Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of [³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.     

Effects of con A and other lectins on pure 5'nucleotidase isolated from lymphocyte plasma membranes.

Inhibition of purified or membrane-bound 5′nucleotidase by various lectins was studied in lymphocytes from pig mesenteric lymph nodes. Con A or $\text{Lens culinaris}$ lectin LcH inhibited (75 %) purified 5′nucleotidase by a non-competitive process without cooperativity. Inhibition by these lectins of 5′ nucleotidase activity in whole lymphocytes, plasma membranes (untreated or solubilized) and LcH-receptor fraction displayed high positive cooperativity, reached higher level (90 %) and was of mixed type. An interaction between lectin receptors and 5′nucleotidase accounted for these differences. Wheat germ agglutinin (WGA) and divalent Con A which are not mitogenic for T lymphocytes had no effect on 5′nucleotidase; pokeweed mitogen (PWM), mitogen of T and B cells, was not inhibitor. When membrane proteins were cross-linked by glutaraldehyde, Con A inhibition of whole lymphocyte 5′nucleotidase presented the same properties as the purified enzyme. Possible correlation between 5′nucleotidase inhibition and lymphocyte stimulation is discussed.     

Calcium dependency of the binding and mitogenicity of phytohemagglutinin: Differentiation between calcium-dependent and independent binding events

The same amount of phytohemagglutinin binds to lymphocytes at 37 °C and at 0 °C. The binding is inhibited by calcium chelators at 37 °C, but not at 0 °C, during the very first minutes of contact between lectin and cells. N-Acetylgalactosamine in high concentrations inhibits binding at both temperatures. The binding at 0 °C is abortive in the sense that it does not result in subsequent DNA synthesis. These findings indicate that the binding of phytohemagglutin (PHA) to the glycoprotein receptor on the cell surface does not in itself activate the immediate transducer of the mitogenic signal. The binding has to be accompanied by some change which involves calcium and occurs very rapidly, but cannot take place at 0 °C, to give a proper mitogenic anchorage of PHA to the cells.     

Response of spleen lymphocytes shortly after thymectomy in adult mouse.

The blastogenesis response to the phytomitogens, PHA-P, Con A and PWM was used to assess the effect of adult thymectomy on the spleen lymphocytes of C57B1 mice. The mitogenic response to the phytomitogens was determined by 3H-thymidine uptake. The changes produced in theta-antigen bearing spleen lymphocytes were also evaluated making use of theta antibodies from AKR/S mice previously injected with splenic and thymic lymphocytes from CBA/J mice. The present results show that the response to mitogens PHA-P and Con A is reduced early after thymectomy while the response to PWM was only slightly reduced. There was not any correlation between the disminished response to mitogens and the changes observed in theta bearing spleen lymphocytes.     

Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of ¹²⁵I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with ¹²⁵I-NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of ¹²⁵I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.     

Preparation and properties of metal ion derivatives of the lentil and pea lectins

Lentil lectin (LcH) and pea lectin (PSA) belong to the class of D-glucose/D-mannose binding lectins and resemble concanavalin A (Con A) closely in physicochemical, structural, and biological properties. LcH and PSA, like Con A, are Ca2+-Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. Studies of the relationship between the metal ions binding and saccharide binding activity in LcH and PSA have been difficult due to the problem of metal ion replacement in these proteins. We now report a method of metal ion replacement in both lectins that allows substitution of the Mn2+ in the native proteins with a variety of transition metal ions, as well as substitution of the Ca2+ with Cd2+ in a particular complex. The following metal ion derivatives of both LcH and PSA have been prepared: Ca2+-Zn2+, Ca2+-Co2+, Ca2+-Ni2+, and Cd2+-Cd2+. All of these derivatives are as active as the native lectins, as demonstrated by precipitation with specific polysaccharides, saccharide inhibition of precipitation, and hemagglutination assays. The yields of these derivatives are good (generally greater than 70%), and the degree of metal ion incorporation is high (generally greater than 90%). The method of preparation is quite different from that for metal ion substitution in Con A, which proceeds via the apoprotein. In contrast, the apoproteins of LcH and PSA are unstable, aggregate above pH 4.0, and cannot be remetallized once formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

