Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.     

Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells

The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.     

Interaction of arterial cells. I. Endothelial cells alter cholesterol metabolism in co-cultured smooth muscle cells

Results of previous in vivo experiments indicated that the presence of arterial endothelium modifies cholesteryl ester (CE) metabolism and the retention of low density lipoproteins (LDL) in injured arteries. We describe herein the effects of bovine arterial endothelial cells (ENDO) on the CE cycle, fluid phase endocytosis, and cell proliferation in co-cultured bovine arterial smooth muscle cells (SMC). Following several days of cultivation on confluent SMC, ENDO were removed from SMC by treatment of the co-cultures with 1.0% collagenase (type II). Removal of only ENDO from the co-culture dishes was confirmed by immunofluorescent staining for Factor VIII antigen, hemotoxylin-eosin staining, and biochemical analyses. We observed that ENDO grown to 75% confluency on confluent SMC induced: 1) a reduction of CE hydrolysis as a result of decreased lysosomal CE hydrolytic activity in SMC as compared to SMC cultured alone; and 2) an increase in the rate of incorporation of labeled oleate into CE as a result of increased acyl CoA:cholesterol O-acyltransferase activity in SMC as compared to SMC cultured alone. Neither endothelial cell-derived culture media (ECDM) nor fibroblasts modulated CE metabolism in co-cultured SMC. Additional experiments showed that the presence of endothelial cells or ECDM decreased the proliferation of co-cultured SMC by 50%, but enhanced the endocytotic rate of labeled sucrose into SMC threefold. Results of experiments described herein demonstrate that, in addition to providing a thrombo-resistant surface and regulating permeability, endothelial cells may also serve to modulate cholesteryl ester metabolism in smooth muscle cells derived from the arterial wall.     

BCG-induced murine effector cells. II. Characterization of natural killer cells in peritoneal exudates.

The cytotoxic activity in peritoneal exudates harvested from C57BL/6 mice 4 to 6 days after they had received viable Bacillus Calmette-Guérin (BCG) organisms i.p. was associatee with a nonadherent, nonphagocytic cell. The cytotoxic cell lacked demonstrable surface immunoglobulin and Thy 1 antigen and bore no readily detectable Fc receptors. Lytic activity was labile at 37 degrees C and was diminished after trypsinization of the effector cells. Preincubating effector cells with immune complexes was without effect on lytic expression. These features make it likely that the cytolytic activity was associated with "natural killer" (NK) cells, previously described in unimmunized mouse spleens and mesenteric lymph nodes. Whether BCG induced "activation" of resident NK cells, the de novo production of NK cells, or "homing" into the peritoneum of cells normally resident in other lymphoid tissue is not known.     

Macrophage-like suppressor cells in rats. I. Inhibition of natural macrophage-like suppressor cells by red blood cells

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal.After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later.Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.     

Stellate cells of aortic intima: II. Arborization of intimal cells in culture.

The present study analyzed effects of different cAMP-elevators on cell morphology in primary culture of human intimal and medial cells from grossly normal and atherosclerotic areas. In primary culture of human aortic cells adenylate cyclase activator forskolin and other cAMP elevators induced arborization of cells, i.e. they reversibly changed the shape of cells. This resulted in the formation of thin branching processes and in the concentration of cytoplasm around the nucleus. In the culture, the shape of the arborized cells resembled that of stellate ones detected in the aortic intima in situ. The arborization of cells was accompanied by destruction of myofilaments. Due to cAMP elevators' effect, most of the arborized cells were exhibited in the cultures isolated from the elastic-hyperplastic layer of the intima. The number of arborized cells was significantly less in the cultures isolated from the musculo-elastic layer and still lesser in those isolated from media. We failed to reveal any significant difference in the number of arborized cells cultured from fatty streaks, atherosclerotic plaques and grossly normal aortic areas. Obtained results suggest that the previously revealed polymorphism of human aortic intimal cells may be accounted for by the cell shape transformations underlined by the mechanism similar to that of arborization in vitro.     

Automaticity in cardiac cells.

Automaticity is the result of dynamic changes in transmembrane currents during electrical diastole. It is readily demonstrated in most cardiac cell types. In all four cardiac cell types studied by the voltage clamp technique (Purkinje, ventricular, atrial, and sino-atrial node fibers), the major change detected during diastolic depolarization is a decrease in outward current. This decrease in a repolarizing current (largely potassium mediated) permits an inward current (sodium and/or calcium mediated) to depolarize the cell.All four cardiac cell types appear to possesess a time-dependent potassium conductance which controls the decrease in outward current over the ?70 to ?30 mV potential range. Purkinje fibers manifest an additional conductance which is responsible for automaticity in this type of cell at potentials between ?100 and ?70 mV.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Growth of mouse ectoplacental cone cells in subcutaneous tissues. Development of placental-like cells.

