Single cell studies on the immunological marker profile of plasmacytoid T-zone cells.

Plasmacytoid T cells (PTC) are known to home to thymic (T) zones in human lymph nodes and are characterized by their abundant, concentrically layered, rough endoplasmic reticulum. These cells have been found in reactive and neoplastic conditions. Three cases of PTC lymphomas have so far been reported. All of them were complicated by a myelomonocytic leukemia leading to the assumption of a functional relationship between PTC and the myeloid system. The immunologic phenotype of PTC, as revealed on frozen tumor tissue sections, comprised the expression of CD5 (T1), CD4 (T4), and HLA-DR, but not CD8 (T8) and CD2 (T11) and suggested an affiliation to the T cell system. Extending our previous report on one of these cases we here present the first study on the immunological marker profile of suspended PTC. The employment of unfractionated or PTC-enriched tumor cell suspensions rendered possible the application of a panel of monoclonal antibodies (moAbs) on both fixed and unfixed cells and enabled us to allocate various markers either to the intracytoplasmic or surface domain of this cell type. Our results suggest that PTC from our case rest in the G0/G1 phase of the cell cycle. They express the transferrin receptor, but not the Il-2 receptor (CD25) or the nuclear antigen Ki-67. No T cell antigen was demonstrated on the surface of unfixed suspended PTC. Under these conditions only HLA-DR and a predominantly monocytic antigen (CD36/moAb 5F1) were identified. Fixed cells, however, showed a weak cytoplasmic reactivity for CD5 and two myelomonocytic antigens (CD15/moAb 1G10 and CD14/moAb My4). Our findings do not sustain positive evidence for a T cell nature of PTC. Whether their phenotypical pattern indicates terminal differentiation with concomitant loss of T cell antigens or points to a cytogenetic relationship of PTC to the myeloid system, remains speculative. Until the cytogenesis of PTC is clarified we propose the noncommitted term "plasmacytoid T-zone cells" for this elusive cell type.

Amino acid control of protein degradation in normal and leukemic human lymphocytes.

Lymphocytes from normal human subjects and from patients with chronic lymphocytic leukemia were found to degrade their endogenous protein at similar rates (2.5-3.0%/h) when incubated in an amino acid-free buffer. Protein degradation was inhibited 20-35% by inhibitors of autophagic sequestration (amino acids, 3-methyladenine) and by inhibitors of intra-lysosomal proteolysis (leupeptin, propylamine), the extent of inhibition being similar in normal and leukemic lymphocytes. The inhibitor effects, together with the electronmicroscopic demonstration of autophagosomes in the lymphocyte cytoplasm, is taken as evidence for the existence of an autophagic-lysosomal pathway in human lymphocytes, potentially responsible for as much as one-third of their overall protein degradation.

Promotive and inhibitory effects of macrophages on the mitogen-induced blastogenesis of lymphocytes: a flow-cytometric study.

The effect of various concentrations of rat-liver macrophages on the Con-A-induced blastogenesis of syngeneic spleen lymphocytes has been studied by means of flow-cytometric methods and by measurement of the uptake of 3H-thymidine. By measuring the distributions of cellular and nuclear volume and cellular DNA to characterize the progression of responding lymphocytes through the cell cycle, we have distinguished a promotive and an inhibitory effect of macrophages. The promotive effect is on the number of cells initiating blastogenesis and on their rate of progression through the cell cycle. The degree of promotion increased strongly with the concentration of macrophages even for concentrations that suppressed the incorporation of 3H-thymidine almost completely from about 30 h of culture. The inhibition observed for macrophage concentrations +/- 10% was a late effect causing stagnation of cell cycle and reduced viability only from about 24 h of culture.

The accessory cell effects of liver macrophages in concanavalin A stimulation of rat spleen lymphocytes.

Liver and peritoneal macrophages under similar test conditions behaved in an identical manner with regard to accessory cell effects in the lymphocyte response to concanavalin A. When present in low concentrations (less than or equal to 3.3%) they stimulate lymphocytes, and when present in high concentrations (greater than or equal to 10%) they inhibit lymphocyte proliferation. These two effects are, however, mediated through totally different mechanisms. Stimulation was an early effect, required viable cells, was not affected by enzymatic treatment of macrophages, and was similar to the effect of 2-mercaptoethanol, allogeneic macrophages, and even non-macrophages. Inhibition occurred at a larger stage of lymphocyte transformation, was sensitive to collagenase and pronase treatment of macrophages, was more specifically due to macrophages, was reduced with allogeneic macrophages, and persisted after freeze-thawing of macrophages. Removal of Fc receptors or related segments of the surface of macrophages greatly reduced their inhibitory capacity, whereas removal of foreign surface receptors apparently had no consequence.