The effects of concanavalin A on the mobility of lymphocyte surface receptors

It has been found that concanavalin A (Con A) bound to the lymphocyte surface can either induce cap formation or inhibit cap formation of various receptors including those for Con A itself. The expression of these antagonistic activities is highly dependent on the conditions under which cells are incubated with Con A. Incubation with Con A at 37 °C resulted in cap formation in only a small percentage of the cells and inhibited patch and cap formation induced by other reagents such as anti-immunoglobulin. In contrast, incubation of cells with Con A at 4 °C, followed by removal of unbound Con A molecules and elevation of the temperature to 37 °C resulted in cap formation in more than 40 % of the cells. Quantitative analyses suggest that these effects involve cross-linkage of Con A receptors, which occur in two states, mobile and relatively immobile. A model is proposed to explain the various effects of Con A in terms of the association of these receptors with colchicine binding proteins.     

Macrophage dependency of T-lymphocyte mitogenesis by Nocardia rubra cell-wall skeleton.

The mitogenic activity of the cell-wall skeleton (CWS) of Nocardia rubra on purified splenic T-cells (thymus-derived lymphocytes) was investigated. N. rubra CWS showed remarkable mitogenic activity on normal spleen cells of C57BL/6J mice at concentrations ranging from 10 to 100 microgram per milliliter of culture medium, while, on purified splenic T-cells, N. rubra CWS did not act as an mitogen at any concentration. However, mitogenic activity of N. rubra CWS on T-cells was restored if purified splenic T-cells was reconstituted with X-irradiated peritoneal exudate cells (macrophages). The above results suggest the necessity of macrophages for T-lymphocyte activation by N. rubra CWS as well as PHA-P or Con A.     

Lectin-mediated sperm agglutination of bufo arenarum and leptodactylus chaquensis spermatozoa

Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination.     

Agglunation of a transformed mouse cell line and a variant subline with concanavalin A: Effect of temperature and time of lectin incubation

Colchicine treatment enhanced Con A-mediated agglutination of erythrocytes to LM cells (LM is a “spontaneously” transformed mouse line) incubated for brief periods with Con A at 22° C. Longer incubations with Con A at 22° C rendered colchicine treated cells less agglutinable than untreated cells. Even short incubation times with Con A at higher temperature (37° C) rendered colchicine treated LM cells less agglutinable than their untreated counterparts. Below 15° C, colchicine treated cells remained more agglutinable than untreated cells even after long periods of Con A treatment. Cells of a variant clone (Rl) isolated from LM by negative selection with concanavalin A exhibited increased substratum adhesiveness and an absolute serum requirement. LM and variant cells exhibited a differential reponse to colchicine treatment, the variant subline reguiring longer periods of colchicine treatment to elicit changes in morphology and agglutinability.     

Accumulation of eosinophils and monocytes in lymphoid organs of chick-embryos. II. Effect of mitogenic stimulation.

The mitogen effect on migration of eosinophils and monocytes was studied in embryonic chickens. On the 13th embryonic day, chickens were injected with mitogens, such as concanavalin A (Con A), phytohemagglutinin-P (PHA-P), and lipopolysaccharide (LPS), into the allantoic cavity, and the mitogenic effect was estimated from the relative frequencies of eosinophils and monocytes by enumerating the number of oxidase positive cells (OPC) in the spleen, thymus, and bursa of Fabricius. Splenic frequencies of OPC increased in the embryos treated with mitogens. Similar influences were also detected in the thymic OPC. Higher responses were seen on the 18th embryonic day in the number of splenic OPC when embryos were treated with Con A or PHA-P than with LPS. These findings suggest that Con A and PHA-P are preferential OPC accumulation promoters. However, bursal frequencies of OPC in the cortex were low after mitogenic stimulations when compared with controls, although appreciable responses were detected in the bursal medulla after LPS stimulation. These results suggest that the migration pattern in the population of eosinophils and monocytes is affected not only by T cell mitogens but is also derived from LPS stimulation.     

