rIL-2-activated killer cell generation is influenced by autologous TNF-alpha production.

Low-density (LD) lymphocytes and their CD16+ and CD16- subsets were cultured with recombinant interleukin-2 (rIL-2) in the presence or absence of tumor necrosis factor-alpha (TNF-alpha)-neutralizing antibodies (Abs). Cultures were evaluated with respect to the cell recovery, K562 cytolytic potential and interferon (IFN)-gamma production. The addition of anti-TNF-alpha neutralizing Abs to rIL-2-activated cultures of LD lymphocytes resulted in greater cell expansion and a decrease in cytolytic potential. In the CD16+ subpopulation of LD lymphocytes, there was an increase in cytolytic potential and no improvement in the cell yield was observed. The CD16- subpopulation of LD lymphocytes was not affected by the Abs. In cultures with anti-TNF alpha Abs (polyclonal or monoclonal), a decrease in the rIL-2-dependent generation of IFN-gamma was observed. Recombinant TNF-alpha, when added to rIL-2-supplemented cultures of LD lymphocytes, enhanced the rIL-2-induced generation of IFN-gamma in a dose-dependent manner. TNF-alpha appeared to up-regulate rIL-2-induced IFN-gamma production by LD lymphocytes and to modulate lymphokine-activated killer cell generation (neutralization of TNF-alpha promoted cell expansion in LD lymphocyte cultures and cytotoxicity in CD16+ lymphocyte cultures).

Phenotypical and functional characterization of Fc gamma receptor I (CD64)-negative monocytes, a minor human monocyte subpopulation with high accessory and antiviral activity.

Fc gamma receptor I-positive (CD64+) and Fc gamma receptor I-negative (CD64-) monocytes were prepared from highly purified (elutriation-derived) human monocytes by cytofluorograph cell sorting, and a phenotypical and functional dissociation of the isolated CD64+ and CD64- monocyte subsets is demonstrated. Surface analyses revealed that the surface antigen pattern of CD64+ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64- monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA-A, -B, -C) and MHC class II antigens (HLA-DR, -DP, -DQ), and low expression of the monocyte-specific marker CD14 which is found on nearly all CD64+ monocytes. However, 75% of the CD64- cells were found to be esterase-positive, and 85% were positive for the the CD64- cells were found to be esterase-positive, and 85% were positive for the monocyte/macrophage-specific intracellular antigen CD68. Furthermore, CD64- monocytes show significantly higher expression of CD45RA and Fc gamma receptor III (CD16) than CD64+ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64- monocyte subset have a higher accessory cell capacity in antigen-driven T cell activation than CD64+ monocytes. CD64- monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon-gamma and also higher proliferation in responding autologous T cells than PPD-pretreated CD64+ monocytes. Similar results were obtained for T cells in mixed leukocyte reaction. Interferon-gamma release and proliferation of allogeneic lymphocytes were consistently higher in the presence of irradiated CD64- monocytes than of irradiated CD64+ monocytes. Furthermore, when CD64- and CD64+ monocytes were stimulated with Newcastle disease virus, we measured an up to 67-fold higher interferon-alpha release from CD64- than from CD64+ monocytes, indicating a higher anti-viral capacity of this subset. CD64- monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan- or latex-induced chemiluminescence than CD64+ monocytes. These findings indicate that CD64- monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon-alpha.

The role of monocytes in the induction and regulation of IFN-gamma production by lectin-activated human T lymphocytes.

The data presented show that the production of interferon gamma (IFN-gamma) by pokeweed mitogen (PWM)-activated T lymphocytes requires monocytes and that the amount of lymphokine produced depends on the number of monocytes present in the culture. Accessory function of monocytes was independent from their ability to secrete IL-1 but required cell-cell contact, since blocking of adhesion molecules reduced the IFN-gamma production. Furthermore, production of IFN by lectin-preactivated T lymphocytes could not be triggered by IL-2 but also required monocyte-T cell interaction.

Augmentation of interleukin 2-activated cytotoxicity after treatment of cells with inhibitors of interferon production.

Low-density human lymphocytes cultured with recombinant interleukin 2 (rIL-2) generated a high level of interferon(s) (IFN). Consistently more IFN including IFN-tau was produced during the first 3 days of culture with rIL-2 than during the subsequent 4 days. That ability was mainly associated with mepacrine+ cells and was decreased by low concentrations of leucine methyl ester (Leu-O-Me) or ammonium chloride. Leu-O-Me was employed either for the pretreatment of cells before the culture or as the additive to culture medium. The decrease in IFN production after pretreatment was associated with enhanced rIL-2-activated cytotoxicity. Similarly, 1 mM of ammonium chloride or 1 mM of Leu-O-Me added to rIL-2 supplemented cultures for 3 days showed an association between inhibition of IFN-tau generation and increased activation of cytotoxic activity. Thus NK cells appear to regulate their own response to rIL-2 activation and the control mechanism seems to be associated with the ability of the cells to produce IFN(s) and possibly other cytokines.

