Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation.

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Activated macrophages use different cytolytic mechanisms to lyse a virally infected or a tumor target.

Murine bone-marrow-culture-derived-macrophages can be differentially activated to lyse either vesicular stomatitis virus infected BALB/c3T3 cells or the tumor target P815. Macrophages were activated in a manner so that they could lyse both targets. The ability of this activated population to lyse either target type was differentially inhibited by varying the assay conditions. The lysis of P815 targets was more sensitive to inhibition by the proteinase inhibitor N-p-tosyl-L-lysine chloromethyl ketone than was the lysis of virally infected cells. On the other hand, reduction of the concentration of glucose in the assay medium, which inhibits the production of oxygen metabolites by the hexose monophosphate shunt, or the addition of anti-tumor necrosis factor (anti-TNF) serum were able to decrease the lysis of virally infected targets but not P815 targets. Thus, the observed differences in the lysis of these two targets were due to both the activation state of the macrophages and the differential susceptibility of the targets to different effector mechanisms.

Membrane channel formation by the lymphocyte pore-forming protein: comparison between susceptible and resistant target cells.

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.

A protein factor that enhances amsacrine-mediated formation of topoisomerase II-DNA complexes in murine mastocytoma cell nuclei.

Extracts of K21 murine mastocytoma cells contain a factor that enhances formation of amsacrine-induced topoisomerase II-DNA complexes (PDCs) when added to isolated K21 nuclei. The PDC-enhancing activity is reduced in extracts from 2 or 6 h cycloheximide or cordycepin-treated cells, implying that continuous protein synthesis is required to maintain the factor. The factor is heat-labile, proteinase-sensitive and has other properties that distinguish it from the two known classes of topoisomerases. The data suggest that the factor is a labile protein with a molecular weight in excess of 50,000. This appears to be the first direct evidence of a protein factor that modulates drug-induced topoisomerase II action.

Induction of granulopoiesis of mastocytoma cells: ultrastructural and cytochemical studies on production of serotonin in a cultured mouse mastocytoma cell line.

Electron microscopic observations of an originally established mouse mastocytoma cell line (BSP-MST-2) revealed that the cytoplasm of many of the MST-2 cells contained small and low osmiophilic granules and a few mature electron-dense granules. Fluorescent- and immuno-histochemical examinations also suggested the immaturity of granules as the cytoplasmic reaction for serotonin (5-HT) was weak. Induction of further maturation of granules was investigated by administration of various chemical agents. Among the chemicals examined, sodium butyrate and hydrocortisone were effective. In the presence of 1 mM sodium butyrate for 24 h, the cytoplasmic granules contained an abundant dense matrix. MST-2 cells incubated with hydrocortisone at 5 micrograms/ml for 24 h showed a somewhat different granulopoietic pattern from those incubated with sodium butyrate, including numerous electron-dense progranules. Fluorescent- and immuno-histochemical studies showed increased reactions of cytoplasmic 5-HT of both butyrate- and hydrocortisone-treated MST-2 cells. The specificity of these morphological and cytochemical changes was confirmed by treatment with reserpine, a drug which depletes cellular 5-HT; electron-dense materials were virtually diminished and cytochemical reactions were significantly decreased. The mode of induced production of 5-HT in mastocytoma granules is discussed, in relation to mastocyte differentiation.

Effects of mast cell-derived mediators on epithelial cells in canine trachea.

We examined the interaction between mast cell-derived mediators and the electrical and ion transport properties of canine tracheal epithelium. We compared the effect of mediators released by immunologic challenge of sensitized lung parenchyma with that of mediators released from canine mastocytoma cells challenged with calcium ionophore A23187. Short-circuit current (Isc) increased by 19.2 +/- 3.0 microA/cm2 in response to mediators released from sensitized lung fragments challenged with ragweed antigen. This effect was not due to histamine. When the epithelial tissues were pretreated with indomethacin, the same mediator supernatant increased Isc by only 3.8 +/- 4.3 microA/cm2. The mediators released from 10(7) mastocytoma cells challenged with calcium ionophore increased Isc by 25.1 +/- 13.6 microA/cm2. In the presence of indomethacin, the Isc increased by 2.0 +/- 0.4 microA/cm2. Mastocytoma-derived mediators produced an increase in net chloride secretion without a significant effect on net sodium absorption. This study provides direct evidence that mast cell-derived mediators can stimulate epithelial ion transport in canine trachea and suggests that the effect is indirect and dependent on intact cyclooxygenase pathways in the tracheal epithelium.