Kupffer cell suspensions and cultures as a tool in experimental carcinogenesis.

Approximately one-third of the cells in the liver are nonhepatocytes. Of these, the Kupffer cells, or phagocytes lining the sinusoids, are of particular significance since environmental carcinogens must first traverse a Kupffer cell barrier before reaching the liver parenchyma. Phagocytosis and subsequent degradation of carcinogens by Kupffer cells lead to their permanent removal. Factors such as membrane receptors, which determine the avidity of Kupffer cells for various substances, would consequently have a decisive role in the primary interaction between carcinogens and Kupffer cells. Likewise, the intracellular lysosomal apparatus, which determines the ability of these cells to degrade various substances, would determine whether these substances can persist in an active form. In vivo data on Kupffer cell clearance of various substances are plentiful. However, to dissect the complex problem of Kupffer cell interaction with carcinogens, a clear-cut in vitro system would certainly be useful. A system for separating Kupffer cells from other types of liver cells and maintaining pure Kupffer cell cultures has been achieved in recent years. Some basic cell biological studies--such as studies of membrane receptors and lysosomal enzyme apparatus--have already been carried out. It could now be rewarding to adopt the system for in vitro studies of Kupffer cell interactions with carcinogens.

Lack of effect of clonazepam on serum levels of diphenylhydantoin, phenobarbital and carbamazepine.

The influence of clonazepam on steady-state serum levels of diphenylhydantoin, phenobarbital and carbamazepine was studied in 22 patients with epilepsy receiving one or two of these drugs. Clonazepam was given in slowly increasing doses for 2 weeks until a maximum dose of 4-6 mg per day was reached; thereafter the dose was kept constant. The serum levels of diphenylhydantoin, phenobarbital and carbamazepine were determined once a week for at least 6 weeks. During clonazepam medication in ordinary doses the serum levels of the drugs in question were unaltered. Thus, it is unlikely that the antiepileptic effect of clonazepam in these patients even partly may be due to increased serum levels of these other drugs mentioned.

Introduction of B-chain-inactivated ricin into mouse macrophages and rat Kupffer cells via their membrane Fc receptors.

Experiments have been made to test whether the toxic lectin ricin can be bound to and introduced into cells by some other mechanism than via its B chain, the natural binding moiety of the toxin, without its toxic effect being neutralized. Complexes consisting of ricin and antibodies specifically directed against ricin B chain were incubated with mouse peritoneal macrophages and rat Kupffer cells, which are known to possess surface receptors for the Fc portion of the immunoglobulin molecule. After incubation for 26 h, cellular protein synthesis, as measured by incorporation of labeled leucine into acid-insoluble material, was completely inhibited. HeLa cells, which do not possess Fc receptors, were unaffected by the complex. The effect of the complex on protein synthesis of macrophages was prevented by soluble antigen-antibody complexes, but not by the presence of lactose which prevents attachment of the ricin B chain to the cell membrane. The [ricin-antiricin B] complex was attached to red cells, and the resulting complex was incubated with rat Kupffer cells. Cellular protein synthesis ceased after 6 h, and phase contrast microscopy studies showed that the complexes were taken up by the Kupffer cells. The data indicate that ricin, when present in the complex with antiricin B, can be introduced into cells through cell membrane receptors other than the B chain receptor, in this case the Fc receptor, and that the internalized toxin retains a least part of its activity.

Mass isolation and culture of rat kupffer cells.

Collagenase perfusion of the liver followed by pronase treatment of the cell suspension thus obtained gave a quantitative recovery of viable nonparenchymal liver cells (NPC). From these NPC, Kupffer (K) cells can be purified by attachment to tissue culture dishes. Tail vein injection of carbon 1-2 h before liver perfusion permitted stepwise calculation as well as visualization of carbon-containing K cells. When these K cells have been put into tissue culture medium with serum and incubated overnight, they exhibit typical macrophage characteristics. Phase-contrast and transmission electron microscopy showed typical macrophage morphology and scanning electron microscopy revealed well-spread cells with cytoplasmic projections and ruffled membranes. Endocytosis studies using radioactive colloidal gold and inert latex particles also indicated that these cells are highly active in pinocytosis and phagocytosis. Further characterization of K cells is the identification of Fc receptor on their membranes. Studies on lysosomal enzymes showed that purified K cells possess higher specific activities in beta-glucuronidase, acid DNase, and cathepsin D than in purified parenchymal cells.