Detection of interleukin 1 with human dermal fibroblasts.

A fibroblast proliferation assay was developed for the detection of interleukin 1 (IL 1). Proliferation was measured by thymidine incorporation and by staining of cellular proteins with crystal violet. Response of fibroblasts was optimal at cell numbers of 4,000 to 9,000 cells/culture and an incubation period of four days. Serum content of the culture medium, ranging from 1 to 10% fetal calf serum (FCS), enhanced the proliferative response in a concentration-dependent manner, while higher concentrations of FCS did not lead to further increase. Both detection methods were equally suitable for the measurement of IL 1 biological activity in purified and crude preparations. In contrast to the conventional thymocyte comitogenic assay, the fibroblasts in this assay did not proliferate in response to IL 2 or IL 6. Fibroblasts were weakly stimulated by recombinant (rec) tumor necrosis factor (rec TNF-alpha); they did, however, not proliferate in response to mitogens, lipopolysaccharide, rec granulocyte-macrophage colony stimulating factor (rec GM-CSF), macrophage-CSF, rec interferon-gamma, insulin or transferrin. The detection of IL 1 activity by crystal violet staining of human dermal fibroblasts was easier and faster than by measurement of thymidine incorporation of fibroblasts or mouse thymocytes; without loss of sensitivity, the sample capacity of the IL 1 assay could be enhanced, and the use of experimental animals was avoided.

The correlation between tissue differentiation and production of mammary tumor virus (MTV) in transplanted murine mammary tumors. Electron microscopic observations.

The spontaneous mammary tumors of the NMRI mouse are well developed microcystic adenocarcinomas. Serial isologous transplantation of the tumors results in nearly complete dedifferentiation to a solid tumor, in which only electron-microscopically rudimentary acinus-like microlumina can be observed. The adenocarcinomas produce A and B particles in abundance, with the A particles appearing intracellularly in the adluminal cytoplasmic regions of the epithelial cells in association with typical cellular structures and the B particles being restricted to closed extracellular compartments such as vacuoles or acini alone. The loss of alveolar organization in the solid tumors is followed by an almost complete reduction in mature B particles, while A particles are still regularly observed and appear to be less reduced in number. This suggests that the production of extracellular B particles is dependent upon the secretory activity of the tumor cells and that in nonsecreting cells it is predominantly a late step in virus release that is inhibited, not the synthesis of intracellular precursors.

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse. II. A freeze-fracture study in conjunction with filipin.

The distribution, size, and configuration of intercellular junctions in the sebaceous tumor of mice were examined using the freeze-fracture technique. Three types of junctions were observed: desmosomes, gap, and tight junctions. Tight junctions in general consisted of short linear unbranched fibrils, and macular or complex tight junctional patterns were present only occasionally. Gap junctions ranged from small sports of 0.9 x 10(-3) micrometer2 to areas of approximately 0.46 micrometers2. Desmosomes were the most frequent junctional specializations, and it is concluded that they are at least partially responsible for this tumor not being metastatic. Filipin in conjunction with freeze-fracture showed filipin-sterol complexes on the plasma membrane, the nuclear envelope, and the membranes of the endoplasmic reticulum and mitochondria. The intercellular junctions were devoid of these complexes indicating that these regions are low in cholesterol.

Structure and function of the junctional complement of spontaneous and transplanted murine mammary carcinomas.

In this study the morphology of intercellular junctions in a murine mammary adenocarcinoma and in a solid carcinoma which resulted from continuous transplantations of this spontaneous tumor are described employing the techniques of ultrathin-sectioning after conventional fixation and tannic acid fixation and freeze-fracturing. The acini of the original adenocarcinoma are separated from the intercellular spaces by tight junctions which form a narrow belt-like zonula around the adluminal parts of all epithelial cells. The apical-to-basal width of the tight junctions varies from 0.4 to 0.8 micrometer. Desmosomes and gap junctions are located close to the zonulae occludentes. The size of gap junctions ranges from small spots 0.2 micrometer in diameter to large areas approximately 1.5 micrometer in diameter. In the solid carcinoma these cellular junctions appear without acinar organization randomly between the cells. In addition, special contact zones can be observed. The contact zones seem to be the precursors of a formation plaque for gap or tight junction formation. The sizes of the gap junctions show a wide range of variation, from as small as 5 particles to as large as 0.5 micrometer in diameter. Tight junctions do not form continuous belt-like zonulae indicating that they have lost their significance as a permeability barrier. They are interpreted as mechanical links. In addition to isolated gap junctions and tight junctions there exist gap junctions adjacent to tight junction fibrils suggesting a probable biogenetic relationship of these two structures.