The isolation and properties of the dimeric subunit of concanavalin A

Concanavalin A (Con A) was dissociated into dimeric and monomeric subunits by incubation at 37°C in acetate buffer of pH 3.8 containing 0.5% sodium dodecyl sulfate. The dimer was isolated in pure form by a density gradient ultracentrifugation method. Several properties of the dimer were determined including the formation of a precipitin with anti-Con A antibodies, the molecular weight, the lack of a binding site for glycogen, the lack of mitogenic activity for spleen lymphocytes, and the lack of inhibition by -methyl d-glucoside. The latter findings differ from results reported by other investigators.     

Mitogenic receptors on human peripheral blood lymphocytes: the interaction of Phaseolus vulgaris erythroagglutinating phytohemagglutinin and anti-thymocyte globulin on the human peripheral blood lymphocyte membrane.

A horse anti-human thymocyte antibody (ATG) obtained from the Upjohn Company was shown to stimulate DNA synthesis in human lymphocytes with a time course and magnitude of radioactive thymidine uptake comparable to that seen with phytohemagglutinin (E-PHA) and concanavalin A (Con A). Low mitogenic or nonmitogenic concentrations of intact ATG or its Fab fragments inhibited E-PHA-induced mitogenesis, whereas the response to Con A was unaffected. Competitive binding studies with ATG and E-PHA revealed mutual inhibition of binding to lymphocytes suggesting that E-PHA and the ATG share a common receptor site on the cell surface. ATG binding was unaffected by Con A. From the analysis of the binding data and the inhibition of mitogenesis, it appears that at least part of the E-PHA response in human lymphocytes involves receptors that are not acted on by Con A.     

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Summary Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides

Summary Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4°C, and internalized the peptide after 10 min at 37°C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4°C had specific surface labelling. After 30 s at 37°C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4°C. After 5 min at 37°C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.     

Correlation of the fluorescent banding pattern and ultrastructure of a human chromosome

After incubation with Con A, cultured melanoma cells B16-C2W stuck firmly to the dish wall and could be detached neither with trypsin and pronase treatment nor with EDTA treatment, whereas the control cells were easily released from the dish wall by the same treatments. This effect became evident within 5 min of incubation at 37 °C with Con A in a greater concentration than 5 μg/ml. Resistance to trypsinization arose more rapidly as the temperature increased up to 22 °C and changed substantially around 15 °C. By addition of α-methyl-d-mannoside which combines specifically with Con A, the resistant cells became de novo susceptible to trypsinization within 5 min of incubation at 37 °C. The reversing effect of the inhibitor was also temperature dependent. The appearance of resistance to trypsinization was observed without divalent cations (Ca²⁺ and Mg²⁺), but was inhibited by pretreatment with 10^?4 M 2,4-dinitrophenol. The temperature-dependence and the concentration of Con A required for agglutination of freed cells was the same as for induction of resistance to trypsinization.     

Binding of concanavalin A to Chinese hamster ovary cells after storage in liquid nitrogen.

The binding of Concanavalin A to the surface of Chinese Hamster Ovary (CHO) cells after storage in liquid nitrogen was studied by a number of techniques. Electron microscope cytochemistry revealed that cells frozen at 1.7 °C/min resulting in high viability had a Con A positive surface coat similar to non-frozen control cells whereas those that had been frozen at 200 °C/min, resulting in low viability, exhibited a range of staining patterns with many cells showing a loss of the coat. However, the binding of I¹²⁵-labeled Con A to the frozen cells revealed no overall loss of binding sites as compared to non-frozen cells although those frozen at 1.7 °C/min appeared to have two types of binding sites whereas the cells frozen at 200 °C/min had only one. Agglutination studies showed that the frozen cells had reduced levels of agglutinability.