Ectoplacental cones of mouse embryos collected on day 8 of pregnancy were grafted into the dorsal subcutaneous tissue of host mice. The grafts were collected between days 3 and 8 after transfer and processed for light and electron microscope morphological analysis as well as for cytochemistry of nonspecific alkaline phosphatase. Fragments of normal mouse placentas collected between days 12 and 18 of pregnancy were processed similarly. About 37% of the grafts were nonhemorrhagic nodules formed by different kinds of trophoblastic cells. These cells had many morphological and cytochemical features of cells present in normal mouse placentas. Nonphagocytic giant cells, glycogen cells, as well as cells with a well-developed granular endoplasmic reticulum were similar to cells found in the placenta and were always present in the grafts. Cells showing features intermediate between the above-mentioned cells and those whose cytoplasm was poor in organelles also were found in the grafts. The latter resembled cells of layer 1 of the labyrinth of the placenta. These results suggest that trophoblastic cells of the ectoplacental cones had differentiated into placental cells following their transfer to the subcutaneous tissue.     

Endocytosis in polarized cells.

Recent biochemical and morphological studies using the MDCK cell line have provided insights into the organization of the endocytic pathways in an epithelial cell. The cytoskeletal organization of these cells has been described and evidence for the involvement of microtubules in facilitating endocytic traffic has been obtained. The findings with this model system are compared to results from in vivo studies of the endocytic pathways from the surface domains of specialized epithelial cells and to studies of endocytosis in neurons.     

Dendritic cells in glomerulonephritis.

Renal biopsies (n = 45) from patients with various forms of glomerulonephritis (GN), comprising mesangial IgA-GN (n = 25), focal glomerular sclerosis (n = 13) and acute GN (n = 7), were examined by double staining immunocytochemistry (APAAP, streptavidin-peroxidase) using unconjugated monoclonal antibodies (Ab) against--(i) the CD1b antigen expressed on dendritic cells (DCs), (ii) the invariant chain (Ii), and (iii) biotin-conjugated Ab against HLA-DR. In normal control kidneys (n = 7) without interstitial inflammation, CD1b-positive DCs were not detected. Glomerular endothelial cells and a few cells in mesangial areas showed double staining with the Ab against HLA-DR in Ii. In GN without active interstitial inflammation (n = 9), CD1b-positive DCs were not found. In biopsies with interstitial inflammation (n = 36) CD1b-positive DCs were found interspersed among other inflammatory cells. In seven of the biopsies showing IgA-GN DCs were seen in the vicinity of those glomeruli that exhibited either crescents or glomerular sclerosis with splitting of Bowman's capsule. In proximal tubular epithelial cells de novo expression of HLA-DR/Ii-chain was only seen when DCs were present. We conclude that in different forms of GN: (i) CD1b-positive DCs play an important role in the development of interstitial inflammation, and (ii) their presence may be related to the de novo coexpression of HLA-DR/Ii in tubular epithelial cells, possibly mediated through the production of interferon gamma and other cytokines.     

Culture of human haemopoietic cells in diffusion chambers. I. Growth of normal peripheral blood cells.

Growth of mononuclear cells from human peripheral blood from 10 normal individuals was tested in diffusion chamber culture over a period up to 17 or 21 days. After an initial decrease during the first few days an increase of the total cell number was observed with maximal values on day 13. In all individuals growth of undifferentiated blast cells, lymphocytes, plasma cells, immature and mature granulopoietic cells, macrophages, and megakaryocytes occurred. In all individuals the different cell types had similar growth patterns in diffusion chamber culture. The considerable numerical variations which were seen in the granulopoietic cells were probably due to different stem cell concentrations in the peripheral blood of the investigated individuals. The results indicate that the diffusion chamber technique is a valuable method for the detection of haemopoietic stem cells and the culture of lymphocytic cells in man.     

Reaggregation of sea urchin blastula cells. I. Intrinsic differences in the blastula cells

The reaggregation process was studied in dissociated blastula cells from sea urchin embryos to characterize the degree of differentiation among them. During the reaggregation process at least four different cell types appear: (a) cells that remain round, do not reaggregate, and can differentiate into pigment cells; (b) epithelial cells that spread on the substratum and join together to form epithelial sheets, which then develop cilia and round up to form blastulalike structures; (c) spindle-shaped cells that send out protoplasmic extensions over long distances to make contact with other cells; (d) single polyfilamentous cells, their cytoplasm extending into filaments and forming a branched network. The polyfilamentous cells also form syncytia and can show regional differentiation into pigment. Partial success in separating the above types of cells has shown that some of them differ intrinsically. The differences are reflected in morphological differentiation in differential response to calcium, and in amount of hyalin produced.     

Gastrointestinal stem cells. II. Intestinal stem cells

Current views of the identity, distribution, and regulation of small intestinal epithelial stem cells and their immediate progeny are discussed. Recent works implicating Wnt signaling in stem and progenitor proliferation, the involvement of Notch signaling in epithelial lineage specification, and the role of hedgehog and bone morphogenetic protein families in crypt formation are integrated. We had the good fortune that many of these papers came in pairs from independent groups. We attempt to identify points of agreement, reinterpret each in the context of the other, and indicate directions for continued progress.