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse.

In this study a transplantable sebaceous adenoma is presented, which maintained its histological structure with similarities to normal sebaceous glands over a long series of transplantations. From the periphery toward the center a basal lamina, an outer layer of undifferenciated stem cells, loosely connected secreting tissue, a tightly packed zone of close cell attachment, and finally a central sebaceous zone can be distinguished. Special reference is made to the belt-like region around the zone of cell lysis and sebum accumulation, which forms a barrier to the surrounding tissue by means of numerous cell adhesions in the form of desmosomes. Relatively few virus particles of the retrovirus group, particularly type A particles, were found in any of the examined tumors.

Electron-microscopic morphology of virus replication in cells of the murine Borstel-leukemia X 429.

Intraperitoneal injections of cell-free filtrates of Borstel-leukemia X 429 (immature-cell myeloid leukemia induced by 200 rad whole-body X-irradiation) were given to 330 1-week-old NMRI mice of both sexes. Leukemia developed in 21% after injection of cell-free filtrates from leukemic spleens and in 20% after injection of cell-free filtrates from leukemic lymph nodes. The mean time lapse from injection to the appearance of the leukemia was 12.8 months. The virus replication in the newly formed leukemic cells, was studied by electron microscopy. Special attention was given to the relation between A-particles and mature type-C viruses. The leukemia cells contain intracytoplasmic and intracisternal A-particles consisting of two concentric spherical shells with an external diameter of 70 nm. A-particles present mainly as closed "virus fields" in the vicinity of the cell nucleus, frequently in the Golgi area. The intracisternal A-particles appear inside the lumen of expanded vesicles of the cytoplasma. Typ-C viruses develop on the cytoplasmic membrane and/or on the outer cell membrane by condensation of crescent-shaped electron-dense zones. All layers of the later mature virus are recognisable in these early morphological stages. After the virus has been detached from the cell it collapses, and the outer membrane appears wrinkled. No spatial correlation between the storage sites of the A-particles and the sites of formation of mature type-C viruses are demonstrable.

[Transplantable sebaceous adenoma of the mouse with virus particles studied by electron microscopy (author's transl)]. / Transplantables Talgdrüsenadenom der Maus mit elektronenmikroskopischem Virusnachweis

Histology and ultrastructure of a sebaceous adenoma of the mouse -- derived from i.p.-injection of 0.1 ml DNA, isolated from cells of a transplantable mouse-leukemia -- were studied. The original tumour and all transplanted tumours display the same tissular differentiation. In all tumours virus particles were found. Structure and behaviour of the intracytoplasmic A-particles resemble those of the mouse mammary tumour virus while the C-particles found extracellular were identical with the leukemia viruses of the mouse. It might be suspected that mammary tumour virus and leukemia viruses are either non specific or that a new virus is present in the sebaceous adenoma.

[Colonization of the mouse liver by transplanted virogenic leukemia cells. Electron microscopic investigations (author's transl)]. / Die Kolonisation transplantierter virusbildender Leukämiezellen in der Leber der Maus. Elektronenmikroskopische Untersuchungen

Cells of a virogenic, immature myelogenic leukemia were injected in i.p. in one week old mice, strain NMRI, each animal receiving 10(5), 10(6) or 13(7) cells respectively in 0.2 ml Hanks' BSS. Mice were sacrificed at different times between 10 hrs and 21 days p.i. and liver specimens were prepared for electronmicroscopic studies. 10 hrs p.i. the leukemic cells are found in the sinusoids of the liver and after 30 hrs in the periportal fields. The leukemia cells migrate into the Disse spaces from the sinusoids. The leukemic cells penetrate with cytoplasmic digitations between connected endothelial cells. There is no lytic disintegration of the endothelial cells. The leukemic cells multiply by mitotic division of the Disse spaces, thereby compressing the liver cells. The cell membranes of both cell-types remain intact. Electronmicroscopically no evidence is found of a secretion of enzymes from the leukemia cells that destroy liver cells. Destruction of the liver cells is the consequence of the growth-pressure exerted by the dividing leukemic cells. Within the liver of the recipients the leukemic cells produce RNA-viruses. In the cytoplasm - frequently in the area of the Golgi-field - groups of immature A-particles are formed. In the virus fields the ribosomes disappear. The immature A-particles consist of two concentric electron dense shells. On the surface of the liver cells budding particles and immature C-particles develop. Extracellular mature C-particles with a homogeneous nucleoid and an irregular outer shell may be seen. The formation of viruses is found to be independent of the stage of the leukemia.