced death. The c‐Myc and TNF‐induced apoptosis was inhibited by ectopic expression of the Bcl2 oncoprotein and by the free oxygen radical scavenging enzyme Mn superoxide dismutase. Furthermore, in highly TNF‐sensitive fibrosarcoma cells, antisense c‐myc oligodeoxynucleotides caused a specific inhibition of TNF cytotoxicity. Our results suggest that the deregulation of c‐Myc, which is common in human tumors and tumor cell lines is one reason why these cells are TNF sensitive.

Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-κB activation

Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF‐sensitive upon deregulation of c‐Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF‐elicited signaling responses in fibroblasts containing a conditionally active c‐Myc protein. In association with cell death, c‐Myc impaired TNF‐induced activation of phospholipase A2, JNK protein kinase and cell survival‐signaling‐associated NF‐κB transcription factor complex. The TNF‐induced death of mouse primary fibroblasts expressing deregulated c‐Myc was inhibited by transient overexpression of the p65 subunit of NF‐κB, which increased NF‐κB activity in the cells. Unlike other TNF‐induced signals, TNF‐induced accumulation of the wild‐type p53 mRNA and protein was not inhibited by c‐Myc. TNF, with c‐Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53‐deficient primary fibroblasts. The C‐terminal domain of p53, which is a transacting dominant inhibitor of wild‐type p53, failed to inhibit apoptosis by c–Myc and TNF, suggesting that the cell death was not dependent on the transcription‐activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein‐expressing cells involves p53 and an impaired signaling for survival in such cells.

The cytotoxic activity of human natural killer cells requires an intact secretory apparatus.

Abstract We have analyzed the effect of various inhibitors of cellular secretion and motility on the cytolytic activity of human natural killer (NK) cells. As effector cells we used highly purified peripheral blood lymphocytes consisting of 75–85% large granular lymphocytes (LGL) that have previously been shown to be responsible for the NK activity in man. Treatment of the effector cells with a carboxylic ionophore monensin inhibited irreversibly the NK-cell-mediated killing. This drug is known to interrupt the vesicular traffic of Golgi-derived vesicles and thus the results strongly suggested that secretory processes are required in the cytolytic activity of human NK cells. In the monensin-treated effector cells large amounts of glycoprotein accumulated in the Golgi area within 24 hr of incubation. The lytic activity did not require intact microtubules since effector cells in which vinblastine-induced tubulin-containing paracrystals were demonstrated still mediated normal NK activity. Energy was required in the human NK-cell-mediated cytolysis. The lethal hit stage of the cytolytic activity was preceded by formation of intimate contacts between effector and target cells and required active cell movement and divalent cations.

Human natural cell-mediated cytotoxicity against fetal fibroblasts. III. Morphological and functional characterization of the effector cells.

Abstract We have isolated and characterized human natural killer cells cytotoxic to human fetal fibroblasts utilizing adsorption-elution of the effector cells from target cell-coated beads. The cell associated with enriched cytotoxicity was slightly larger than small- to medium-sized lymphocytes, the cytoplasm was pale and characteristically granular. In direct surface marker analysis the cell was Fc-receptor-positive, formed E-rosettes, and displayed strong either diffuse or granular ANAE reactivity in the cytoplasm. The ANAE reactivity could not be inhibited with sodium fluoride and in mitogen and antigen stimulation experiments the cell had T-cell characteristicis. The cell type was termed large granular lymphocyte and we suggest that it is the main direct effector cell for natural killer activity against human fetal fibroblasts.

Parotid clear-cell adenoma of possible myoepithelial origin.

A case of a parotid tumor occurring in a 67‐year‐old man, who had noted a slowly gowing swelling under the left ear 4 years prior to admission to the hospital, is described. At operation, the tumor was pale and firm, encapsulated, and did not affect the facial nerve. Histologically, it consisted of polyhedral cells with clear cytoplasm and regular nuclei surrounding remnants of the acinar secretory system of the gland. Histologically and electron microscopically, the picture was compatible with the diagnosis of myoepithelial clear‐cell adenoma. The tumor is considered to be a rare variant of the pleomorphic adenoma but with sufficiently characteristic features to be separated as a diagnostic entity. At follow‐up 22 months after resection of the superficial lobe containing the tumor, the patient is symptom free.

CARCINOEMBRYONIC ANTIGEN IN MALIGNANT AND NONMALIGNANT GYNECOLOGIC TUMORS Circulating Levels and Tissue Localization

Carcinoembryonic antigen (CEA) was determined in the serum and tissues of patients with gynecologic tumors utilizing radioimmunoassay and immunoperoxidase techniques. Among 328 patients with gynecologic cancer the serum CEA concentration was elevated over the normal background level of 5 ng/ml in 32 cases (9.8%), and the frequency of elevated CEA levels increased with advancing clinical stage. Among 84 patients with benign tumors CEA was elevated in 5 cases (6%). Elevated levels were most commonly seen in patients with ovarian cancer (20%). In squamous cell carcinoma of the uterine cervix elevated levels occurred in 10%, and in cervical adenocarcinoma CEA elevation took place in 19% of the cases. In endometrial cancer CEA became less frequently elevated (7%). After radical surgery the CEA concentration decreased and remained low in 20 of 24 cancer patients (83%). Fifteen of these remained clinically tumor free during the follow‐up period of 4–24 months. Three of four patients whose CEA concentration did not significantly decrease after operation were found to have residual tumor. Radiotherapy resulted in an increased CEA concentration in 3 of 5 patients with advanced cancer. Immunoperoxidase staining of CEA was carried out in formalin‐fixed paraffin‐embedded sections of 64 tumors and 33 specimens from normal genital tract. Tissue CEA was demonstrable in early as well as in advanced stages of malignant tumors. All normal tissue specimens from the genital tract were CEA‐negative, but the brushborder of normal colon was CEA‐positive. CEA activity in tissue was found in 11 of 14 squamous cell carcinomas of the cervix (79%) and in 7 of 9 cervical adenocarcinomas (78%). By contrast, endometrial adenocarcinoma showed CEA staining in one of 11 tumors only, and in ovarian adenocarcinoma positive CEA staining was never seen. CEA reactions were sometimes found in benign mucinous cystadenomas of the ovary, but not in serous cysts. The serum CEA concentration was normal or sometimes slightly elevated in cases where the tumor was CEA‐negative, whereas normal or even highly elevated serum CEA concentrations were seen in CEA‐positive tumors. On the basis of these results the following conclusions can be made: 1) Serum CEA measurement is likely to be more effective in cervical and ovarian than in endometrial cancer for the detection of recurrent CEA‐positive tumors. 2) It is possible by immunoperoxidase technique to identify certain CEA‐positive cervical carcinomas before the serum CEA concentration becomes elevated. 3) Some benign ovarian tumors apparently produce high amounts of CEA without any clinical evidence of malignancy.