Quantification of the strength of cell-cell adhesion: the capture of tumor cells by activated murine macrophages proceeds through two distinct stages.

The binding of tumor cells by macrophages activated with Bacillus Calmette-Guerin is a necessary step toward destruction of those cells. Although several characteristics of the interaction have been defined, little is known of how the actual binding process develops. We used a technique to quantify the forces required to disrupt cell-cell interactions. Over a range of applied relative centrifugal forces, the majority of targets that bound to the activated macrophages fell on two distinct plateaus. Approximately 90% of added targets were bound to the monolayers of macrophages over the range of 1 to 100 X G; 25 to 30% remained bound from 1200 X G to 1500 X G. Two strengths of binding, termed weak and strong binding, respectively, were thus defined on the basis of these curves. Strong binding developed only between activated macrophages and tumor cells. By contrast, weak interactions occurred between either activated or nonactivated macrophages and neoplastic or non-neoplastic target cells. The strong binding required time (60 to 90 min), metabolic activity by the macrophages, and trypsin-sensitive surface structures on the macrophages for development, whereas the weak interaction occurred rapidly and required none of these. Additional evidence indicated the weak binding developed into strong when activated macrophages bound neoplastic cells. This stabilization increased the strength of force to separate tumor cells from the macrophages at least approximately 15 fold (i.e., from approximately 16 mu dynes/cell to approximately 240 mu dynes/cell). Of note, the development of strong binding of antibody-coated targets had distinct requirements for establishment. Taken together, the data suggest the stabilization of binding (i.e., the development of weak into strong binding) leading to effective cell-cell interaction is a complex and dynamic process that may vary depending upon the recognition system involved.

The antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay.

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.

[Sensitivity of mice to systemic hyperthermia: effect of ascites tumor cell transplants]. / Sensibilité de la Souris à l'hyperthermie systémique: influence d'une transplantation des cellules de la tumeur ascitique.

The effect of a transplantation of mastocytoma cells in the abdominal cavity on the sensitivity of mice to a systemic hyperthermia was studied. The systemic hyperthermia was induced by exposing whole-body of animals to 2,450 MHz waves under anesthesia. Core body temperature was raised up to 42.0 +/- 0.2 degrees C in 15 min and maintained constant at the temperature for variable length of time. Thermosensitivity of animal was expressed with LD50, 42 degrees which was the length of heating time at the temperature of 42 degrees C lethal for 50% of the animals examined. The transplants were mastocytoma FMA3 cells. They were transplanted at a dose of 10(5) cells per mouse. The LD50, 42 degrees observed 3, 12 hrs, 1, 2, 3 and 6 days after the transplantation was 33, 23, 17, 24 and 35 min, respectively. In mice without tumor it was 43 min.

Adherence of tumor cells to endothelial monolayers: inhibition by lymphokines.

Tumor cells can adhere to endothelial cell monolayers in vitro. The kinetics of this reaction are rapid; 50% of maximal binding occurs by 30 min of incubation. In the case of the P815 mastocytoma, the maximal percentage of binding is approximately 70%, suggesting that there are both binding and nonbinding tumor cell populations. Binding is independent of tumor cell dose over a 200-fold range of cell concentrations. Lymphokine-containing preparations were found to markedly suppress the binding of either P815 mastocytoma or Ehrlich ascites cells to endothelium. This effect appeared to be due to both diminished attachment and enhanced dissociation. The activity is found in the same molecular weight range as tumor migration inhibition factor (TMIF), and is not found in preparations lacking TMIF activity. Thus, the factor may prove to be TMIF itself or a lymphokine related to it. Of equal interest is the possibility that it represents a previously undescribed factor.